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 from the abdominal vena cava under general anesthesia (ketamine/xylazine, 100/6 mg/kg intraperitoneal). 
	NOTE: In order to avoid RNA degradation, use RNase-free instruments and materials, wear gloves and work quickly avoiding storage of samples at RT throughout the whole experiment.</li>
	<li>Remove whole femurs rapidly, clean them of surrounding soft tissues using scalpel and paper towels. Pour optimal cutting temperature (OCT) compound into the embedding mold, and put the femur into the bottom of the embedding mold. Snap-freeze the samples in liquid nitrogen. OCT is transparent at RT and white when frozen.</li>
	<li>Once entirely frozen, wrap the samples in foil and transfer them on dry ice to -80 &#176;C. Store samples at -80 &#176;C until further processing. 
	NOTE: The protocol can be paused here.</li>
</ol>
2. Section Preparation
<ol>
	<li>Set the temperature in the cryostat to -19 &#176;C and of the block holder to -17 &#176;C. Wipe the cryostat interior using 70% ethanol. Place the disposable blade for hard tissues, the glass slides and a fitting tool into the cryostat to cool. Keep them inside the cryostat for the duration of sectioning.</li>
	<li>Transfer the frozen tissue block on dry ice to the cryostat and allow it to equilibrate for at least 10 min. 
	NOTE: Avoid repetitive thawing and frequent cycling of one block from -80 &#176;C to -19 &#176;C for cryosectioning.</li>
	<li>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. Wait until the OCT medium is fully frozen.</li>
	<li>Place the block holder in the object holder and tighten it in place. Adjust the blade position and trim the block using 15 &#181;m cutting increments to remove the OCT covering the sample. If the sample has been cut earlier and the surface exposed to air, discard the first 2&#8722;3 tissue sections from the block.</li>
	<li>Adjust the cryostat to generate 8 &#181;m sections and cut 2&#8722;3 cryosections that will be discarded (the first few will typically be thicker than 8 &#181;m). Place the adhesive film on the block and use a fitting tool to adhere the film to the block. Make the cut slowly and at a constant speed holding the section by the film.</li>
	<li>Place the film (sample facing up) immediately on a precooled 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. Proceed immediately with the staining protocol.</li>
</ol>
3. Rapid Staining Protocol
<ol>
	<li>Prepare 40 mL of the following solutions in 50 mL tubes and put them on ice: 95% ethanol, RNase-free water, 100% ethanol, 100% ethanol and 100% xylene. Perform all steps on ice (except the staining). For each experimental day, prepare all aqueous reagents fresh with RNase-free water. 
	CAUTION: Work in the hood to avoid intoxication by xylene.</li>
	<li>Incubate sections for 30 s in 95% ethanol, then dip sections 30 s in RNase-free water to remove OCT carefully and completely which may interfere with LCM and downstream applications.</li>
	<li>Dispense 50 &#181;L of commercial LCM frozen section stain (Table of Materials) onto the section, incubate for 10 s at RT and drain it by placing the edge of the slide on absorbent tissue paper. Remove excess stain by rinsing 30 s in 100% ethanol.</li>
	<li>Immerse bone sections in a second tube with 100% ethanol for 30 s and transfer them to 100% xylene for 30 s.</li>
	<li>Put adhesive film (sample facing up) on dry glass slide as a support. Take care that the film is not impacted and placed as flat as possible. Do not allow the sample to dry completely.</li>
	<li>Place a PET membrane frame slide on it. Shortly press a gloved finger on the membrane to attach it to the film. The sample is then 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. Proceed immediately with the LCM.</li>
</ol>
4. Laser Capture Microdissection
<ol>
	<li>Clean the stage and cap holder from RNase using surface decontaminant (Table of Materials). Load the slide and the caps in the slide holder and the cap holder, respectively.</li>
	<li>Adjust the focus and acquire slide overview with 1.25x objective. Change to the 40x objective and adjust the focus. Choose the area of interest using the slide overview. Adjust laser parameters as follows: aperture, 7; laser power, 60; speed, 5; pulse frequency, 201; specimen balance, 20. Optimize these parameters for each objective.</li>
	<li>If the laser fails to cut the sample, increase the laser power. Alternatively, the laser can be applied more than once. In addition, inspect the target for spots of incomplete cuts and re-drawn the line in these spots using the Move and cut option. Save laser settings for the dissection of subsequent slides.</li>
	<li>Select osteoblasts, osteocytes and bone lining cells in distal femoral cancellous or cortical bone based on morphologic criteria. Draw a line for laser path further away from the target cells to minimize the damage by the UV laser. Perform all microdissections within less than 1 h after the staining.</li>
	<li>Collect each cell type in a separate cap of a 0.5 mL tube. 
	NOTE: Dry capturing instead of liquid recovery may avoid RNA degradation. In addition, very small volumes of buffer contained in the cap of the microcentrifuge tube can either evaporate or crystallize (depending on its composition) during the LCM.</li>
	<li>Confirm capture success by observation of the collection tube cap after the LCM where applicable. Proceed immediately with the RNA extraction.</li>
</ol>
5. RNA Extraction
<ol>
	<li>Dispense 50 &#181;L of the lysis buffer containing &#946;-mercaptoethanol into the cap of the collection tube and lyse the sample by pipetting up and down in the cap for 1 min. Spin down the lysate and add 300 &#181;L of the lysis buffer containing &#946;-mercaptoethanol into the tube. If several caps are going to be pooled, take care that the total volume of buffer is as recommended for the RNA extraction kit (Table of Materials). 
	CAUTION: &#946;-mercaptoethanol must be added to protect RNA from degradation, but it is considered toxic. Wear protective clothes and gloves and work in the hood.</li>
	<li>In addition, for each slide prepare one labeled 1.5 mL microcentrifuge tube with 350 &#181;L of the lysis buffer containing &#946;-mercaptoethanol. Use the sections remaining after the LCM to extract RNA. Carefully separate the film from the membrane and lyse the sample by slowly pipetting the lysis buffer onto the section several times. 
	NOTE: Total amount of lysis buffer should be 350 &#181;L. Do not use all of it at once for digestion as it will flow off the slide.</li>
	<li>Put the lysate samples on dry ice and store them at -80 &#176;C. 
	NOTE: The protocol can be paused here.</li>
	<li>Thaw the lysates at RT. Transfer lysates from LCM-harvested cells from collection tubes into the 1.5 mL microcentrifuge tubes. If more than one cap was used to harvest the sample, pool several lysates.</li>
	<li>Extract RNA according to the manufacturer&#8217;s instructions. Treat columns with DNase I to remove genomic DNA. Elute RNA with 14 &#181;L of RNase-free water, resulting in a 12 &#181;L eluate. Store RNA at -80 &#176;C. 
	NOTE: The protocol can be paused here.</li>
</ol>
6. Measurement of RNA Yield and Integrity
<ol>
	<li>Thaw RNA on wet ice. Measure the yield and integrity of isolated RNA using micro-capillary electrophoresis. Load total RNA (1 &#181;L per sample) into a chip (Table of Materials) according to the manufacturer&#8217;s instructions.</li>
</ol>

<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>

The authors have nothing to disclose.

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 resolution and the laser may not be able to cut through. It is likely that optimal tissue thickness depends on the LCM system (and the laser and slide type) used, as well as on the tissue in question8,25. In the present study, cryosections of different thicknesses (4, 8 or 12 &#181;m) were tested; 8 &#181;m-thick sections were found to be ideal for LCM, while 12 &#181;m bone sections were more difficult to cut with the laser and 4 &#181;m sections gave lower amounts of RNA.
The endogenous RNase activity varies between different tissues. Therefore, staining protocols developed for tissues with very low RNase activity could be unsuitable for other tissue types. Nuclear fast red26 and cresyl violet24 were suggested to be the best in terms of preserving RNA integrity. In addition, alcohol-based methods were superior to aqueous stains for maintaining RNA integrity. However, they suffered from irreproducible staining intensity27. Time is an important parameter to consider. On the one hand, the time required to search and identify cells of interest in the section, and to perform LCM is often relatively long. On the other hand, time available for LCM is limited, since, in some cases, 20% RNA degradation was reached after 30 min28. Therefore, different methods were applied in order to stabilize RNA, such as exposure of sections to argon flux during the microdissection28, or use of buffered alcohol-based cresyl violet staining and keeping the humidity level in the laboratory low27. In addition, a maximum of 15 min for the microdissection step was suggested2. In this study, frozen bone sections were stained using a rapid protocol for a commercial LCM stain, and even after 1 h at RT, RIN values decreased from 8.3 to 9.1. Therefore, all microdissections were performed within less than 1 h after staining. In addition, different protocols for staining were tested (with shorter incubations in aqueous reagents or without the xylene step). No significant improvement in RIN values was achieved.
In LCM studies, two different types of RNA extraction methods have been compared: a phase separation method vs. a column-based method. It was found that the RNA extraction method based on columns led to better RNA quality and higher yield compared to the phase separation method8. In another study, a commercial kit that uses minimal digestion time and temperature (5 min at RT) resulted in superior RNA quality compared to an RNA isolation kit that needs longer digestion time at higher temperature (30 min at 42 &#176;C)7. In the present study, it was possible to extract high quality RNA (RIN &#62; 8) of sufficient concentration from LCM samples using fresh-frozen mouse bones and a column-based RNA extraction method. Thorough lysis (1 min pipetting in the cap) is essential for good RNA yield. The effect of the RNA extraction method on the RNA yield and integrity obtained after LCM was investigated using several different RNA isolation kits according to the manufacturers&#8217; instructions. However, better quality RNAs and increased quantity were obtained with the kit used in the optimized protocol. In the pilot study, microdissected tissue regions of 1 mm2 final area were cut, and it was possible to isolate approximately 8.5 ng of RNAs using 8-&#181;m-thick bone sections (approximately 800 pg/&#181;L). Typically, osteoblasts, osteocytes, and bone lining cells were captured in 2&#8722;3 sections per sample.
Direct comparisons between different LCM instruments are scarce. In one study, two common LCM instruments were tested, which differ in the type of laser used (UV and infrared [IR], respectively) and method of capturing tissue (adhesive isolation cap vs. caps coated with transparent thermoplastic film, respectively). It was found that for thinly-sectioned fresh-frozen mouse brain sections, the IR system resulted in modestly higher RNA quality7. In the present study, two LCM systems that allow contact-free sample collection were compared. In one system, the microdissected sample falls by gravity into the cap placed just below29. Another system uses a de-focused laser pulse, which catapults the material into the overhanging cap30. For fresh frozen bones, higher RNA amounts and RIN values were obtained when gravity was used for tissue capturing. In addition, the catapulting technology is not compatible with the &#8220;sandwich&#8221; composed of adhesive film, cryo-section, and PET membrane, due to the additional weight of the PET membrane.
LCM requires physical access to the tissue surface. Therefore, mounting and glass covering of the specimen is inapplicable, with the consequence of impaired visualization of morphology. To overcome this limitation, a fluid cover medium was developed31. Alternatively, 10&#8722;15 &#181;L of ethanol can be added directly on the tissue section, enabling better morphological analysis before evaporation2. In the present study, frozen bone sections were cut on an adhesive film and, before the sample was allowed to dry completely, it was sandwiched between the PET membrane and the film. This &#8220;sandwich&#8221; method gave good morphological contrast and specific bone cells were clearly identified.

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 color 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&amp;nbsp;Laser&amp;nbsp;Mikrodissektion&amp;nbsp;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 &amp;mu;m</td><td>Molecular Machines &amp;amp; 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 used to obtain a high-quality RNA of sufficient yield. The yield and integrity of isolated RNA was measured using micro-capillary electrophoresis (Figure 1).
The difference in RNA quality and quantity obtained using different lysis protocols can be seen in the representative gel and electropherograms. When the sample was lysed by pipetting up and down in the cap for 1 min, it was possible to isolate approximately 8.5 ng of RNA from 1 mm2 microdissected bone tissue (8 &#181;m-thick section). RIN value was 8.60 (Figure 2).
Alternatively, LCM was performed using an LCM system that uses de-focused laser pulse, which catapults the material into the overhanging adhesive cap. RNA quality and quantity can be estimated in the representative gel and electropherograms. For fresh frozen bones, it was possible to isolate approximately 1.6 ng of RNA from 1 mm2 microdissected bone tissue (8 &#181;m-thick section). The RIN value was 1 (Figure 3).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60197/60197fig01.jpg" /> 
Figure 1: Flowchart of laser capture microdissection protocol for fresh-frozen bones. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60197/60197fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60197/60197fig02.jpg" /> 
Figure 2: Representative gel (top) and electropherograms (bottom) of RNA samples.&#160;LCM was performed using an LCM system that uses gravity for sample collection. RNA samples were retrieved from LCM-harvested tissue (samples 1, 3, 5, 7, and 9) and corresponding control sections (samples 2, 4, 6, 8, and 10, respectively). One RNA sample was retrieved from the control section that was stained but was not used for LCM (sample 11). A total area of 1 mm2 was microdissected, and different lysis protocols were used. Sample 1: 350 &#181;L of the lysis buffer containing &#946;-mercaptoethanol were added into the tube. The cap was closed carefully, and the tube inverted. LCM-harvested cells were lysed by vortexing 1 min, incubating at RT for 10 min and vortexing 1 min. Sample 3: 350 &#181;L of the lysis buffer containing &#946;-mercaptoethanol was added into the collection tube, the cap was carefully closed, and the tube inverted. LCM-harvested cells were lysed by incubating collection tubes upside down for 30 min at RT. Sample 5: 50 &#181;L of the lysis buffer containing &#946;-mercaptoethanol was added into the cap of the collection tube and the sample was lysed by pipetting up and down in the cap for 1 min. The lysate was centrifuged and 300 &#181;L of the lysis buffer containing &#946;-mercaptoethanol was added into the tube. Sample 7: 350 &#181;L of the lysis buffer containing &#946;-mercaptoethanol was added into the tube. The cap was closed carefully, and the tube inverted. LCM-harvested cells were lysed by vortexing and inverting several times. Sample 9: 50 &#181;L of the lysis buffer containing &#946;-mercaptoethanol was added into the cap of the collection tube, and the sample was lysed by incubation for 5 min at RT in the cap. The lysate was centrifuged, and 300 &#181;L of the lysis buffer containing &#946;-mercaptoethanol was added to the tube. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60197/60197fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60197/60197fig03.jpg" /> 
Figure 3: Representative gel (top) and electropherograms (bottom) of RNA samples.&#160;LCM was performed using an LCM system that uses de-focused laser pulse, which catapults the material into the adhesive cap positioned above the section. RNA samples were retrieved from LCM-harvested tissue (samples 5 and 6) and corresponding control section (sample 7). One RNA sample was retrieved from the control section that was stained but was not used for LCM (sample 8). A total area of 0.5 mm2 or 1 mm2 was microdissected (samples 5 and 6, respectively). 350 &#181;L of the lysis buffer containing &#946;-mercaptoethanol was added into the collection tube, the cap was carefully closed and the tube inverted. LCM-harvested cells were lysed by incubating collection tubes upside down for 30 min at RT. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60197/60197fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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Nanyang Technological University

Research

JoVE Journal

Biology

9.5K Views.  University of Veterinary Medicine Vienna. Heb je ooit nagedacht over het benutten van de kracht van laser capture microdissection voor analyse van genexpressie in specifieke botcellen binnen hun natuurlijke omgeving? Hier beschrijven we een protocol waarmee u voldoende hoeveelheden RNA van hoge kwaliteit verkrijgen uit cryosections van muisbotten. Deze technologie is een geweldig hulpmiddel voor het onderzoeken van veranderingen in genexpressie in specifieke botcellen in genetisch gemanipuleerde muismodellen of in ziektemodellen....

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

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

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

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

Murine Hind Limb Long Bone Dissection and Bone Marrow Isolation

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, biomechanics, and stem cell biology. Despite the importance of the bone in healthy and pathologic states, however, it is a largely under-researched organ due to lack of specialized knowledge of bone dissection and bone marrow isolation. Mice are a common model organism to study effects on bone and bone marrow, necessitating a standardized and efficient method for long bone dissection and bone marrow isolation for processing of large experimental cohorts. We describe a straightforward dissection procedure for the removal of the femur and tibia that is suitable for downstream applications, including but not limited to histomorphologic analysis and strength testing. In addition, we outline a rapid procedure for isolation of bone marrow from the long bones via centrifugation with limited handling time, ideal for cell sorting, primary cell culture, or DNA, RNA, and protein extraction. The protocol is streamlined for rapid processing of samples to limit experimental error, and is standardized to minimize user-to-user variability.

Here we present a protocol for the dissection of hind limb long bones (femurs and tibiae) from the laboratory mouse. We further describe a rapid technique for bone marrow isolation from these bones that utilizes centrifugation for removal of bone marrow from the bone marrow space.

Investigation of the bone and the bone marrow is critical in many research fields including basic bone biology, immunology, hematology, cancer metastasis, ...

Immunology and Infection

Protocol to Prepare Demineralized FFPE Bone Tissue Samples with RNA Integrity 

Methods to Enable Spatial Transcriptomics of Bone Tissues

Understanding the relationship between the cells and their location within each tissue is critical to uncover the biological processes associated with normal development and disease pathology. Spatial transcriptomics is a powerful method that enables the analysis of the whole transcriptome within tissue samples, thus providing information about the cellular gene expression and the histological context in which the cells reside. While this method has been extensively utilized for many soft tissues, its application for the analyses of hard tissues such as bone has been challenging. The major challenge resides in the inability to preserve good quality RNA and tissue morphology while processing the hard tissue samples for sectioning. Therefore, a method is described here to process freshly obtained bone tissue samples to effectively generate spatial transcriptomics data. The method allows for the decalcification of the samples, granting successful tissue sections with preserved morphological details while avoiding RNA degradation. In addition, detailed guidelines are provided for samples that were previously paraffin-embedded, without demineralization, such as&#160;samples collected from tissue banks. Using these guidelines, high-quality spatial transcriptomics data generated from tissue bank samples of primary tumor and lung metastasis of bone osteosarcoma are shown.

Author Spotlight: Exploring Advanced Therapeutic Targets in Osteosarcoma Through Spatial Transcriptomics

Here, we describe a method that allows for the decalcification of freshly obtained bone tissues and the preservation of high-quality RNA. A method is also illustrated for sectioning Formalin Fixed Paraffin Embedded (FFPE) samples of non-demineralized bones to obtain good quality results if fresh tissues are not available or cannot be collected.

Understanding the relationship between the cells and their location within each tissue is critical to uncover the biological processes associated with ...

A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (&#8805;8) as needed for transcriptional profiling experiments.

This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or ...

Collection of Frozen Rodent Brain Regions for Downstream Analyses

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex (mPFC) or nucleus accumbens. The challenge in this work is to dissect the correct area while preserving the microenvironment to be examined. In this paper, we describe a simple, low-cost method using resources readily available in most labs. This method preserves nucleic acid and proteins by keeping the tissue frozen throughout the process. Brains are cut into 0.5&#8211;1.0 mm sections using a brain matrix and arranged on a frozen glass plate. Landmarks within each section are compared to a reference, such as the Allen Mouse Brain Atlas, and regions are dissected using a cold scalpel or biopsy punch. Tissue is then stored at -80 &#176;C until use. Through this process rat and mouse mPFC, nucleus accumbens, dorsal and ventral hippocampus and other regions have been successfully analyzed using qRT-PCR and Western assays. This method is limited to brain regions that can be identified by clear landmarks.

This procedure describes the collection of discrete frozen brain regions to obtain high-quality protein and RNA using inexpensive and commonly available tools.

As our understanding of neurobiology has progressed, molecular analyses are often performed on small brain areas such as the medial prefrontal cortex ...

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 ...

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.

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 ...

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

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