Name: dissection of adult mouse stria vascularis for single-nucleus sequencing or immunostaining
Uploaded: 2023-04-21T12:50:00.000+00:00
Duration: 8 min 51 s
Description: Watch this Scientific Journal Video about Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining at JoVE.com

This research was supported in part by the Intramural Research Program of the NIH, NIDCD to M.H. (DC000088)

All animal experiments and procedures were performed according to protocols approved by the Animal Care and Use Committee of the National Institute of Neurological Diseases and Stroke and the National Institute on Deafness and Other Communication Disorders, National Institutes of Health. All experimental protocols were approved by the Animal Care and Use Committee of the National Institute of Neurological Diseases and Stroke and the National Institute on Deafness and Other Communication Disorders, National Institutes of Health. All methods were carried out in accordance with relevant guidelines and regulations of the Animal Care and Use Committee of the National Institute of Neurological Diseases and Stroke and the National Institute on Deafness and Other Communication Disorders, National Institutes of Health.
1. Animal euthanasia
<ol>
	<li>Use C57BL/6J or Kcnj10-ZsGreen transgenic mice, postnatal day 30+ (P30+), that weigh between 15-20 g.</li>
	<li>Euthanize an adult mouse (age P30 or older) using an approved protocol (here CO2 asphyxiation). Perform decapitation as a secondary step. 
	NOTE: If immunostaining, some antibodies may have background signal due to nonspecific binding to blood in the capillaries of the SV. Cardiac perfusion with fixative can address this issue by removing blood from the capillaries of the SV. However, most antibodies can achieve good results without this method, and it will not be covered in this protocol.</li>
</ol>
2. Exposing bony labyrinth
<ol>
	<li>Clean the mouse by spraying with 70% ethanol and wiping with a paper towel. Pay particular attention to the head region to reduce the risk of contamination. Remove the skin from the skull by pulling the skin toward the nose.</li>
	<li>Using sharp scissors, split the skull along the midsagittal plane to separate the left and right portions.</li>
	<li>Remove the brain from the skull to expose the bony labyrinth.</li>
</ol>
3. Inner ear extraction
<ol>
	<li>Identify the inner ear within the temporal bone (Figure 2A) and scrape away the cranial nerves using #55 forceps. 
	NOTE: The user should stabilize the specimen with their nondominant hand using a second set of #55 forceps. Different areas of the specimen may be grabbed with the stabilizing forceps throughout the dissection if critical areas of the specimen are avoided (e.g., do not crush the cochlea).</li>
	<li>Using #55 forceps, dissect out the bulla and capsule, detaching them from surrounding bone. Remove any remaining soft tissue from the inner ear surface.</li>
	<li>Transfer the specimen to a clear tissue culture dish containing 1x cold PBS (phosphate-buffered saline), pH 7.4, and place under a dissection scope (Figure 2B).</li>
</ol>
4. SV dissection
NOTE: With practice, it is possible to dissect the SV as one long piece resembling a ribbon. The SV is fragile, so if it breaks into pieces, these may be stored together. Alternatively, these can be stored in separately labeled wells according to their turn (e.g., basal, middle, apical).
<ol>
	<li>If the dissection scope light sources can be moved, place one light above the dish and the second light source from the side, parallel to the dissecting surface. This allows for a better contrast of the tissue under the cochlear bone.</li>
	<li>Using #55 forceps, hold the specimen by the vestibular portion with the cochlea facing up.</li>
	<li>Using #55 forceps, pierce through the cochlear bone at the apex. 
	NOTE: #5 forceps are thicker than #55 and may be used for breaking bone, but the increase in size/strength sacrifices the fineness of the forceps and the ability to manipulate small tissues. Consider using forceps made with a material such as inox or dumostar to avoid damage that may occur to more delicate forceps. Avoid pushing the forceps too deep under the bone to avoid damaging the SV.</li>
	<li>Using #55 forceps, scrape along the apical turn (Figure 2C), applying gentle force to break away small pieces of the outer bone layer. Gently lift the bone and detach it from the lateral wall. 
	NOTE: It may be helpful to think about this dissection as &#34;removing everything away from the SV&#34;, rather than &#34;dissecting the SV out of the cochlea&#34;.</li>
	<li>Continue using #55 forceps to remove cochlear bone pieces from the apical and middle turns (Figure 2D,E). Gently pushing down the lateral wall, pry and remove bone wall pieces toward the middle turn to expose the lateral wall of the apical and middle turns.</li>
	<li>Continuing using #55 forceps, gently push the apical turn lateral wall aside to expose the spiral ganglion. Detach the lateral wall of apical and middle turns from the spiral ganglion along the outer hair cell layer. Remove pieces of spiral ganglion from inside the cochlea. 
	NOTE: #55 forceps are smaller and provide an advantage when manipulating small and delicate tissues. Extra care should be taken to avoid bending or damaging them.</li>
	<li>To have better access to the lateral wall of the basal turn, detach the cochlea from the vestibular portion of the temporal bone using #55 forceps. Put the #55 forceps into the round and oval window and push down toward the vestibule. The cochlea will detach. Remove the vestibular portion of the inner ear.</li>
	<li>Continuing using #55 forceps, now remove pieces of basal cochlear bone, starting from the middle turn area. Gently push the lateral wall layer under the bone to detach it.</li>
	<li>Remove the farthest basal part of the lateral wall by prying and gently pulling the tissue. The bone in this region may be too thick to be removed without damaging the soft tissue below.</li>
	<li>Now, with the basal part of the lateral wall detached, separate as much of the lateral wall from the cochlea as possible. This can be done by tracing the #55 forceps along the lateral wall. Gently brush the forceps between the bone and the remaining lateral wall. This can be done all the way to the apex in one piece if the user is experienced. Move the lateral wall to fresh PBS. 
	NOTE: Although larger pieces of SV may be easier to image, given the delicate nature of the tissues, smaller pieces can be taken (Figure 2F,G), and depending on the size, can also work for imaging.</li>
	<li>Use #55 forceps to lay the lateral wall flat. The SV should be visible as a darker layer of tissue on the internal side of the lateral wall. 
	NOTE: Users may find, particularly closer to the apical end, that some of the SV tissue incidentally detaches from the lateral wall throughout the dissection.</li>
	<li>Gently pry the SV layer to detach it from the lateral wall at its basal end (Figure 2H). Gently push aside the detached SV and move the forceps further toward the apical end of the lateral wall, detaching the SV as one long ribbon if possible (Figure 2I). Do not squeeze the SV with forceps to pull it. If the tissue is too fragile to be grabbed, it can alternatively be collected using a tissue scoop, such as a chalazion curette. 
	NOTE: Moving the SV should always be done under light microscopy to ensure it is not lost. Always be careful when grabbing using forceps due to the risk of damage. Users may find that using chalazion curette mitigates the risk of damage to SV tissue. For single-nucleus sequencing, proceed to section 5. For SV immunostaining, proceed to section 7.</li>
</ol>
5. SV single-nucleus suspension
NOTE: This protocol is adapted for SV tissue specifically from a published manufacturer&#39;s single-nucleus suspension protocol. Platforms from different manufacturers may be used31. Given platform-specific variation, it is recommended to review the manufacturer-specific protocol provided with the equipment. To achieve optimal results for sNuc-Seq and minimize RNA degradation, the faster the tissue dissection the better (recommended within 15-20 min from euthanasia). It may be helpful to euthanize one animal at a time and only when ready to dissect. Having multiple people simultaneously work on the dissections can also eliminate degradation time (e.g., one lab personnel working on the left ear while another works on right ear).
<ol>
	<li>If the SV tissue will be used for sNuc-Seq, place the tissue in chilled lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.005% nonidet P40 in nuclease free water). 
	NOTE: If sequencing is to be done immediately following dissection, continue the protocol. If it is desired to pause the protocol, SV preservation is possible by placing the SV into RNAlater. Store overnight at 4 &#176;C, then flash freeze in liquid nitrogen and store at -80 &#176;C until ready to use. When ready to use, thaw and wash twice in PBS for 5 min each, then proceed with homogenization (step 5.2).</li>
	<li>Homogenize the tissue in a 2 mL dounce homogenizer with 10-20 strokes on ice. 
	NOTE: The glass cylinder will manually contact the bottom of the glass tube, fragmenting the SV. The SV is delicate, so 20 strokes usually achieves successful homogenization.</li>
	<li>Lyse the tissue on ice for 25 min. 
	NOTE: The lysis time varies according to the tissue type. A 25 min lysis in SV tissue may achieve a low cell viability (less than 4%), but the time may be adapted if the cell viability is too high (step 5.8).</li>
	<li>Filter through a 30 &#181;m filter and spin the filtrate at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Remove the supernatant and resuspend the cell pellet in 1 mL of nuclei wash and resuspension buffer (1x PBS, 1% bovine serum albumin [BSA], 0.2U/&#181;L RNase inhibitor).</li>
	<li>Filter the cells through a 10 &#181;m filter and centrifuge at 500 x g for 5 min at 4 &#176;C.</li>
	<li>Remove the supernatant and resuspend in 50 &#181;L of nuclei wash and resuspension buffer.</li>
	<li>Count the nuclei using a cell counter and trypan blue staining. Dilute 5 &#181;L of the cell suspension into 50 &#181;L of 1x PBS for cell counting; The viability should be minimal (3%-4%) at this point. If the viability is found to be higher, adapt the protocol for step 5.3 (lysis) and standardize the lysis time to ensure adequate lysis of the cells. 
	NOTE: A high cell viability in a single nucleus preparation means there are more intact cells than desired, and that additional digestion may be required.</li>
	<li>Load the sample with the desired nuclear density onto the manufacturer&#39;s apparatus/chip (see manufacturers guide). Single-nuclei captures are now ready. 
	&#8203;NOTE: From one adult mouse SV, 50 &#181;L of the suspension at a concentration of 150,000 nuclei/mL is usually achieved.</li>
</ol>
6. SV single-nucleus sequencing
<ol>
	<li>Send the samples to the core facility for sequencing. 
	NOTE: This part of the protocol follows the general steps of (1) gel beads-in emulsion (GEM) generation and barcoding, (2) post-GEM-RT cleanup and cDNA amplification, and (3) 3&#39; gene expression library construction. The remainder of the sNuc-Seq protocol is relatively lengthy, and it is recommended that readers use their manufacturer-specific protocol. The manufacturer guide will likely describe steps with specific details and accompanying images. There may also be &#39;how-to&#39; videos on the manufacturer website. Datasets generated may be represented in a dimensional reduced fashion, such as a uniform manifold approximation and projection (UMAP; Figure 3). Many labs utilize a sequencing core that may perform this protocol for users.</li>
</ol>
7. SV immunostaining and tissue mounting
<ol>
	<li>If the tissue will be used for immunostaining, transfer it to a 24-well plate, submerged in 200 &#181;L of 4% paraformaldehyde (PFA) in 1x PBS, and incubate for 20 min at room temperature. 
	NOTE: It is helpful to perform all liquid removal, washing, and immunostaining underneath a microscope. Visualization while pipetting the tissue will ensure it is not accidently lost during liquid removal. The volumes of the antibodies and washes may be adjusted as long as the volume is large enough to submerge the SV tissue within the solution.</li>
	<li>After the fixation step, remove the PFA by performing two short washes in 1x PBS (approximately 500 &#181;L) at 4 &#176;C for 5 min each on an orbital shaker at low (30-50) RPM. If storing, the tissue can be stored in 1x PBS at 4 &#176;C.</li>
	<li>Remove the PBS and permeabilize and block for a minimum of 1 h at room temperature in approximately 300 &#181;L of PBS-T solution (0.05 Tween20 in 1x PBS with 10% fetal bovine serum). 
	NOTE: Primary and secondary antibodies should be diluted in this blocking solution.</li>
	<li>Stain the tissue with primary antibody according to the specifications of the particular antibody used, usually overnight at 4 &#176;C . Remove the remaining PBS and add the diluted primary antibody. 
	NOTE: The dilution should be chosen based on the manufacturer suggestions, published data, previous experience, etc. Usually, the first concentration to test is a 1:100-200 dilution. For the example presented in this paper, the GS-IB4 and DAPI antibodies were each diluted at 1:200. If staining for more than one protein, multiple primary antibodies can be combined into the same solution, as long as each primary antibody has a different host to avoid cross reactivity of secondary antibodies.</li>
	<li>Wash the primary antibody off the tissue using approximately 500 &#181;L of 1x PBS twice for 10 min each on an orbital shaker at low RPM. 
	NOTE: Continue to ensure that the SV tissue is submerged in the solution, and not stuck on the side of the well.</li>
	<li>Stain with a secondary antibody according to the specifications of the particular antibody used, usually for 2 h at room temperature on an orbital shaker at low RPM. Cover the 24-well plate so the fluorescently tagged secondary antibody is protected from light. 
	NOTE: If more than one secondary antibody is needed, combine the secondary antibodies into the same solution. Ensure to avoid cross labeling.</li>
	<li>Wash the secondary antibody off the tissue using approximately 500 &#181;L of 1x PBS four times for 5 min each on an orbital shaker at low RPM. Keep the 24-well plate covered to protect it from light.</li>
	<li>Prepare the larger microslide (75 mm x 25 mm) by placing two small streaks of glue along the 25 mm axis, just smaller than the 18 mm x 18 mm smaller glass coverslip. Place one drop of mounting reagent (approximately 50 &#181;L) in between the glue streaks. 
	NOTE: The glue creates a small amount of vertical space so that when the second coverslip is placed on top, the SV tissue sample can safely exist, but continue to remain in place. While the stria thickness is 30-40 microns, squeezing the tissue between the glass surfaces should be avoided to preserve morphology. Alternatively, clear tape may also be used.</li>
	<li>Using #55 forceps, grab as little tissue as possible on one end of the SV and transfer from the PBS to the drop of mounting reagent on the larger microslide (75 mm x 25 mm). Place one end of a smaller glass coverslip (18 mm x 18 mm) on the slide where one streak of glue was placed and gently release to avoid creating air bubbles. The coverslip will fall to the other glue streak and will cover the SV tissue.</li>
	<li>Seal the mount by dabbing a drop of transparent nail polish on each corner of the mount. Label the specimen accordingly on the glass coverslip away from the visualization field. It is now ready for confocal microscopy (Figure 4).</li>
	<li>Visualize the SV tissue using a dissection microscope to ensure it is laying flat and not near any air bubbles. If the SV tissue is not oriented correctly, the user can attempt to push out air bubbles or carefully remove the smaller glass coverslip and reposition the SV. This must be done gently to avoid breaking the glass coverslips.</li>
</ol>

Stria Vascularis Microdissection for Single-Nucleus Sequencing or Immunostaining

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J., Nevill, G., Forge, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane+properties+of+cochlear+root+cells+are+consistent+with+roles+in+potassium+recirculation+and+spatial+buffering.">The membrane properties of cochlear root cells are consistent with roles in potassium recirculation and spatial buffering.</a> Journal of the Association for Research in Otolaryngology. 11 (3), 435-448 (2010).</li><li>Ito, T., Kurata, N., Fukunaga, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-resident+macrophages+in+the+stria+vascularis.">Tissue-resident macrophages in the stria vascularis.</a> Frontiers in Neurology. 13, 818395 (2022).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGFA165+gene+therapy+ameliorates+blood-labyrinth+barrier+breakdown+and+hearing+loss.">VEGFA165 gene therapy ameliorates blood-labyrinth barrier breakdown and hearing loss.</a> JCI Insight. 6 (8), e143285 (2021).</li><li>Shi, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiology+of+the+cochlear+intrastrial+fluid-blood+barrier+(review).">Pathophysiology of the cochlear intrastrial fluid-blood barrier (review).</a> Hearing Research. 338, 52-63 (2016).</li><li>Gu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+potential+Meniere's+disease+targets+in+the+adult+stria+vascularis.">Identification of potential Meniere's disease targets in the adult stria vascularis.</a> Frontiers in Neurology. 12, 630561 (2021).</li><li>Fischer, J., Ayers, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+nucleus+RNA-sequencing:+how+it's+done,+applications+and+limitations.">Single nucleus RNA-sequencing: how it's done, applications and limitations.</a> Emerging Topics in Life Sciences. 5 (5), 687-690 (2021).</li><li>Korrapati, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+and+single+nucleus+RNA-Seq+reveal+cellular+heterogeneity+and+homeostatic+regulatory+networks+in+adult+mouse+stria+vascularis.">Single cell and single nucleus RNA-Seq reveal cellular heterogeneity and homeostatic regulatory networks in adult mouse stria vascularis.</a> Frontiers in Molecular Neuroscience. 12, 316 (2019).</li><li>Pyle, M. P., Hoa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+single-cell+sequencing+for+the+field+of+otolaryngology:+A+contemporary+review.">Applications of single-cell sequencing for the field of otolaryngology: A contemporary review.</a> Laryngoscope Investigative Otolaryngology. 5 (3), 404-431 (2020).</li><li>Hwang, B., Lee, J. H., Bang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA+sequencing+technologies+and+bioinformatics+pipelines.">Single-cell RNA sequencing technologies and bioinformatics pipelines.</a> Experimental &amp; Molecular Medicine. 50 (8), 1-14 (2018).</li><li>Shafer, M. E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-species+analysis+of+single-cell+transcriptomic+data.">Cross-species analysis of single-cell transcriptomic data.</a> Frontiers in Cell and Developmental Biology. 7, 175 (2019).</li><li>Chen, G., Ning, B., Shi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+RNA-Seq+technologies+and+related+computational+data+analysis.">Single-cell RNA-Seq technologies and related computational data analysis.</a> Frontiers in Genetics. 10, 317 (2019).</li><li>Longo, S. K., Guo, M. G., Ji, A. L., Khavari, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+single-cell+and+spatial+transcriptomics+to+elucidate+intercellular+tissue+dynamics.">Integrating single-cell and spatial transcriptomics to elucidate intercellular tissue dynamics.</a> Nature Reviews Genetics. 22 (10), 627-644 (2021).</li><li>Kim, N., Kang, H., Jo, A., Yoo, S. A., Lee, H. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+single-nucleus+RNA+sequencing+in+different+cell+types+and+tissues.">Perspectives on single-nucleus RNA sequencing in different cell types and tissues.</a> Journal of Pathology and Translational Medicine. 57 (1), 52-59 (2023).</li><li>Grindberg, R. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-sequencing+from+single+nuclei.">RNA-sequencing from single nuclei.</a> Proceedings of the National Academy of Sciences. 110 (49), 19802-19807 (2013).</li><li>Montgomery, S. C., Cox, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+mount+dissection+and+immunofluorescence+of+the+adult+mouse+cochlea.">Whole mount dissection and immunofluorescence of the adult mouse cochlea.</a> Journal of Visuazlied Experiments. (107), e53561 (2016).</li></ol>

The authors have nothing to disclose.

Prior to the advent of single-cell sequencing, many researchers used bulk tissue analysis, which only made it possible to analyze transcriptomes averaged across cells. In particular, single-cell and sNuc-Seq made it possible to isolate the transcriptome of a single cell or single nucleus, respectively32. In this instance, single-nucleus transcriptomes can be identified for marginal, intermediate, and basal cells, as well as spindle cells30. This enables the investigation of transcriptional heterogeneity amongst SV cell types and can be used in the future to investigate the contribution of these cell types to SV function, including the generation and maintenance of EP. Datasets generated using sNuc-Seq can be displayed in a dimensionally reduced fashion to facilitate the understanding of overall transcriptional differences and compare different cell populations using a UMAP plot Figure 4. There are numerous ways to conduct bioinformatic analysis for the interpretation of sNuc-Seq datasets. These include but are not limited to: differential expression analysis, single-cell regulatory network inference and clustering (SCENIC), heatmap or grin violin plot construction, and regulon analysis18. Further computational analysis techniques for single-cell RNA sequencing are similar to sNuc-Seq, and are discussed in more detail in reviews by Hwang et al.32, Shafer33, and Chen et al.34.
The other major application of this dissection technique is immunostaining to better study selected structures within the SV. We can observe this in the setting of an SV whole mount that was imaged using confocal microscopy (Figure 3). The nuclei of all cells are visualized with DAPI labeling. The capillaries are seen via endothelial cell staining with GS-IB4 in red. Intermediate cells, in this case, have been genetically modified using an intermediate cell-specific promoter to express ZsGreen.
Limitations of sNuc-Seq include: (1) lack of spatial information, (2) biases against immune cell populations, and (3) exclusion of cytoplasmic RNA. Spatial transcriptomic techniques can provide more specific information about cellular subpopulations within tissue35. Given protocol differences between sNuc-Seq and single-cell RNA sequencing, such as tissue dissociation and storage, the sNuc-Seq-generated transcriptional profile may bias against immune cells while being more powerful at characterizing attached cell types36. Also, single-cell RNA sequencing includes cytoplasmic RNA such as mitochondrial RNA, while sNuc-Seq does not. Despite these instances of transcriptional differences between the two methods, it has been demonstrated that, by and large, sNuc-Seq has a similar transcriptional profile to whole single-cell RNA sequencing37, and in the SV in particular30.
For successful microdissection, there are several critical steps to be aware of. In the first few steps, one must properly extract the inner ear to avoid crushing the underlying cochlea. Observation of proper temporal bone extraction is shown in Figure 2A. Once the cochlea is properly exposed, care must be taken to gently scrape and break the cochlear bone to expose the underlying lateral wall and SV. Once the lateral wall and SV are extracted successfully, the user must carefully detach the SV from the lateral wall. Enough force in the correct plane should be applied to pry the SV from the lateral wall, while avoiding damage to the fragile SV. For SV microdissection, it may be helpful to think of "dissecting other structures away from the SV", rather than "dissecting the SV out of the cochlea".
This protocol can be modified for different ages of mice and for different mammalian species, such as rats. For younger mice, tissues will differ in texture and structures will be smaller in general. For different mammalian species, the cochlear anatomy will be relatively similar, with the size of the animal determining the majority of the difference.
In general, drawbacks of this protocol are similar to other cochlear microdissection techniques38. Isolating the delicate SV can be technically challenging and it can be broken into smaller pieces.
This technique is not optimal for saving the organ of Corti for whole mounting as it is often damaged with this protocol. There exists other dissection techniques specifically aimed at preserving the organ of Corti for whole mounting and immunostaining38, but these have their own drawbacks, considering sNuc-Seq must be done with fresh samples to avoid RNA degradation, not in the setting of tissue fixation and decalcification.
With time and repetition, users will become more comfortable with this SV microdissection protocol.

The cochlea consists of three fluid filled chambers, the scala vestibuli, scala media, and scala tympani. The scala vestibuli and scala tympani each contain perilymph, which has a high concentration of sodium (138 mM) and a low concentration of potassium (6.8 mM)1. The scala media contains endolymph, which has a high concentration of potassium (154 mM) and a low concentration of sodium (0.91 mM)1,2,3. This difference in ion concentration can be referred to as the endocochlear potential (EP), and is primarily generated by the movement of potassium ions through various ion channels and gap junctions in the stria vascularis (SV) along the lateral wall of the cochlea4,5,6,7,8,9,10,11. The SV is a heterogenous, highly vascularized tissue that lines the medial aspect of the lateral wall of the cochlea and contains three main cell types: marginal, intermediate, and basal cells12 (Figure 1).
Marginal cells are connected by tight junctions to form the most medial surface of the SV. The apical membrane faces the endolymph of the scala media and contributes to potassium ion transport into the endolymph using various channels, including KCNE1/KCNQ1, SLC12A2, and Na+-K+-ATPase (NKA)5,10,13,14. Intermediate cells are pigmented cells that reside between marginal and basal cells and facilitate potassium transport through the SV using KCNJ10 (Kir 4.1)15,16. Basal cells lie in close proximity to the lateral wall of the cochlea and are closely associated with fibrocytes of the spiral ligament to promote potassium recycling from the perilymph12. Pathology of the SV has been implicated in numerous otologic disorders17,18. Mutations in genes expressed in the major SV cell types, such as Kcnq1, Kcne1, Kcnj10, and Cldn11, can cause deafness and SV dysfunction, including the loss of EP19,20,21,22,23. In addition to the three major cell types, there are other less-studied cell types in the SV, such as spindle cells22, root cells12,24, macrophages25, pericytes26, and endothelial cells27, that have incompletely defined roles involving ionic homeostasis and the generation of EP28.
In comparison to bulk RNA-sequencing, single-nuclei RNA-sequencing (sNuc-Seq) provides information about cell heterogeneity, rather than an average of mRNA across a group of cells29, and can be particularly useful when studying the heterogenous SV30. For example, sNuc-Seq has produced transcriptional analysis that suggests there may be a role for spindle and root cells in EP generation, hearing loss, and Meniere&#39;s disease18. Further transcriptional characterization of the various SV cell types can provide us with invaluable information on the pathophysiology underlying different mechanisms and subtypes of SV-related hearing fluctuation and hearing loss. The harvest of these delicate inner ear structures is of paramount importance to optimal tissue analysis.
In this study, the microdissection approach to access and isolate the stria vascularis from the adult mouse cochlea for sNuc-Seq or immunostaining is described. Dissection of the adult mouse SV is required to understand various SV cell types and further characterize their role in hearing.

<table><tbody><tr><td>10-&amp;micro;m filter (Polyethylenterephthalat)</td><td>PluriSelect</td><td>#43-50010-01</td><td>Filter tissue during sNuc-Seq</td></tr><tr><td>18 x 18 mm cover glass</td><td>Fisher Scientific</td><td>12-541A</td><td>Cover slip to mount SV</td></tr><tr><td>30-&amp;micro;m filter (Polyethylenterephthalat)</td><td>PluriSelect</td><td>#43-50030-03</td><td>Filter tissue during sNuc-Seq</td></tr><tr><td>75 x 25 mm Superfrost Plus/Colorforst Plus Microslide</td><td>Daigger</td><td>EF15978Z</td><td>Microslide to mount SV on</td></tr><tr><td>C57BL/6J Mice</td><td>The Jackson Laboratory</td><td>RRID: IMSR_JAX:000664</td><td>General purpose mouse strain that has pigment more easily seen in the intermediate cells of the SV.</td></tr><tr><td>Cell Counter</td><td>Logos Biosystems</td><td>L20001</td><td>Used for cell counting</td></tr><tr><td>Chalizon curette 5&#039;&#039;, size 3 2.5 mm</td><td>Biomedical Research Instruments</td><td>15-1020</td><td>Used to transfer SV</td></tr><tr><td>Chromium Next GEM single Cell 3&#039; GEM Kit v3.1</td><td>Chromium</td><td>PN-1000141</td><td>Generates single cell 3&#039; gene expression libraries</td></tr><tr><td>Clear nail polish</td><td>Fisher Scientific</td><td>NC1849418</td><td>Used for sealing SV mount</td></tr><tr><td>Corning Falcon Standard Tissue Culture Dishes, 24 well</td><td>Corning</td><td>08-772B</td><td>Culture dish used to hold specimen during dissection</td></tr><tr><td>DAPI</td><td>Invitrogen</td><td>D1306, RRID: AB_2629482</td><td>Stain used for nucleus labeling</td></tr><tr><td>Dounce homogenizer</td><td>Sigma-Aldrich</td><td>D8938</td><td>Used to homogenize tissue for sNuc-seq</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11252-30</td><td>General forceps for dissection</td></tr><tr><td>Dumont #55 Forceps</td><td>Fine Science Tools</td><td>11255-20</td><td>Forceps with fine tip that makes SV manipulation easier</td></tr><tr><td>Fetal Bovine Serum</td><td>ThermoFisher</td><td>16000044</td><td>Used for steps of sNuc-Seq</td></tr><tr><td>Glue stick</td><td>Fisher Scientific</td><td>NC0691392</td><td>Used for mounting SV</td></tr><tr><td>GS-IB4 Antibody</td><td>Molecular Probes</td><td>I21411, RRID: AB-2314662</td><td>Antibody used for capillary labeling</td></tr><tr><td>KCNJ10-ZsGreen Mice</td><td>n/a</td><td>n/a</td><td>Transgenic mouse that expresses KCNJ10-ZsGreen, partiularly in the intermediate cells of the SV.</td></tr><tr><td>MgCl2</td><td>ThermoFisher</td><td>AM9530G</td><td>Used for steps of sNuc-Seq</td></tr><tr><td>Mounting reagent</td><td>ThermoFisher</td><td>#S36940</td><td>Mounting reagent for SV</td></tr><tr><td>Multiwell 24 well plate</td><td>Corning</td><td>#353047</td><td>Plate used for immunostaining</td></tr><tr><td>NaCl</td><td>ThermoFisher</td><td>AAJ216183</td><td>Used for steps of sNuc-Seq</td></tr><tr><td>Nonidet P40</td><td>Sigma-Aldrich</td><td>9-16-45-9</td><td>Used for steps of sNuc-Seq</td></tr><tr><td>Nuclease free water</td><td>ThermoFisher</td><td>4387936</td><td>Used for steps of sNuc-Seq</td></tr><tr><td>Orbital shaker</td><td>Silent Shake</td><td>SYC-2102A</td><td>Used for steps of immunostaining</td></tr><tr><td>PBS</td><td>ThermoFisher</td><td>J61196.AP</td><td>Used for steps of immunostaining and dissection</td></tr><tr><td>RNA Later</td><td>Invitrogen</td><td>AM7021</td><td>Used for preservation of SV for sNuc-Seq</td></tr><tr><td>Scizzors</td><td>Fine Science Tools</td><td>14058-09</td><td>Used for splitting mouse skull</td></tr><tr><td>Tris-HCl</td><td>Sigma-Aldrich</td><td>15506017</td><td>Used for steps of sNuc-Seq</td></tr><tr><td>Trypan blue stain</td><td>Gibco</td><td>15250061</td><td>Used for cell counting</td></tr><tr><td>Tween20</td><td>ThermoFisher</td><td>AAJ20605AP&amp;nbsp;</td><td>Used for steps of sNuc-Seq</td></tr><tr><td>Zeiss STEMI SV 11 Apo stereomicroscope</td><td>Zeiss</td><td>n/a</td><td>Microscope used for dissections</td></tr></tbody></table>

dissection of adult mouse stria vascularis for single-nucleus sequencing or immunostaining

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell mechanotransduction and ultimately hearing. Pathologies of the stria vascularis can result in a decreased hearing. Dissection of the adult stria vascularis allows for focused single-nucleus capture and subsequent single-nucleus sequencing and immunostaining. These techniques are used to study stria vascularis pathophysiology at the single-cell level. 
Single-nucleus sequencing can be used in the setting of transcriptional analysis of the stria vascularis. Meanwhile, immunostaining continues to be useful in identifying specific populations of cells. Both methods require proper stria vascularis dissection as a prerequisite, which can prove to be technically challenging.

We present a method to isolate the SV to be used for either sNuc-Seq or immunostaining. The relevant anatomy (Figure 1) of the cochlea relative to the SV can help users better understand the organization of the SV and steps of the dissection protocol.
Each step of this microdissection of SV from a P30 mouse is detailed in the associated video, and snapshots of the key steps of this dissection and isolation of SV are presented in Figure 2.
sNuc-Seq is useful in investigating the transcriptional profile of the various cells in the heterogenous SV. One way this may be visualized is by clustering on a 2D UMAP (Figure 3). The dataset generated from sNuc-Seq may be evaluated using different data analysis techniques, further outlined in the discussion.
SV whole mounting with GS-IB4 and DAPI immunolabeling, along with Kcnj10-ZsGreen fluorescence, are presented in Figure 4. Using florescence, the SV can be visualized after removing the surrounding tissues and mounting. In these mice, ZsGreen is expressed particularly in the SV intermediate cells. The vasculature of the SV can be visualized in red (endothelial cells, GS-IB4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65254/65254fig01.jpg" /> 
Figure 1: Schematic of the stria vascularis cellular heterogeneity and organization. Stria vascularis cellular heterogeneity and organization. Schematic of the stria vascularis (SV) and its relationship to structures in the cochlea. The SV is composed of three layers of cells and is responsible for generating EP and high potassium concentration in the endolymph-containing scala media. The relationship between the marginal, intermediate, and basal cells are demonstrated with the marginal extending basolateral projections to interdigitate with the intermediate cells, which have bidirectional cellular projections that interdigitate with both the marginal and basal cells. In addition to these cell types, other cell types, including spindle cells (yellow), endothelial cells, pericytes, and macrophages (not shown) are present in the SV. Used with permission from Korrapati et al.30. <a href="https://www.jove.com/files/ftp_upload/65254/65254fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65254/65254fig02.jpg" /> 
Figure 2: Dissection of an adult mouse cochlea at greater than or equal to postnatal day 30 (P30+). (A) Specimen during inner ear extraction from the surrounding temporal bone during step 3.2. The inner ear is outlined in purple. (B) Specimen during step 4.2 showing the surface of the cochlea and underlying pigmented SV. (C) Specimen during step 4.4 showing bone removed from the apical turn and the exposed SV (yellow arrow). (D) Specimen during step 4.5 showing the cochlea with more bone removed from the apical and middle turns. (E) Specimen during step 4.5 showing the removal of bone covering the middle turn, showing the underlying SV (yellow arrow). (F) Example of smaller pieces of SV (yellow arrow) and the lateral wall (blue arrow) during step 4.10. (G) Example of fluorescent ZsGreen SV while the remaining lateral wall remains dark. Kcnj10-ZsGreen is particularly expressed in the intermediate cells of the SV. (H) Example of larger pieces of SV (yellow arrow) and the lateral wall (blue) being separated during step 4.12. (I) The SV fully separated from the lateral wall during step 4.12. <a href="https://www.jove.com/files/ftp_upload/65254/65254fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65254/65254fig03.jpg" /> 
Figure 3: UMAP plot of sNuc-Seq datasets from an RNAlater-treated adult stria vascularis. Samples preserved with RNAlater treatment of adult SV tissue with subsequent nuclei isolation were clustered by a modularity-based clustering method with the Leiden optimization algorithm and visualized in a dimensionally reduced fashion by a 2D UMAP plot. Each dot represents a single cell, and cells with similar transcriptional profiles are clustered together. Cell types are colored based on their expression of known genes expressed by adult SV cell types. The samples were stored in RNAlater for no more than 6 months. The SV from about five CBA/J P30 mice were used to generate this dataset. <a href="https://www.jove.com/files/ftp_upload/65254/65254fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65254/65254fig04.jpg" /> 
Figure 4. Stria vascularis whole mounting from a P30 Kcnj10-ZsGreen mouse. Confocal image of SV whole mounting from a P30 mouse, demonstrating ZsGreen expression in the intermediate cells, GS-IB4 (red) labeling endothelial cells, and DAPI (white) labeling nuclei. <a href="https://www.jove.com/files/ftp_upload/65254/65254fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining at JoVE.com

Author Spotlight: Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining  

The stria vascularis is vital to the generation of endocochlear potential. Here, we present the dissection of the adult mouse stria vascularis for single-nucleus sequencing or immunostaining.

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining

Endocochlear potential, which is generated by the stria vascularis, is essential to maintain an environment conducive to appropriate hair cell ...

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University of Pittsburgh

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2.6K Views.  National Institutes of Health. The stria vascularis is vital to the generation of endocochlear potential. Here, we present the dissection of the adult mouse stria vascularis for single-nucleus sequencing or immunostaining.

Video: Author Spotlight: Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining  

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Developmental Biology

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

Neuroscience

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death. Furthermore, these methods are generally restricted to fresh tissue, thus limiting studies on archival and bio-bank material. Single nucleus RNA sequencing (snRNA-Seq) is an appealing alternative for transcriptional studies, given that it accurately identifies cell types, permits the study of tissue that is frozen or difficult to dissociate, and reduces dissociation-induced transcription. Here, we present a high-throughput protocol for rapid isolation of nuclei for downstream snRNA-Seq. This method enables isolation of nuclei from fresh or frozen spinal cord samples and can be combined with two massively parallel droplet encapsulation platforms.

Here, we present a protocol to rapidly isolate high-quality nuclei from the fresh or frozen tissue for downstream massively parallel RNA sequencing. We include detergent-mechanical and hypotonic-mechanical tissue disruption and cell lysis options, both of which can be used for isolation of nuclei.

Probing an individual cell&#39;s gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone (SEZ) is a thin and distinct region exposed to the ventricles and cerebrospinal fluid. The isolation of this niche allows the analysis of a neurogenic stem cell microenvironment. However, extraction of small tissues for proteome analysis&#160;is challenging, especially for the maintenance of considerable measurement depth and the achievement of reliable robustness. A new method termed cryo-section-dissection (CSD), combining high precision with minimal tissue perturbation, was developed to address these challenges. The method is compatible with state-of-the-art mass spectrometry (MS) methods that allow the detection of low-abundant niche regulators. This study compared the CSD and its proteome data to the method and data obtained by laser-capture-microdissection (LCM) and a standard wholemount dissection. The CSD method resulted in twice the quantification depth in less than half the preparation time compared to the LCM and simultaneously clearly outperformed the dissection precision of the wholemount dissection. Hence, CSD is a superior method for collecting the SEZ for proteome analysis.

Cryo-section-dissection allows fresh, frozen preparation of the largest neurogenic niche in the murine brain for deep quantitative proteome analysis. The method is precise, efficient, and causes minimal tissue perturbation. Therefore, it is ideally suited for studying the molecular microenvironment of this niche, as well as other organs, regions, and species.

The subependymal neurogenic niche consists of a paraventricular ribbon of the lateral ventricular wall of the lateral ventricle. The subependymal zone ...

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by crossing the &#39;Tet-On&#39; system oTet-Cre mouse with a novel reverse tetracycline transactivator (rtTA) transgene linked to the Tert promoter, which we have demonstrated marks a novel population of adult brain stem cells. Here, administration of the tetracycline derivative doxycycline to mTert-rtTA::oTet-Cre mice will indelibly mark a population of cells that express a 4.4 kb fragment of the promoter region of the gene Tert. When combined the Rosa-mTmG reporter, mTert-rtTA::oTet-Cre::Rosa-mTmG mice will express membrane tdTomato (mTomato) until doxycycline treatment induces the replacement of mTomato expression with membrane EGFP (mGFP) in cells that also express Tert. Therefore, when these triple-transgenic lineage tracing mice receive doxycycline (the &#34;pulse&#34; period during which TERT expressing cells are marked), these cells will become indelibly marked mGFP+ cells, which can be tracked for any desirable amount of time after doxycycline removal (the &#34;chase&#34; period), even if Tert expression is subsequently lost. Brains are then perfusion-fixed and processed for immunofluorescence and other downstream applications in order to interpret changes to stem cell activation, proliferation, lineage commitment, migration to various brain niches, and differentiation to mature cell types. Using this system, any rtTA mouse can be mated to oTet-Cre and a Rosa reporter to conduct doxycycline-inducible &#34;pulse-chase&#34; lineage&#160;tracing experiments using markers of stem cells.

The ability to permanently mark stem cells and their progeny with a fluorophore using an inducible transgenic lineage tracing mouse line allows for spatial and temporal analysis of activation, proliferation, migration, and/or differentiation in vivo. Lineage tracing can reveal novel information about lineage commitment, response to intervention(s), and multipotency.

A telomerase reverse transcriptase (Tert) lineage-tracing mouse line was developed to investigate the behavior and fate of adult tissue stem cells, by ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing

The cardiac autonomic nervous system is crucial in controlling cardiac function, such as heart rate and cardiac contractility, and is divided into sympathetic and parasympathetic branches. Normally, there is a balance between these two branches to maintain homeostasis. However, cardiac disease states such as myocardial infarction, heart failure, and hypertension can induce the remodeling of cells involved in cardiac innervation, which is associated with an adverse clinical outcome.
Although there are vast amounts of data for the histological structure and function of the cardiac autonomic nervous system, its molecular biological architecture in health and disease is still enigmatic in many aspects. Novel technologies such as single-cell RNA sequencing (scRNA-seq) hold promise for the genetic characterization of tissues at single-cell resolution. However, the relatively large size of neurons may impede the standardized use of these techniques. Here, this protocol exploits droplet-based single-nucleus RNA sequencing (snRNA-seq), a method to characterize the biological architecture of cardiac sympathetic neurons in health and disease. A stepwise approach is demonstrated to perform snRNA-seq of the bilateral superior cervical (SCG) and stellate ganglia (StG) dissected from adult mice.
This method enables long-term sample preservation, maintaining an adequate RNA quality when samples from multiple individuals/experiments cannot be collected all at once within a short period of time. Barcoding the nuclei with hashtag oligos (HTOs) enables demultiplexing and the trace-back of distinct ganglionic samples post sequencing. Subsequent analyses revealed successful nuclei capture of neuronal, satellite glial, and endothelial cells of the sympathetic ganglia, as validated by snRNA-seq. In summary, this protocol provides a stepwise approach for snRNA-seq of sympathetic extrinsic cardiac ganglia, a method that has the potential for broader application in studies of the innervation of other organs and tissues.

This protocol describes the detailed, low-input sample preparation for single-nucleus sequencing, including the dissection of mouse superior cervical and stellate ganglia, cell dissociation, cryopreservation, nucleus isolation, and hashtag barcoding.

Dissection of Adult Mouse Stria Vascularis for Single-Nucleus Sequencing or Immunostaining

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Chapters in this video

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

Cryo-section Dissection of the Adult Subependymal Zone for Accurate and Deep Quantitative Proteome Analysis

Lineage Tracing of Inducible Fluorescently-Labeled Stem Cells in the Adult Mouse Brain

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

Low-input Nucleus Isolation and Multiplexing with Barcoded Antibodies of Mouse Sympathetic Ganglia for Single-nucleus RNA Sequencing