Name: a laser capture microdissection protocol that yields high quality rna from fresh-frozen mouse bones
Uploaded: 2019-09-16T15:00:00
Description: Watch this Scientific Journal Video about A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones at JoVE.com

The authors thank Ute Zeitz and Nikole Ginner for their excellent technical help as well as the Vetcore and animal care staff for their support.

Bone tissue from mice was used in strict accordance with prevailing guidelines for animal care and all efforts were made to minimize animal suffering.
1. Animals and Freeze Embedding
<ol>
	<li>House animals in conditions of constant room temperature (RT; 24 &#176;C) and a 12 h light/12 h dark cycle with free access to food and water. 
	NOTE: Bone tissues in this study were obtained from 3-month-old male wild type C57BL/6 mice.</li>
	<li>At necropsy, exsanguinate mice fr...

<ol>
	<li>Emmert-Buck, M. R., 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=Laser+capture+microdissection.">Laser capture microdissection.</a> Science. 274 (5289), 998-1001 (1996).</li><li>Legres, L. G., Janin, A., Masselon, C., Bertheau, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+laser+microdissection+technology:+follow+the+yellow+brick+road+for+cancer+research.">Beyond laser microdissection technology: follow the yellow brick road for cancer research.</a> American journal of Cancer Research. 4 (1), 1-28 (2014).</li><li>Datta, 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=Laser+capture+microdissection:+Big+data+from+small+samples.">Laser capture microdissection: Big data from small samples.</a> Histology and histopathology. 30 (11), 1255-1269 (2015).</li><li>Kerman, I. A., Buck, B. J., Evans, S. J., Akil, H., Watson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+laser+capture+microdissection+with+quantitative+real-time+PCR:+effects+of+tissue+manipulation+on+RNA+quality+and+gene+expression.">Combining laser capture microdissection with quantitative real-time PCR: effects of tissue manipulation on RNA quality and gene expression.</a> Journal of Neuroscience Methods. 153 (1), 71-85 (2006).</li><li>Adiconis, X., 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=Comparative+analysis+of+RNA+sequencing+methods+for+degraded+or+low-input+samples.">Comparative analysis of RNA sequencing methods for degraded or low-input samples.</a> Nature Methods. 10 (7), 623-629 (2013).</li><li>Golubeva, Y. G., Warner, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Microdissection+Workflow+for+Isolating+Nucleic+Acids+from+Fixed+and+Frozen+Tissue+Samples.">Laser Microdissection Workflow for Isolating Nucleic Acids from Fixed and Frozen Tissue Samples.</a> Methods in Molecular Biology (Clifton, N.J.). 1723, 33-93 (2018).</li><li>Farris, S., Wang, Y., Ward, J. M., Dudek, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+Method+for+Robust+Transcriptome+Profiling+of+Minute+Tissues+Using+Laser+Capture+Microdissection+and+Low-Input+RNA-Seq.">Optimized Method for Robust Transcriptome Profiling of Minute Tissues Using Laser Capture Microdissection and Low-Input RNA-Seq.</a> Frontiers in Molecular Neuroscience. 10, 185 (2017).</li><li>Garrido-Gil, P., Fernandez-Rodr&#237;guez, P., Rodr&#237;guez-Pallares, J., Labandeira-Garcia, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+capture+microdissection+protocol+for+gene+expression+analysis+in+the+brain.">Laser capture microdissection protocol for gene expression analysis in the brain.</a> Histochemistry and Cell Biology. 148 (3), 299-311 (2017).</li><li>Kawamoto, T., Kawamoto, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+thin+frozen+sections+from+nonfixed+and+undecalcified+hard+tissues+using+Kawamot's+film+method.">Preparation of thin frozen sections from nonfixed and undecalcified hard tissues using Kawamot's film method.</a> Methods in Molecular Biology (Clifton, N.J.). 1130, 149-164 (2014).</li><li>Streicher, C., 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=Estrogen+Regulates+Bone+Turnover+by+Targeting+RANKL+Expression+in+Bone+Lining+Cells.">Estrogen Regulates Bone Turnover by Targeting RANKL Expression in Bone Lining Cells.</a> Scientific Reports. 7 (1), 6460 (2017).</li><li>Vaidya, M., 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=Osteoblast-specific+overexpression+of+amphiregulin+leads+to+transient+increase+in+femoral+cancellous+bone+mass+in+mice.">Osteoblast-specific overexpression of amphiregulin leads to transient increase in femoral cancellous bone mass in mice.</a> Bone. 81, 36-46 (2015).</li><li>Jay, F. F., 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=Amphiregulin+lacks+an+essential+role+for+the+bone+anabolic+action+of+parathyroid+hormone.">Amphiregulin lacks an essential role for the bone anabolic action of parathyroid hormone.</a> Molecular and Cellular Endocrinology. 417, 158-165 (2015).</li><li>Murali, S. K., Andrukhova, O., Clinkenbeard, E. L., White, K. E., Erben, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excessive+Osteocytic+Fgf23+Secretion+Contributes+to+Pyrophosphate+Accumulation+and+Mineralization+Defect+in+Hyp+Mice.">Excessive Osteocytic Fgf23 Secretion Contributes to Pyrophosphate Accumulation and Mineralization Defect in Hyp Mice.</a> PLoS Biology. 14 (4), e1002427 (2016).</li><li>Andrukhova, O., Sch&#252;ler, C., Bergow, C., Petric, A., Erben, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Augmented+Fibroblast+Growth+Factor-23+Secretion+in+Bone+Locally+Contributes+to+Impaired+Bone+Mineralization+in+Chronic+Kidney+Disease+in+Mice.">Augmented Fibroblast Growth Factor-23 Secretion in Bone Locally Contributes to Impaired Bone Mineralization in Chronic Kidney Disease in Mice.</a> Frontiers in Endocrinology. 9, 311 (2018).</li><li>Golubeva, Y. G., Smith, R. M., Sternberg, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+Frozen+Sample+Preparation+for+Laser+Microdissection:+Assessment+of+CryoJane+Tape-Transfer+System&#174;.">Optimizing Frozen Sample Preparation for Laser Microdissection: Assessment of CryoJane Tape-Transfer System&#174;.</a> PLoS ONE. 8 (6), e66854 (2013).</li><li>Pacheco, E., Hu, R., Taylor, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Capture+Microdissection+and+Transcriptional+Analysis+of+Sub-Populations+of+the+Osteoblast+Lineage+from+Undecalcified+Bone.">Laser Capture Microdissection and Transcriptional Analysis of Sub-Populations of the Osteoblast Lineage from Undecalcified Bone.</a> Methods in Molecular Biology (Clifton, N.J.). 1723, 191-202 (2018).</li><li>Nioi, P., 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=Transcriptional+Profiling+of+Laser+Capture+Microdissected+Subpopulations+of+the+Osteoblast+Lineage+Provides+Insight+Into+the+Early+Response+to+Sclerostin+Antibody+in+Rats.">Transcriptional Profiling of Laser Capture Microdissected Subpopulations of the Osteoblast Lineage Provides Insight Into the Early Response to Sclerostin Antibody in Rats.</a> Journal of Bone and Mineral Research. 30 (8), 1457-1467 (2015).</li><li>Taylor, 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=Differential+time-dependent+transcriptional+changes+in+the+osteoblast+lineage+in+cortical+bone+associated+with+sclerostin+antibody+treatment+in+ovariectomized+rats.">Differential time-dependent transcriptional changes in the osteoblast lineage in cortical bone associated with sclerostin antibody treatment in ovariectomized rats.</a> Bone Reports. 8, 95-103 (2018).</li><li>Taylor, 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=Time-dependent+cellular+and+transcriptional+changes+in+the+osteoblast+lineage+associated+with+sclerostin+antibody+treatment+in+ovariectomized+rats.">Time-dependent cellular and transcriptional changes in the osteoblast lineage associated with sclerostin antibody treatment in ovariectomized rats.</a> Bone. 84, 148-159 (2016).</li><li>Martin, L. B. B., 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=Laser+microdissection+of+tomato+fruit+cell+and+tissue+types+for+transcriptome+profiling.">Laser microdissection of tomato fruit cell and tissue types for transcriptome profiling.</a> Nature Protocols. 11 (12), 2376-2388 (2016).</li><li>Imbeaud, 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=Towards+standardization+of+RNA+quality+assessment+using+user-independent+classifiers+of+microcapillary+electrophoresis+traces.">Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces.</a> Nucleic Acids Research. 33 (6), e56 (2005).</li><li>Schroeder, A., 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=The+RIN:+an+RNA+integrity+number+for+assigning+integrity+values+to+RNA+measurements.">The RIN: an RNA integrity number for assigning integrity values to RNA measurements.</a> BMC Molecular Biology. 7, 3 (2006).</li><li>Gallego Romero, I., Pai, A. A., Tung, J., Gilad, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-seq:+impact+of+RNA+degradation+on+transcript+quantification.">RNA-seq: impact of RNA degradation on transcript quantification.</a> BMC Biology. 12, 42 (2014).</li><li>Bevilacqua, C., Makhzami, S., Helbling, J. C., Defrenaix, P., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+RNA+integrity+in+a+homogeneous+population+of+mammary+epithelial+cells+isolated+by+Laser+Capture+Microdissection.">Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection.</a> BMC Cell Biology. 11, 95 (2010).</li><li>Mahalingam, M., Murray, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+Capture+Microdissection:+Insights+into+Methods+and+Applications.">Laser Capture Microdissection: Insights into Methods and Applications.</a> Laser Capture Microdissection: Methods and Protocols. , 1-17 (2018).</li><li>Burgemeister, R., Gangnus, R., Haar, B., Sch&#252;tze, K., Sauer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+quality+RNA+retrieved+from+samples+obtained+by+using+LMPC+(laser+microdissection+and+pressure+catapulting)+technology.">High quality RNA retrieved from samples obtained by using LMPC (laser microdissection and pressure catapulting) technology.</a> Pathology, Research and Practice. 199 (6), 431-436 (2003).</li><li>Cummings, M., 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=A+robust+RNA+integrity-preserving+staining+protocol+for+laser+capture+microdissection+of+endometrial+cancer+tissue.">A robust RNA integrity-preserving staining protocol for laser capture microdissection of endometrial cancer tissue.</a> Analytical Biochemistry. 416 (1), 123-125 (2011).</li><li>Cl&#233;ment-Ziza, M., Munnich, A., Lyonnet, S., Jaubert, F., Besmond, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stabilization+of+RNA+during+laser+capture+microdissection+by+performing+experiments+under+argon+atmosphere+or+using+ethanol+as+a+solvent+in+staining+solutions.">Stabilization of RNA during laser capture microdissection by performing experiments under argon atmosphere or using ethanol as a solvent in staining solutions.</a> RNA. 14 (12), 2698-2704 (2008).</li><li>K&#246;lble, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+LEICA+microdissection+system:+design+and+applications.">The LEICA microdissection system: design and applications.</a> Journal of Molecular Medicine. 78 (7), B24-B25 (2000).</li><li>B&#246;hm, M., Wieland, I., Sch&#252;tze, K., R&#252;bben, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbeam+MOMeNT:+non-contact+laser+microdissection+of+membrane-mounted+native+tissue.">Microbeam MOMeNT: non-contact laser microdissection of membrane-mounted native tissue.</a> The American Journal of Pathology. 151 (1), 63-67 (1997).</li><li>Micke, P., 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=A+fluid+cover+medium+provides+superior+morphology+and+preserves+RNA+integrity+in+tissue+sections+for+laser+microdissection+and+pressure+catapulting.">A fluid cover medium provides superior morphology and preserves RNA integrity in tissue sections for laser microdissection and pressure catapulting.</a> The Journal of Pathology. 202 (1), 130-138 (2004).</li></ol>

Both RNA quality and quantity can be affected negatively at all stages of the sample preparation such as tissue manipulation, LCM process, and RNA extraction. Therefore, an LCM protocol was developed to obtain sufficient amount of high-quality RNA for subsequent gene expression analysis.
For LCM, most laboratories use sections 7&#8722;8 &#181;m thick2. Thicker sections would allow more material to be harvested. However, if they are too thick, this could reduce the microscopic resolu...

Tissues are made up of heterogeneous and spatially distributed cell types. Different cell types in a given tissue may respond differently to the same signal. Therefore, it is essential being able to isolate specific cell populations for the assessment of the role of different cell types in both physiological and pathological conditions. Laser capture microdissection (LCM) offers a relatively rapid and precise method for isolating and removing specified cells from complex tissues1. LCM systems use the power of a laser beam to separate the cells of interest from histological tissue sections without the need for enzymatic processing or growth in culture. This means that the cells are in their natural tissue habitat, and that tissue architecture including the spatial relationship between different cells is retained. Morphology of both the captured cells and the residual tissue is well preserved, and several tissue components can be sampled sequentially from the same slide. Isolated cells can then be used for subsequent analysis of their RNA, DNA, protein or metabolite content2,3.
In order to analyze gene expression in different cell populations, or after different treatments, it is necessary to obtain mRNAs of sufficient quality and quantity for the subsequent analysis4,5. In contrast to DNA, RNAs are more sensitive to fixation and the use of frozen tissue is recommended when the objective is to study RNA. Since mRNAs are quickly degraded by ubiquitous ribonucleases (RNase), stringent RNase-free conditions during specimen handling and preparation and avoiding storage of samples at room temperature are required. In addition, rapid techniques without any prolonged aqueous phase steps are crucial to prevent RNA degradation6. The RNA yield and integrity can also be affected by the LCM process and the LCM system used7,8. Currently, four LCM systems with different operating principles are available2. The method of RNA extraction can also be important, since different RNA isolation kits have been tested with significant differences in RNA quantity and quality7,8.
Any tissue preparation method requires finding a balance between obtaining good morphological contrast and maintaining RNA integrity for further analyses. For preparing frozen sections from bone, an adhesive film was developed and continuously improved9. The bone sections are cut and stained directly on the adhesive film. This adhesive film is applicable to many types of staining, and can be employed to isolate the cells of interest from bone cryosections using LCM9,10,11,12,13,14. All the steps including the surgical removal, embedding, freezing, cutting and staining can be completed within less than one hour. Importantly, cells such as osteoblasts, bone lining cells, and osteoclasts can be clearly identified9,10,11,12,13,14. This method has the advantage of being rapid and simple. An alternative method for generating bone cryosections is to use the tape transfer system15. However, the latter technique is more time-consuming and requires additional instrumentation, since the sections have to be transferred from the adhesive tape onto precoated membrane slides by ultraviolet (UV) cross-linking. Although the tape transfer system has been successfully coupled with LCM16,17,18,19, it should be noted that the cross-linked coating can create a background pattern that can interfere with cell-type identification20.
Typically, only small amounts of RNA are extracted from microdissected cells, and RNA quality and quantity are often assessed by micro-capillary electrophoresis21. A computer program is used to assign an index of quality to RNA extracts called RNA integrity number (RIN). A RIN value of 1.0 indicates completely degraded RNA, whereas a value of 10.0 suggests that the RNA is fully intact22. Usually, indexes over 5 are considered sufficient for RNA studies. Gene expression patterns in samples with an RIN value of 5.0&#8722;10.0 have been reported to correlate well with each other23. Although the sensitivity of this method is high, since as little as 50 pg/&#181;L of total RNA can be detected, it can be very difficult to obtain a quality assessment if the RNA concentration in the sample is very low. Therefore, in order to assess RNA quality, the tissue section remaining after the LCM is often used to extract RNA, by pipetting buffer onto the slide24.
Although LCM has been used extensively on different frozen tissues, RIN values of extracted RNAs are rarely reported. Furthermore, there are no comparative studies to clarify the most appropriate method to study RNA in mouse bones. In the present study, frozen sections from adult mouse femurs were used to optimize sample preparation, LCM protocol and RNA extraction in order to obtain high quality RNAs. The present protocol was optimized particularly for the LCM system that uses gravity for sample collection.

<table>
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>63689-25ML-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Absolute ethanol EMPLURA</td>
			<td>Merck Millipore</td>
			<td>8,18,76,01,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive film (LMD film)</td>
			<td>Section-Lab</td>
			<td>C-FL001</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer System</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent RNA 6000 Pico Chip Kit</td>
			<td>Agilent Technologies</td>
			<td>5067-1513</td>
			<td></td>
		</tr>
		<tr>
			<td>Arcturus HistoGene Staining Solution</td>
			<td>Applied Biosystems</td>
			<td>12241-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryofilm fitting tool</td>
			<td>Section-Lab</td>
			<td>C-FT000</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat Leica CM 1950</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>glass microscope slides, cut colour frosted orange</td>
			<td>VWR Life Science</td>
			<td>631-1559</td>
			<td></td>
		</tr>
		<tr>
			<td>Histology tissue molds PVC</td>
			<td>MEDITE</td>
			<td>48-6302-00</td>
			<td></td>
		</tr>
		<tr>
			<td>LMD7&#160;Laser&#160;Mikrodissektion&#160;System</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Low profile Microtome Blades Leica DB80 XL</td>
			<td>Leica Biosystems</td>
			<td>14035843496</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclease-free water</td>
			<td>VWR Life Science</td>
			<td>E476-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>PET membrane slides 1.4 mircon</td>
			<td>Molecular Machines &#38; Industries GmbH</td>
			<td>50102</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Away surface decontaminant</td>
			<td>Molecular BioProduct</td>
			<td>7002</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Micro Kit</td>
			<td>Qiagen</td>
			<td>74004</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek optimal cutting temperature (OCT) compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR Life Science</td>
			<td>2,89,73,363</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

An LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells of mouse femurs. In the optimized protocol, 8 &#181;m-thick frozen bone sections were cut on an adhesive film and stained using a rapid protocol for a commercial LCM frozen section stain. The sample was sandwiched between the PET membrane and the adhesive film. Mouse bone cells were microdissected using an LCM system that uses gravity for sample collection. A column-based RNA extraction method was us...

Watch this Scientific Journal Video about A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones at JoVE.com

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

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 ...

Have you ever thought about harnessing the power of laser capture microdissection for analysis of gene expression in specific bone cells within their natural environment? Here, we describe a protocol that will enable you to obtain sufficient quantities of high quality RNA from cryosections of mouse bones. This technology is a great tool for examining changes in gene expression in specific bone cells in genetically engineered mouse models or in disease models.The protocol described here is focused on mouse bones, but can be used to study in situ a gene expression in cells of any hard tissue in any species. Helping to demonstrate this procedure, will be Christiane Schueler, a technician from my laboratory. After euthanizing mice by exsanguination under general anesthesia, remove whole femurs rapidly.Use a scalpel and paper towels to clean the femurs of the surrounding soft tissues. Next, pour optimal cutting temperature compound into the embedding mold and place the femurs into the bottom of the embedding molds. Snap freeze the samples in liquid nitrogen.Once the samples are entirely frozen, wrap them in foil and transfer them on dry ice to a freezer. Store them at minus 80 degrees Celsius until ready for further processing. First, set the temperature in the cryostat to minus 19 degrees Celsius and the temperature of the block holder to minus 17 degrees Celsius.Next, wipe down the interior of the cryostat with 70%ethanol. Place the disposable blade for hard tissues, the glass slides, and a fitting tool into the cryostat to cool, and keep them inside the cryostat for the duration of sectioning. Transfer the frozen tissue block on dry ice to the cryostat and allow it to equilibrate for at least 10 minutes.Then, press the bottom of the embedding mold to push the OCT block out of the mold. Apply enough OCT medium to the block holder to adhere the block to it and wait until the OCT medium freezes completely. After this, place the block holder in the object holder and tighten it in place.Adjust the blade position and trim the block in 15-micrometer cutting increments to remove the OCT covering the sample. Adjust the cryostat to generate eight-micrometer sections and cut two to three cryosections that will be discarded. Place adhesive film on the block and use the fitting tool to adhere the film to the block.Now, make a cut slowly and at constant speed, while holding the section by the film. Place the film with the sample facing up on a pre-cooled glass slide on the cryobar within the cryostat to avoid thawing of the sample. Use tape to fix the film to the glass slide for easier staining.In a fume hood, prepare the necessary solutions of ethanol, RNase-free water, and xylene on ice as outlined in the text protocol. Incubate the sections in 95%ethanol for 30 seconds. Then, carefully dip the sections in RNase-free water for 30 seconds to completely remove the OCT.Next, dispense 50 microliters of commercial LCM frozen section stain onto the section and incubate at room temperature for 10 seconds. Drain the section by placing the edge of the slide on absorbent tissue paper. Rinse the section in 100%ethanol for 30 seconds to remove any excess stain.Immerse the bone sections in a second tube containing 100%ethanol for 30 seconds and then, transfer them to 100%xylene for 30 seconds. Put adhesive film on a dry glass slide as a support, taking care to place the film as flat as possible. After this, place a PET membrane frame slide on the film and briefly press a gloved finger on the membrane to attach it to the film.This sample will be sandwiched between the membrane and the adhesive film. The adhesive film should not be folded or wrinkled and there should be no air bubbles between the film and the membrane. Start by using surface decontaminant to clean the stage end cap holder.Load the slide into the slide holder and the caps into the cap holder. Next, adjust the focus and acquire a slide overview with the 1.25x objective. Change to the 40x objective and adjust the focus.Using the slide overview, choose the area of interest. Then, adjust the laser parameters as shown here, making sure to optimize these parameters for each objective. If the laser fails to cut the sample, increase the laser power.Select osteoblast, osteocites, and bone lining cells in distal femoral cancellous or cortical bone based on morphologic criteria. Draw a line for the laser path further away from the target cells to minimize the damage by the UV laser. If the laser fails to cut the sample, apply the laser more than once or inspect the target for spots of incomplete cuts and use the move and cut option to cut the tissue in these spots.Collect each cell type in a separate 0.5-milliliter tube cap. First, dispense 50 microliters of the lysis buffer containing beta mercaptoethanol into the cap of the collection tube. Lyse the sample by pippetting it up and down in the cap for one minute.Next, spin down the lysate and add 00 microliters of the lysis buffer containing beta-mercaptoethanol to the tube. For each slide, prepare a labeled microcentrifuge tube with 350 microliters of the lysis buffer containing-beta mercaptoethanol. Use the sections remaining after the LCM to extract RNA.Carefully separate the film from the membrane and lyse the sample by slowly pippetting the lysis buffer onto the section several times. Then, put the lysate samples on dry ice and store them at minus 80 degrees Celsius. When ready to continue, thaw the lysates at room temperature.After this, transfer the lysates from LCM-harvested cells in the collection tubes to new 1.5-milliliter microcentrifuge tubes and extract RNA according to the manufacturer's instructions. In this study, an LCM protocol is developed to obtain sufficient quantity of high quality RNA for gene expression analysis in bone cells of mouse femurs. LCM is performed using an LCM system that uses gravity for sample collection.The yield and integrity of isolated RNA was measured using microcapillary electrophoresis. The difference in RNA quality and quantity obtained using different lysis protocols can be seen here in the representative gel and electropherograms. When the sample is lysed by pippetting up and down in the cap for one minute, it is possible to isolate approximately 8.5 nanograms of RNA from one square millimeter of microdissected bone tissue.The RIN value is 8.60. Alternatively, LCM is performed using an LCM system that uses a defocused laser pulse, which catapults the material into the overhanging adhesive cap. For fresh frozen bones, it is possible to isolate approximately 1.6 nanograms of RNA from one square millimeter of microdissected bone tissue.The RIN value is one. In conclusion, the most important things to remember in this procedure are apply the standing protocol in the correct manner, prevent complete dryout of sections in the sandwich complex, use an appropriate LCM system, and do not exceed the time limits for harvesting the cells. Once you have isolated RNA from the captured cells, you can use the RNA for expression profiling, for example, by RNA seek.Finally, we would like to remind you that xylene and beta-mercaptoethanol are hazardous substances that need to be handled under a hood. In addition, please take appropriate precautions when handling liquid nitrogen and also the cryotome blades.

a-laser-capture-microdissection-protocol-that-yields-high-quality-rna

Research

JoVE Journal

Biology

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...

A Web Tool for Generating High Quality Machine-readable Biological Pathways

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological information. The pathways generated by this online application are machine-readable and fully compatible with essentially all web-browsers and computer operating systems. It uses a specially developed, web-enabled pathway drawing interface that permits the selection and placement of different combinations of pre-drawn biological or biochemical entities to depict reactions, interactions, transport processes and binding events. This palette of entities consists of chemical compounds, proteins, nucleic acids, cellular membranes, subcellular structures, tissues, and organs. All of the visual elements in it can be interactively adjusted and customized. Furthermore, because this tool is a web server, all pathways and pathway elements are publicly accessible. This kind of pathway &#34;crowd sourcing&#34; means that PathWhiz already contains a large and rapidly growing collection of previously drawn pathways and pathway elements. Here we describe a protocol for the quick and easy creation of new pathways and the alteration of existing pathways. To further facilitate pathway editing and creation, the tool contains replication and propagation functions. The replication function allows existing pathways to be used as templates to create or edit new pathways. The propagation function allows one to take an existing pathway and automatically propagate it across different species. Pathways created with this tool can be &#34;re-styled&#34; into different formats (KEGG-like or text-book like), colored with different backgrounds, exported to BioPAX, SBGN-ML, SBML, or PWML data exchange formats, and downloaded as PNG or SVG images. The pathways can easily be incorporated into online databases, integrated into presentations, posters or publications, or used exclusively for online visualization and exploration. This protocol has been successfully applied to generate over 2,000 pathway diagrams, which are now found in many online databases including HMDB, DrugBank, SMPDB, and ECMDB.

PathWhiz is a comprehensive, online pathway drawing tool for generating biochemical and biological pathways. It uses publicly accessible databases and easily expandable palettes consisting of pre-drawn pathway components. This protocol describes how to easily build new pathways, replicate and edit existing pathways, and propagate previously drawn pathways to different organisms.

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...