Madison Grant, Steven Foltz, Halie Zastre and Junna Luan provided technical assistance. Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under award number AR065077. The content is solely the responsibility of the author and does not necessarily represent the official views of the National Institutes of Health.

<ol><li>Foltz, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Abnormal+skeletal+muscle+regeneration+plus+mild+alterations+in+mature+fiber+type+specification+in+Fktn-Deficient+Dystroglycanopathy+Muscular+Dystrophy+Mice%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Abnormal skeletal muscle regeneration plus mild alterations in mature fiber type specification in Fktn-Deficient Dystroglycanopathy Muscular Dystrophy Mice.</a> PLoS One. 11 (1), 0147049(2016).</li>
<li>Bartoli, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Noninvasive+monitoring+of+therapeutic+gene+transfer+in+animal+models+of+muscular+dystrophies%5BTitle%5D)+AND+%22Gene+Ther%22%5BJournal%5D)">Noninvasive monitoring of therapeutic gene transfer in animal models of muscular dystrophies.</a> Gene Ther. 13, 20-28 (2006).</li>
<li>Ip, W. T., Huggins, C. E., Pepe, S., Delbridge, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluating+RNA+preparation+options+for+archived+myocardial+biopsies%5BTitle%5D)+AND+%22Heart+Lung+Circ%22%5BJournal%5D)">Evaluating RNA preparation options for archived myocardial biopsies.</a> Heart Lung Circ. 20 (5), 329-331 (2011).</li>
<li>Beedle, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+fukutin+deletion+impairs+dystroglycan+processing+and+recapitulates+muscular+dystrophy%5BTitle%5D)+AND+%22J.+Clin.+Invest%22%5BJournal%5D)">Mouse fukutin deletion impairs dystroglycan processing and recapitulates muscular dystrophy.</a> J. Clin. Invest. 122 (9), 3330-3342 (2012).</li>
<li>Foltz, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Four-week+rapamycin+treatment+improves+muscular+dystrophy+in+a+fukutin-deficient+mouse+model+of+dystroglycanopathy%5BTitle%5D)+AND+%22Skeletal+Muscle%22%5BJournal%5D)">Four-week rapamycin treatment improves muscular dystrophy in a fukutin-deficient mouse model of dystroglycanopathy.</a> Skeletal Muscle. 6, 20(2016).</li>
<li>AVMA Guidelines for the euthanasia of animals: 2013 edition. , American Veterinary Medical Association (AVMA). Available from: 
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<li>Liu, L., Cheung, T. H., Charville, G. W., Rando, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+skeletal+muscle+stem+cells+by+fluorescence-activated+cell+sorting%5BTitle%5D)+AND+%22Nat.+Protoc%22%5BJournal%5D)">Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting.</a> Nat. Protoc. 10, 1612-1624 (2015).</li>
<li>Rump, L. V., Asamoah, B., Gonzalez-Escalona, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+commercial+RNA+extraction+kits+for+preparation+of+DNA-free+total+RNA+from+Salmonella+cells%5BTitle%5D)+AND+%22BMC+Res.+Notes%22%5BJournal%5D)">Comparison of commercial RNA extraction kits for preparation of DNA-free total RNA from Salmonella cells.</a> BMC Res. Notes. 3, 211(2010).</li>
<li>Desjardins, P., Conklin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((NanoDrop+microvolume+quantitation+of+nucleic+acids%5BTitle%5D)+AND+%22J.+Vis.+Exp%22%5BJournal%5D)">NanoDrop microvolume quantitation of nucleic acids.</a> J. Vis. Exp. (45), e2565(2010).</li>
<li>Valdez, M. R., Richardson, J. A., Klein, W. H., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Failure+of+Myf5+to+support+myogenic+differentiation+without+myogenin%2C+MyoD%2C+and+MRF4%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">Failure of Myf5 to support myogenic differentiation without myogenin, MyoD, and MRF4.</a> Dev Biol. 219 (2), 287-298 (2000).</li>
<li>Pavlath, G. K., Dominov, J. A., Kegley, K. M., Miller, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regeneration+of+transgenic+skeletal+muscles+with+altered+timing+of+expression+of+the+basic+helix-loop-helix+muscle+regulatory+factor+MRF4%5BTitle%5D)+AND+%22Am.+J.+Pathol%22%5BJournal%5D)">Regeneration of transgenic skeletal muscles with altered timing of expression of the basic helix-loop-helix muscle regulatory factor MRF4.</a> Am. J. Pathol. 162 (5), 1685-1691 (2003).</li>
<li>Chomczynski, P., Mackey, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Substitution+of+chloroform+by+bromo-chloropropane+in+the+single-step+method+of+RNA+isolation%5BTitle%5D)+AND+%22Anal.+Biochem%22%5BJournal%5D)">Substitution of chloroform by bromo-chloropropane in the single-step method of RNA isolation.</a> Anal. Biochem. 225 (1), 163-164 (1995).</li>
<li>Lee, J. T. Y., Cheung, K. M. C., Leung, V. Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extraction+of+RNA+from+tough+tissues+with+high+proteoglycan+content+by+cryosection%2C+second+phase+separation+and+high+salt+precipitation%5BTitle%5D)+AND+%22J.+Biol.+Methods%22%5BJournal%5D)">Extraction of RNA from tough tissues with high proteoglycan content by cryosection, second phase separation and high salt precipitation.</a> J. Biol. Methods. 2 (2), 20(2015).</li>
<li>Guo, D., Catchpoole, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+of+intact+RNA+following+cryosections+of+archived+frozen+tissue%5BTitle%5D)+AND+%22BioTechniques%22%5BJournal%5D)">Isolation of intact RNA following cryosections of archived frozen tissue.</a> BioTechniques. 34, 48-50 (2003).</li>
<li>Goodpaster, T., Randolph-Habecker, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+flexible+mouse-on-mouse+immunohistochemical+staining+technique+adaptable+to+biotin-free+reagents%2C+immunofluorescence%2C+and+multiple+antibody+staining%5BTitle%5D)+AND+%22J.+Histochem.+Cytochem%22%5BJournal%5D)">A flexible mouse-on-mouse immunohistochemical staining technique adaptable to biotin-free reagents, immunofluorescence, and multiple antibody staining.</a> J. Histochem. Cytochem. 62 (3), 197-204 (2014).</li>
<li>Ikezawa, M., Minami, N., Takahashi, M., Goto, Y., Miike, T., Nonaka, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dystrophin+gene+analysis+of+130+patients+with+Duchenne+muscular+dystrophy+with+a+special+reference+to+muscle+mRNA+analysis%5BTitle%5D)+AND+%22Brain+Dev%22%5BJournal%5D)">Dystrophin gene analysis of 130 patients with Duchenne muscular dystrophy with a special reference to muscle mRNA analysis.</a> Brain Dev. 20 (3), 165-168 (1998).</li>
<li>Majumdar, G., Vera, S., Elam, M. B., Raghow, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+streamlined+protocol+for+extracting+RNA+and+genomic+DNA+from+archived+human+blood+and+muscle%5BTitle%5D)+AND+%22Anal.+Biochem%22%5BJournal%5D)">A streamlined protocol for extracting RNA and genomic DNA from archived human blood and muscle.</a> Anal. Biochem. 474, 25-27 (2015).</li>
<li>Espina, V., Heiby, M., Pieroban, M., Liotta, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Laser+capture+microdissection+technology%5BTitle%5D)+AND+%22Expert+Rev+Mol+Diagnostics%22%5BJournal%5D)">Laser capture microdissection technology.</a> Expert Rev Mol Diagnostics. 7 (5), 647-657 (2007).</li>
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The author declares that she has no competing financial interests.

To achieve best results with this method, keep embedding resin restricted to the lower third or half of the muscle during tissue cryopreservation because excess resin will slow the collection of the pooled cryosections and may increase embedding resin contamination in the RNA isolation. Also, careful attention during needle homogenization is important to maximize yield and minimize the probability of clogging the needle. The protocol may be modified by using a Luer-Lok syringe to protect against sample loss if the needle...

The goal of this technique is to make multiple experimental analyses by different modalities, such as histology and gene expression, accessible from a single small skeletal muscle source tissue. Microscopy applications are the most sensitive to sample preservation methods, which must be carefully controlled to limit the formation of ice crystal artifacts during cryopreservation. Thus, method development is based on the tibialis anterior (TA) muscle frozen partially covered with embedding resin in a -140 &#176;C liquid nitrogen-cooled 2-methylbutane bath as the source material for both immunofluorescence microscopy and gene expression analyses.
The need to use the same source material for diverse technical approaches is particularly important for intramuscular injection-based experiments where the left and right muscles represent different conditions, one experimental and one control. For example, in muscle regeneration studies, one muscle is injected with a toxin to cause widespread tissue damage while the contralateral muscle serves as a vehicle-injected control1. Similarly, gene therapy studies for muscle disorders typically begin with validation of the gene therapy vector by intramuscular injection to be compared with empty vector, unrelated vector or vehicle control on the contralateral side2. Therefore, it is not possible to source each TA muscle to a different application.
Common strategies to deal with this issue are: i) to use a different muscle group for each application, ii) to use additional mice, or iii) to cut off a piece of the muscle for each application. However, substantial differences between muscle groups make it difficult to compare data from separate applications, and additional animals increase expense and are poorly justified if other alternatives exist. Dividing the muscle after dissection to source different applications is the best option in many cases. However, the muscle pieces are often too small to use pulverization under liquid nitrogen or mechanical grinding techniques for homogenization2-5. As muscle is a highly structural tissue packed with extracellular matrix and contractile proteins, inadequate mechanical homogenization leads to a low yield of subsequent DNA, RNA or protein. The method detailed here allows small quantities of tissue from one source muscle for use in multiple applications, and the inclusion of cryosectioning and needle trituration improves mechanical homogenization for better RNA yield.

<table><tbody><tr><td>Cork</td><td>VWR Scientific</td><td>23420-708</td><td>Cut into small squares with a sharp blade.</td></tr><tr><td>Plastic coverslip</td><td>Fisher Scientific</td><td>12-547</td><td>Used to orient the muscle during freezing.</td></tr><tr><td>Low temperature thermometer</td><td>VWR Scientific</td><td>89370-158</td><td></td></tr><tr><td>2-methylbutane</td><td>Sigma</td><td>M32631-4L</td><td>Caution: hazardous chemical. Store in flammable cabinet.</td></tr><tr><td>Embedding resin: &quot;cryomatrix&quot;</td><td>Thermo Fisher Scientific</td><td>6769006</td><td>Other embedding resins can be substituted for cryomatrix.</td></tr><tr><td>Cryostat</td><td>Thermo Fisher Scientific</td><td>microm HM550 with disposable blade carrier</td><td>Any working cryostat should be sufficient for the protocol.</td></tr><tr><td>Disposable cryostat blade</td><td>Thermo Fisher Scientific</td><td>3052835</td><td>Use an appropriate blade or knife for the cryostat to be used.</td></tr><tr><td>RNAse decontamination solution: &quot;RNase Zap&quot;</td><td>Thermo Fisher Scientific</td><td>AM9780</td><td></td></tr><tr><td>Analytical balance</td><td>Mettler Toledo</td><td>XS64</td><td></td></tr><tr><td>Paint brush</td><td>Daler Rowney</td><td>214900920</td><td>Use to handle cryosections. Can be found with in stores with simple art supplies.</td></tr><tr><td>Razor blade</td><td>VWR Scientific</td><td>55411-050</td><td></td></tr><tr><td>Microscope slide</td><td>VWR Scientific</td><td>48311600</td><td></td></tr><tr><td>RNA organic extraction reagent: TRIzol</td><td>Thermo Fisher Scientific</td><td>15596026</td><td>Caution: TRIzol is a hazardous chemical. Note: Only organic extraction reagents are recommended for RNA extraction from skeletal muscle.</td></tr><tr><td>18 gauge needle</td><td>VWR Scientific</td><td>BD305185</td><td></td></tr><tr><td>22 gauge needle</td><td>VWR Scientific</td><td>BD305155</td><td></td></tr><tr><td>26 gauge needle</td><td>VWR Scientific</td><td>BD305115</td><td>Optional. Can be used for a third round of sample trituration in the RNA extraction protocol.</td></tr><tr><td>1 ml syringe</td><td>VWR Scientific</td><td>BD309659</td><td>For very high value samples, a Luer-Lok syringe is recommended (&lt;i&gt;e.g.&lt;/i&gt;, VWR BD309628).</td></tr><tr><td>1-bromo-3-chloropentane (BCP)</td><td>Sigma</td><td>B9673</td><td></td></tr><tr><td>For 70% ethanol in DEPC water: 200 proof alcohol</td><td>Decon Laboratories, Inc.</td><td>+M18027161M</td><td>Mix 35 ml 200 proof alcohol + 15 ml DEPC water.&amp;nbsp;</td></tr><tr><td>For 70% ethanol in DEPC water: DEPC-treated water</td><td>Thermo Fisher Scientific</td><td>AM9922</td><td>Mix 35 ml 200 proof alcohol + 15 ml DEPC water.</td></tr><tr><td>RNA purification kit: PureLink RNA minikit</td><td>Thermo Fisher Scientific</td><td>12183018A</td><td>Final steps of RNA preparation.</td></tr><tr><td>DNase/Rnase-free water&amp;nbsp;</td><td>Gibco</td><td>10977</td><td>DEPC-treated water can also be used.</td></tr><tr><td>Spectrophotometer: Nanodrop 2000</td><td>Thermo Fisher Scientific</td><td>NanoDrop 2000</td><td></td></tr><tr><td>Dnase I</td><td>Thermo Fisher Scientific</td><td>AM2222</td><td>Treat purified RNA to remove any DNA contamination before downstream appications.</td></tr><tr><td>Hydrophobic pen</td><td>Thermo Fisher Scientific</td><td>8899</td><td></td></tr><tr><td>Dulbecco&#039;s PBS</td><td>Gibco</td><td>14190</td><td>PBS for immunofluorescence protocol.</td></tr><tr><td>Donkey serum</td><td>Jackson ImmunoResearch Laoratories, Inc</td><td>017-000-121</td><td>Rehydrate normal donkey serum stock according to the manufacturer&#039;s instructions, then dilute an aliquot to 5% for immunofluorescence.&amp;nbsp; Normal goat serum can also be used.</td></tr><tr><td>eMHC antibody</td><td>University of Iowa Developmental Studies Hybridoma Bank</td><td>F1.652</td><td></td></tr><tr><td>Collagen VI antibody</td><td>Fitzgerald Industries</td><td>#70R-CR009x</td><td></td></tr><tr><td>Donkey anti-rabbit AlexaFluor488</td><td>Thermo Fisher Scientific</td><td>A21206</td><td></td></tr><tr><td>Goat anti-mouse IgG1 AlexaFluor546</td><td>Thermo Fisher Scientific</td><td>A21123</td><td></td></tr><tr><td>DAPI (4&#039;,6-diamidino-2-phenylindole)</td><td>Thermo Fisher Scientific</td><td>D1306</td><td></td></tr><tr><td>Aqueous mounting media: Permafluor</td><td>Thermo Fisher Scientific</td><td>TA-030-FM</td><td>Only use mounting media designed for fluorescent applications with anti-fade properties.</td></tr><tr><td>Glass coverslip</td><td>VWR Scientific</td><td>16004-314</td><td>Use for mounting slides at the end of immunofluorescence protocl</td></tr><tr><td>Image analysis software: ImageProExpress</td><td>Media Cybernetics, Inc.</td><td>Image-Pro Express, or more advanced products</td><td>Freeware ImageJ should also work for manual counting. More advanced software with segmentation abiities may allow partial automation of the process; &lt;em&gt;e.g.&lt;/em&gt;, ImageProPremier.</td></tr><tr><td>Merge and map section images: Photoshop</td><td>Adobe</td><td>Photoshop</td><td></td></tr><tr><td>Cardiotoxin</td><td>Sigma</td><td>C9659</td><td>Sigma C9659 has been discontinued. Other options for cardiotoxin are EMD Millipore #217503; American Custom Chemicals Corp. # BIO0000618; or Ge Script # RP17303; but these have not been validated.</td></tr><tr><td>reverse transcription kit: Superscript III First-strand synthesis system</td><td>Thermo Fisher Scientific</td><td>18080051</td><td>Any validated, high quality reverse transcription reagents can be used.</td></tr><tr><td>Standard PCR: GoTaq Flexi polymerase system</td><td>Promega</td><td>M8298</td><td>Any validated, high quality Taq polymerase system can be used. If DNA sequencing is to be used for any application downstream of the PCR, then a high fidelity PCR system should be used instead.</td></tr><tr><td>SYBR green</td><td>Thermo Fisher Scientific</td><td>S7585</td><td>For use in qPCR when not using a dedicated qPCR master mix. Use with SuperROX (for Applied Biosystems instruments) and GoTaq Flexi polymerase and buffers.</td></tr><tr><td>ROX: SuperROX, 15 mM</td><td>BioResearch Technologies, Inc. Novato CA</td><td>SR-1000-10</td><td>SuperROX is more stable in the PCR reaction, so it is preferred for use as a qPCR passive reference dye over ROX (carboxy-X-rhodamine). For qPCR with Applied Biosystems instruments</td></tr><tr><td>Real-time PCR</td><td>Applied Biosystems</td><td>7900HT</td><td></td></tr></tbody></table>

cryosectioning of contiguous regions of a single mouse skeletal muscle for gene expression and histological analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Muscle cryosection RNA is high in quality and provides sufficient yield for most applications
Analyses of sixteen skeletal muscle RNA preparations are shown in Table 1 using 19.4 to 41 mg of pooled tibialis anterior (TA) muscle from 8 control mice. Both left (L) and right (R) TA muscles were prepared in regeneration experiments with muscles collected 3 days after longitudinal intramuscular injection of 25 &#956;l o...

Watch this Scientific Journal Video about Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses at JoVE.com

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

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Biology

15.7K Views.  University of Georgia. Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

Video: Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

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

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Developmental Biology

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of neuromuscular disorders. However, in order to do so, proper care needs to be taken when excising and preserving tissues to achieve optimal staining. One method of tissue preservation involves fixing tissues in formaldehyde and then embedding them with paraffin wax. This method preserves morphology well and allows for long-term storage at RT but is cumbersome and requires handling of toxic chemicals. Further, formaldehyde fixation results in antigen cross-linking, which necessitates antigen retrieval protocols for effective immunostaining. On the contrary, frozen sectioning does not require fixation and thus retains biological antigen conformation. This method also provides a distinct advantage in quick turn around time, making it especially useful in situations needing quick histological evaluation like intraoperative surgical biopsies. Here we describe the most effective method of preparing muscle biopsies for visualization with different histological and immunological stains.

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

Medicine

Simultaneous Cryosectioning of Multiple Rodent Brains

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In neuroscience, as well as a plethora of other scientific fields, these techniques are used. Immunohistochemistry can be done on slide mounted tissue or free-floating sections. Preparing slide-mounted samples is a time intensive process. The following protocol for a technique, called the Megabrain, reduced the time taken to cryosection and mount brain tissue by up to 90% by combining multiple brains into a single frozen block. Furthermore, this technique reduced variability seen between staining rounds, in a large histochemical study. The current technique has been optimized for using rodent brain tissue in downstream immunohistochemical analyses; however, it can be applied to different scientific fields that use cryosectioning.

Here, we present a protocol to freeze and section brain tissue from multiple animals as a timesaving alternative to processing single brains. This reduces staining variability during immunohistochemistry and reduces time cryosectioning and imaging.

Histology and immunohistochemistry are routine methods of analysis to visualize microscopic anatomy and localize proteins within biological tissue. In ...

Microdissection of Mouse Brain into Functionally and Anatomically Different Regions

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain consists of an outer covering of grey matter over the hemispheres known as the cerebral cortex. Underneath this layer reside many other specialized structures that are essential for multiple phenomenon important for existence. Acquiring samples of specific gross brain regions requires quick and precise dissection steps.&#160;It is understood that at the microscopic level, many sub-regions exist and likely cross the arbitrary regional boundaries that we impose for the purpose of this dissection.
Mouse models are routinely used to study human brain functions and diseases. Changes in gene expression patterns may be confined to specific brain areas targeting a particular phenotype depending on the diseased state. Thus, it is of great importance to study regulation of transcription with respect to its well-defined structural organization. A complete understanding of the brain requires studying distinct brain regions, defining connections, and identifying key differences in the activities of each of these brain regions. A more comprehensive understanding of each of these distinct regions may pave the way for new and improved treatments in the field of neuroscience. Herein, we discuss a step-by-step methodology for dissecting the mouse brain into sixteen distinct regions. In this procedure, we have focused on male mouse C57Bl/6J (6-8 week old) brain removal and dissection into multiple regions using neuroanatomical landmarks to identify and sample discrete functionally-relevant and behaviorally-relevant&#160;brain regions. This work will help lay a strong foundation in the field of neuroscience, leading to more focused approaches in the deeper understanding of brain function.

We present a hands-on, step-by-step, rapid protocol for mouse brain removal and dissection of discrete regions from fresh brain tissue. Obtaining brain regions for molecular analysis has become routine in many neuroscience labs. These brain regions are immediately frozen to obtain high quality transcriptomic data for system level analysis.

The brain is the command center for the mammalian nervous system and an organ with enormous structural complexity. Protected within the skull, the brain ...

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 ...

Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative prognostic factor for several other disorders such as cardiovascular disease and cancer. Excessive lipid infiltration decreases muscle mass and strength. It also results in lipotoxicity and insulin resistance depending on total intramyocellular lipid content, lipid droplet (LD) morphology, and subcellular distribution. Fiber type (oxidative vs glycolytic) is also important, since oxidative fibers have a greater capacity to utilize lipids. Because of their crucial implications in pathophysiology, in-depth studies on LD dynamics and function in a fiber type-specific manner are warranted.
Herein, a complete protocol is presented for the quantification of intramyocellular lipid content and analysis of LD morphology and subcellular distribution in a fiber type-specific manner. To this end, serial muscle cryosections were stained with the fluorescent dye Bodipy and antibodies against myosin heavy chain isoforms. This protocol enables the simultaneous processing of different muscles, saving time and avoiding possible artifacts and, thanks to a personalized macro created in Fiji, the automatization of LD analysis is also possible.

Increasing evidence indicates that excessive infiltration of lipids inside skeletal muscle results in lipotoxicity and diabetes. Here, we present a complete protocol, including tissue processing, staining with Bodipy, image acquisition, and analysis, to quantify the size, density, and subcellular distribution of lipid droplets in a fiber-type specific manner.

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative ...

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...

Enzymatic Isolation and Characterization of Variable Length Mouse Muscle Fibers

Intact Short, Intermediate, and Long Skeletal Muscle Fibers Obtained by Enzymatic Dissociation of Six Hindlimb Muscles of Mice: Beyond Flexor Digitorum Brevis

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal with the short fibers of the flexor digitorum brevis (FDB), which restrains the scope of results dealing with fiber types, limits the amount of biological material available, and impedes a clear connection between cellular physiological phenomena and previous biochemical and dynamical knowledge obtained in other muscles.
This paper describes how to obtain intact fibers from six muscles with different fiber type profiles and lengths. Using C57BL/6 adult mice, we show the muscle dissection and fiber isolation protocol and demonstrate the suitability of the fibers for Ca2+ transient studies and their morphometric characterization. The fiber type composition of the muscles is also presented. When dissociated, all muscles rendered intact, living fibers that contract briskly for more than 24 h. FDB gave short (&#60;1 mm), peroneus digiti quarti (PDQA) and peroneus longus (PL) gave intermediate (1-3 mm), while extensor digitorum longus (EDL), extensor hallucis longus (EHL), and soleus muscles released long (3-6 mm) fibers.
When recorded with the fast dye Mag-Fluo-4, Ca2+ transients of PDQA, PL, and EHL fibers showed the fast, narrow kinetics reminiscent of the morphology type II (MT-II), known to correspond to type IIX and IIB fibers. This is consistent with the fact that these muscles have over 90% of type II fibers compared with FDB (~80%) and soleus (~65%). Moving beyond FDB, we demonstrate for the first time the dissociation of several muscles, which render fibers spanning a range of lengths between 1 and 6 mm. These fibers are viable and give fast Ca2+ transients, indicating that the MT-II can be generalized to IIX and IIB fast fibers, regardless of their muscle source. These results increase the availability of models for mature skeletal muscle studies.

Author Spotlight: Isolation of Long Muscle Fibers from Mouse Hindlimb Muscles for Studying Excitation-Contraction Coupling Across Fiber Types

We describe a protocol to obtain enzymatically dissociated fibers of different lengths and types from six muscles of adult mice: three of them already described (flexor digitorum brevis, extensor digitorum longus, soleus) and three of them successfully dissociated for the first time (extensor hallucis longus, peroneus longus, peroneus digiti quarti).

Skeletal muscle fibers obtained by enzymatic dissociation of mouse muscles are a useful model for physiological experiments. However, most papers deal ...