This work was supported by funds from the Pittsburgh Cure Sarcoma (PCS) and the Osteosarcoma Institute (OSI).

All animal procedures described below were approved in compliance with the Guide for the Care and Use of Laboratory Animals at the University of Pittsburgh School of Dental Medicine.
1. Method to prepare FFPE blocks of bone tissue samples that require demineralization
<ol>
	<li>Preparation of reagents and materials
	<ol>
		<li>Prepare EDTA 20% pH 8.0. For 1 L, dissolve 200 g of EDTA in 800 mL of ultrapure water, adjust pH to 8.0&#160;with sodium hydroxide 10 N and finally bring the volume to 1L with ultrapure water. Store at room temperature. 
		NOTE: Always use freshly prepared EDTA 20% pH 8.0. Sodium hydroxide (NaOH) 10 N is prepared by dissolving 400 g of NaOH in 1 L of ultrapure water.</li>
		<li>Prepare paraformaldehyde (PFA) 4% in a fume hood utilizing 1x PBS without calcium and magnesium. 
		NOTE: Designate an area in a fume hood for working with concentrated paraformaldehyde solutions, or paraformaldehyde solids, and label it as such. To avoid exposure, keep containers closed as much as possible. Use in the smallest practical quantities needed for the experiment being performed. If weighing paraformaldehyde powder and the weighing scale cannot be used in a fume hood, first take a container, then add PFA powder in the hood and cover before returning to the scale to weigh the powder. Always use freshly prepared 4% PFA.</li>
		<li>Use a suitable container for each specimen. 
		NOTE: For instance, for 1 mg of bone tissue, use a container that allows for at least 1 mL of any solution to be in contact with the bone.</li>
		<li>Sterilize all the surgical equipment needed for dissection.</li>
		<li>Clean all the areas with a decontaminating solution to remove RNases.</li>
		<li>Collect the remaining experimental materials, including ice containers, 70% ethanol spray, and have access to an orbital shaker.</li>
	</ol>
	</li>
	<li>Isolation of bone tissue samples from mice
	<ol>
		<li>Euthanize the mouse by CO2 exposure, then perform cervical dislocation and lay it on its back. Spray the mouse skin with 70% ethanol.</li>
		<li>Using sterile dissecting scissors, open the skin and expose the leg muscle. Cut the muscle alongside the leg to obtain a clear view of the position of the bone, and gently disarticulate the bone to remove it without damage. 
		NOTE: It is not necessary to remove entirely all the muscle attached to the bone. Try to be as fast as possible to limit RNA degradation.</li>
		<li>Immediately place the bone sample in a tube containing 4% PFA and place the tube on ice.</li>
		<li>Repeat the procedure to collect other bone samples as desired.</li>
		<li>Put samples with 4% PFA in agitation on an orbital shaker at 140 rpm&#160;at 4 &#176;C&#160;for at least 24 h to allow proper fixation.</li>
	</ol>
	</li>
	<li>Decalcification of bone tissue samples
	<ol>
		<li>Retrieve the bone tissue from the orbital shaker after fixation.</li>
		<li>Wash the bone tissue 2 times with 1x PBS for 3 min on an orbital shaker at 140 rpm to remove any PFA left.</li>
		<li>Using sterile dissecting scissors, remove any remaining muscles still attached to the bone.</li>
		<li>Wash the bone tissue one more time with 1x PBS for 3 min.</li>
		<li>Place the bone tissue in a new container with 20% EDTA pH 8.0. Then, place the container on an orbital shaker at 140 rpm for 30 min at room temperature (RT).</li>
		<li>Discard the solution, add fresh 20% EDTA pH 8.0 in the container, and place it on an orbital shaker at 140 rpm for 30 min at RT.</li>
		<li>Repeat step 1.3.6 10 times.</li>
		<li>Discard the solution, add fresh 20% EDTA pH 8.0 in the container, and place it at 4 &#176;C in a cold room on an orbital shaker at 140 rpm overnight.</li>
	</ol>
	</li>
	<li>Dehydration of bone tissue samples
	<ol>
		<li>Retrieve the bone tissue sample from the container.</li>
		<li>Inspect the tissue and verify the efficiency of decalcification by gently turning and twisting the bone. Alternatively, perform an X-ray of the bone using an X-ray machine. 
		NOTE: If the specimen flexes easily, a good grade of decalcification has been achieved. If not, decalcification parameters (time, speed of agitation, volume of 20% EDTA pH 8.0. used, etc.) should be increased.</li>
		<li>Wash the bone tissue 2 times with 1x PBS for 3 min to remove residues of EDTA.</li>
		<li>Place the bone tissue in a new container and initiate dehydration with 70% ethanol. Incubate for 15 min at 140 rpm.</li>
		<li>Discard the 70% ethanol solution, put the sample in 90% ethanol solution, and incubate for 15 min at 140 rpm.</li>
		<li>Discard the 90% ethanol solution, put the sample in 100% ethanol, and incubate for 15 min at 140 rpm.</li>
		<li>Discard the 100% ethanol, put the sample in new 100% ethanol, and incubate for 15 min at 140 rpm.</li>
		<li>Discard the 100% ethanol, put the sample in new 100% ethanol, and incubate for 15 min at 140 rpm.</li>
		<li>Discard the 100% ethanol, put the sample in new 100% ethanol, and incubate for 30 min at 140 rpm.</li>
		<li>Discard the 100% ethanol, put the sample in new 100% ethanol, and incubate for 45 min at 140 rpm. 
		&#8203;NOTE: Dehydration can also be performed using an automatic processor. In this case, set up the program with the same conditions reported for the manual dehydration. The reported conditions are for specimens no thicker than 4 mm (i.e., bone tissue samples obtained from mice). For specimens thicker than 4 mm, dehydration timing must be determined experimentally.</li>
	</ol>
	</li>
	<li>Clearing of bone tissue samples
	<ol>
		<li>Place the bone tissue sample in a new container and initiate the clearing process using xylene. Incubate for 20 min at 140 rpm.</li>
		<li>Discard the used xylene, add new xylene, and incubate for an additional 20 min at 140 rpm.</li>
		<li>Discard the used xylene, add new xylene, and incubate for 45 min at 140 rpm. 
		&#8203;NOTE: Clearing can also be performed using an automatic processor. In this case, set up the program with the same conditions reported for the manual clearing. The reported conditions are for specimens no thicker than 4 mm (i.e., bone tissue samples obtained from mice). For specimens thicker than 4 mm, the timing must be determined experimentally.</li>
	</ol>
	</li>
	<li>Infiltration and embedding of bone tissue samples
	<ol>
		<li>Retrieve the bone tissue sample from the orbital shaker/automatic processor after clearing.</li>
		<li>Place the bone tissue sample in an embedding cassette and initiate infiltration with wax (see Table of Materials). Incubate for 30 min at 60 &#176;C.</li>
		<li>Discard the used wax, add new wax, and incubate for 30 min at 60&#160;&#176;C.</li>
		<li>Discard the used wax, add new wax, and incubate for 45 min at 60&#160;&#176;C.</li>
		<li>Carefully place the bone tissue sample in a mold with the desired orientation.</li>
		<li>Allow the specimen block to solidify on a cold surface.</li>
		<li>Store the obtained FFPE at 4 &#176;C until ready to start sectioning. 
		NOTE: FFPE block can be stored for many years before sectioning is carried out.</li>
	</ol>
	</li>
</ol>
2. Guidelines for sectioning un-demineralized FFPE samples
NOTE: The following guidelines provide valuable tips and instructions useful to greatly improve the quality of the tissue sections while avoiding RNA degradation especially in cases of small undecalcified bone specimens. These guidelines may also be applied to decalcified bone specimens when available. For larger bone tissue samples, demineralization should be performed to obtain better quality sections; in such cases, the method reported above should be followed, and parameters (timing, volumes of solutions, etc.) should be adjusted accordingly. The following guidelines are suitable either when FFPE bone tissues have already been processed (e.g., FFPE block collected from tissue banks or other labs) or when the collected sections will be utilized to perform any type of analysis that requires good quality RNA, high-quality tissue sections, or both.
<ol>
	<li>Equipment preparation
	<ol>
		<li>Spray all work surfaces and instruments with decontaminating solutions to remove RNases.</li>
		<li>Prepare a water bath with double distilled water and set the temperature to 42 &#176;C.</li>
		<li>Take a microtome blade, spray it with 70% ethanol to remove oil residues, then spray it with decontaminating solutions to remove RNases.</li>
		<li>Secure the blade on the microtome. Ensure that the clearance angle is set to 10&#176;. 
		NOTE: Always use new blades for sectioning.</li>
		<li>Prepare two buckets of ice.</li>
		<li>Get tweezers, brushes, and probes, spray them with decontaminating solutions to remove RNases, and place them in one of the two ice buckets.</li>
		<li>Prepare an ice bath by adding double distilled water to the other ice bucket. 
		NOTE: the FFPE block should not float in the added water; therefore, the amount of added water to the ice bucket must be just enough to allow the sample to be wet without floating.</li>
	</ol>
	</li>
	<li>Sectioning
	<ol>
		<li>Retrieve the FFPE block from storage at 4 &#176;C and place it on the microtome. Set the command to Trim or to 14 &#181;m of scroll thickness, and start trimming the sample until the tissue is exposed. 
		NOTE: If the FFPE block has already been trimmed and the tissue is already exposed, skip step 2.2.1. Verify that the block is without holes and cracks. If so, proceed directly to step 2.2.2. If not, cut a few sections to flatten the surface and avoid the holes and cracks, and then proceed to step 2.2.2.</li>
		<li>Place the FFPE block on the ice bath to hydrate the sample. The time of hydration must be determined experimentally.
		<ol>
			<li>Start with 2 min and increase the time if hydration is not enough. Do not exceed 10 min. The hydration will expand the tissue and reduce the formation of &#34;Venetian blinds&#34; and lines. Longer hydration times could cause cracks in the paraffin block and, possibly, tissue detachment.</li>
			<li>If 10 min is insufficient to hydrate the sample, then perform hydration cycles no longer than 10 min and attempt sectioning again.</li>
			<li>If the surface of the block is not smooth and clean due to cracks or cuts, consider re-embedding the sample. RNA quality will not be affected by the re-embedding process.</li>
		</ol>
		</li>
		<li>Put the sample in the specimen clamp of the microtome, align it with the blade, and start sectioning. Set the scroll thickness to 5 &#181;m.</li>
		<li>Collect the section and place it in the water bath at 42 &#176;C using cold tweezers and brushes. 
		NOTE: Alternatively, if RNA isolation of tissue sections will be performed, collect the sections in a centrifuge tube and follow the manufacturing specifications for RNA preparation (see Table of Materials).</li>
		<li>Incubate the section in the water bath to stretch the tissue and eliminate any residual wrinkles and folds. 
		NOTE: The time of floating in the water bath for each section must be determined experimentally. Consider leaving the bone sections an extra minute if tissue detachment issues occur. Do not leave the section floating too long since long floating times could damage the tissue.</li>
		<li>Place the section on a glass slide, then incubate it for 3 h at 42 &#176;C.</li>
		<li>Place the glass slide in a desiccator or an oven overnight at RT for proper drying.</li>
		<li>Store the glass slide with the attached section at RT in a slide box. 
		NOTE: Slides can be stored for many years at RT. If spatial transcriptomic will be performed, instead of a glass slide, place the section on the spatial gene expression slide provided by the manufacturer and follow the reported recommendations (see the Table of Materials).</li>
	</ol>
	</li>
</ol>

Protocol to Prepare Demineralized FFPE Bone Tissue Samples with RNA Integrity 

<ol>
	<li>Baig, M. A., Bacha, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histology. Bone. , (2024).</li><li>Currey, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanical+consequences+of+variation+in+the+mineral+content+of+bone.">The mechanical consequences of variation in the mineral content of bone.</a> J Biomech. 2 (1), 1-11 (1969).</li><li>Goldschlager, T., Abdelkader, A., Kerr, J., Boundy, I., Jenkin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Undecalcified+bone+preparation+for+histology,+histomorphometry+and+fluorochrome+analysis.">Undecalcified bone preparation for histology, histomorphometry and fluorochrome analysis.</a> J Vis Exp. (35), e1707 (2010).</li><li>Wallington, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Histological Methods for Bone. , (1972).</li><li>Callis, G. M., Sterchi, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decalcification+of+bone:+Literature+review+and+practical+study+of+various+decalcifying+agents.+methods,+and+their+effects+on+bone+histology.">Decalcification of bone: Literature review and practical study of various decalcifying agents. methods, and their effects on bone histology.</a> J Histotechnol. 21 (1), 49-58 (1998).</li><li>Zhang, P., Lehmann, B. D., Shyr, Y., Guo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utilization+of+formalin+fixed-paraffin-embedded+specimens+in+high+throughput+genomic+studies.">The utilization of formalin fixed-paraffin-embedded specimens in high throughput genomic studies.</a> Int J Genomics. 2017, 1926304 (2017).</li><li>Trinks, 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=Robust+detection+of+clinically+relevant+features+in+single-cell+RNA+profiles+of+patient-matched+fresh+and+formalin-fixed+paraffin-embedded+(FFPE)+lung+cancer+tissue.">Robust detection of clinically relevant features in single-cell RNA profiles of patient-matched fresh and formalin-fixed paraffin-embedded (FFPE) lung cancer tissue.</a> Cell Oncol (Dordr). , (2024).</li><li>Xu, Z., 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=High-throughput+single+nucleus+total+RNA+sequencing+of+formalin-fixed+paraffin-embedded+tissues+by+snRandom-seq.">High-throughput single nucleus total RNA sequencing of formalin-fixed paraffin-embedded tissues by snRandom-seq.</a> Nat Commun. 14 (1), 2734 (2023).</li><li>10x Genomics. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visium+Spatial+Gene+Expression+Reagent+Kits+for+FFPE+-+User+Guide.,+Document+Number+C+CG000407+Rev+C.">Visium Spatial Gene Expression Reagent Kits for FFPE - User Guide., Document Number C CG000407 Rev C.</a> 10x Genomics. , (2021).</li><li>10x Genomics. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpreting+Space+Ranger+Web+Summary+Files+for+Visium+Spatial+Gene+Expression+for+FFPE+F+FFPEAssay.,+Document+Number+CG000499+Rev+A.">Interpreting Space Ranger Web Summary Files for Visium Spatial Gene Expression for FFPE F FFPEAssay., Document Number CG000499 Rev A.</a> 10x Genomics. , (2022).</li><li>10x Genomics. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visium+Spatial+Gene+Expression+for+FFPE-Tissue+Preparation+Guide.,+Document+Number+C+G+CG000408+Rev+D.">Visium Spatial Gene Expression for FFPE-Tissue Preparation Guide., Document Number C G CG000408 Rev D.</a> 10x Genomics. , (2022).</li></ol>

The authors have no conflicts of interest to disclose.

Here, a detailed method is provided to prepare FFPE blocks of decalcified bones and preserve RNA integrity for sequencing (i.e., next-generation sequencing (NGS)) or for other RNA-related techniques (i.e., in situ hybridization, quantitative reverse transcription polymerase chain reaction (qRT-PCR), etc.).
The method utilizes EDTA to decalcify bone tissue samples; the EDTA incubation allows for slow but fine demineralization of the samples, thus preserving the histological features of...

Bone is a specialized connective tissue comprised mainly of fibers of collagen type 1 and inorganic salts1. As a result, bone is incredibly strong and stiff while being, at the same time, light and trauma-resistant. The great strength of bone derives from its mineral content. In fact, for any given increase in the percentage of mineral content, stiffness increases by five-fold2. Consequently, investigators face significant problems when they analyze, by means of histological sectioning, the biology of a bone specimen.
Undecalcified bone histology is feasible and sometimes required, depending on the type of investigation (e.g., to study the micro-architecture of bone); it is, however, very challenging, especially if the specimens are large. In these cases, tissue processing for histological purposes requires several modifications of the standard protocols and techniques3. In general, to perform common histological evaluations, bone tissues are decalcified right after fixation, a process that may require a few days to several weeks, depending on the size of the tissue and the decalcifying agent utilized4. Decalcified sections are often used for the examination of bone marrow, the diagnosis of tumors, etc. There are three main types of decalcifying agents: strong acids (e.g., nitric acid, hydrochloric acid), weak acids (e.g., formic acid), and chelating agents (e.g., ethylenediaminetetracetic acid or EDTA)5. Strong acids can decalcify bone very rapidly, but they can damage the tissues; weak acids are very common and suitable for diagnostic procedures; chelating agents are by far the most used and appropriate for research application since, in this case, the demineralization process is slow and gentle, allowing for retention of high-quality morphology and preservation of gene and protein information, as required by many procedures (e.g., in situ hybridization, immunostaining). However, when the whole transcriptome needs to be preserved, such as for gene expression analyses, even a slow and gentle demineralization may be detrimental. Therefore, better approaches and methods are needed when the morphological analysis of the tissues needs to be paired with gene expression analyses of the cells.
Thanks to recent improvements in single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics, it is now possible to study the gene expression of a tissue specimen even when formalin fixation paraffin embedding (FFPE) was used to store the tissue samples6,7,8. This opportunity has unlocked access to a larger number of samples, such as those stored in tissue banks worldwide. If scRNA-seq is to be employed, RNA integrity is the most important requirement; however, in the case of spatial transcriptomics of FFPE samples, both high-quality tissue sections and high-quality RNA are necessary to visualize the gene expression within the histological context of each tissue section. While this has been easily achieved with soft tissues, the same cannot be said for hard tissues like bone. In fact, to the best of our knowledge, no study using spatial transcriptomics has ever been performed on FFPE bone samples. This is because of the lack of protocols that can effectively process FFPE bone tissues while preserving their RNA content. Here, a method to process and decalcify freshly obtained bone tissue samples while avoiding RNA degradation is provided first. Then, recognizing the need for transcriptomics analysis of the FFPE samples collected in tissue banks worldwide, developed guidelines to properly handle FFPE samples of non-demineralized bones are also presented.

<table><tbody><tr><td>Advanced orbital shaker</td><td>VWR</td><td>76683-470</td><td>Use to keep tissues under agitation during incubation as reported in the method instructions.</td></tr><tr><td>Camel Hair Brushes</td><td>Ted Pella</td><td>11859</td><td>Use to handle FFPE sections as reported in the guidelines.</td></tr><tr><td>Dual Index Kit TS Set A 96 rxns</td><td>10X Genomics</td><td>PN-1000251</td><td>Use to perform spatial transcriptomics.</td></tr><tr><td>Ethanol 200 Proof</td><td>Decon Labs Inc</td><td>2701</td><td>Use to perform tissue dehydration as reported in the method instructions.</td></tr><tr><td>Ethylenediaminetetraacetic Acid, Disodium Salt Dihydrate (EDTA)</td><td>Thermo Fisher Scientific</td><td>S312-500</td><td>Use to prepare EDTA 20% pH 8.0.&amp;nbsp;</td></tr><tr><td>Fisherbrand Curved Medium Point General Purpose Forceps</td><td>Fisher Scientific</td><td>16-100-110</td><td>Use to handle FFPE sections as reported in the guidelines.</td></tr><tr><td>Fisherbrand Fine Precision Probe</td><td>Fisher Scientific</td><td>12-000-153</td><td>Use to handle FFPE sections as reported in the guidelines.</td></tr><tr><td>Fisherbrand&amp;nbsp;Superfrost Plus Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td>Use to attach sectioned scrolls as reported in the guidelines.</td></tr><tr><td>High profile diamond microtome blades</td><td>CL Sturkey</td><td>D554DD</td><td>Use to section FFPE blocks as reported in the guidelines.</td></tr><tr><td>Novaseq 150PE</td><td>Novogene</td><td>N/A</td><td>Sequencer.</td></tr><tr><td>Paraformaldehyde (PFA) 32% Aqueous Solution EM Grade</td><td>Electron Microscopy Sciences</td><td>15714-S</td><td>Dilute to final concentration of 4% with 1x PBS&amp;nbsp; to perform tissue fixation.</td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Thermo Fisher Scientific</td><td>10010-049</td><td>Ready to use. Use to dilute PFA and to perform washes as reported in the method instructions.</td></tr><tr><td>Premiere Tissue Floating Bath&amp;nbsp;</td><td>Fisher Scientific</td><td>A84600061</td><td>Use to remove wrinkles from FFPE sections as reported in the guidelines.</td></tr><tr><td>RNase AWAY Surface Decontaminant</td><td>Thermo Fisher Scientific</td><td>7002</td><td>Use to clean all surfaces as reported in the method instructions.</td></tr><tr><td>RNeasy DSP FFPE Kit</td><td>Qiagen</td><td>73604</td><td>Use to isolate RNA from FFPE sections once they have been generated as reported in the guidelines.</td></tr><tr><td>Semi-Automated Rotary Microtome</td><td>Leica Biosystems</td><td>RM2245</td><td>Use to section FFPE blocks as reported in the guidelines.</td></tr><tr><td>Sodium hydroxide</td><td>Millipore Sigma</td><td>S8045-500</td><td>Prepare 10 N solution by slowly dissolving 400 g in 1 liter of Milli-Q water.</td></tr><tr><td>Space Ranger</td><td>10X Genomics</td><td>2.0.1</td><td>Use to process sequencing data output .</td></tr><tr><td>Surgipath Paraplast</td><td>Leica Biosystems</td><td>39601006</td><td>Use to perform tissue infliltration and embedding as reported in the method instructions.</td></tr><tr><td>Visium Accessory Kit</td><td>10X Genomics</td><td>PN-1000194</td><td>Use to perform spatial transcriptomic experiments.</td></tr><tr><td>Visium Human Transcriptome Probe Kit Small&amp;nbsp;</td><td>10X Genomics</td><td>PN-1000363</td><td>Use to perform spatial transcriptomic experiments.</td></tr><tr><td>Visium Spatial Gene Expression Slide Kit 4 rxns&amp;nbsp;</td><td>10X Genomics</td><td>PN-1000188</td><td>Use to place the sections if performing spatial transcriptomic experiments.</td></tr><tr><td>Xylene</td><td>Leica Biosystems</td><td>3803665</td><td>Use to perform tissue clearing as reported in the method instructions.</td></tr></tbody></table>

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.

The method presented here describes how to process freshly isolated bones to obtain demineralized FFPE samples that can be easily sectioned with a microtome while preserving the RNA integrity (Figure 1). The method has been successfully employed on murine femurs but can be followed for other bone tissue samples of similar dimensions, or it can be adapted for larger bone specimens (e.g., human samples) by increasing all the parameters (timing, volumes of solutions, etc.).
<p class="jove_c...

Read this Scientific Article Protocol about Author Spotlight: Exploring Advanced Therapeutic Targets in Osteosarcoma Through Spatial Transcriptomics at JoVE.com

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.

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 ...

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2.5K Views.  University of Pittsburgh School of Dental Medicine. Ons doel is om betere therapeutische benaderingen te vinden voor de behandeling van een menselijke botkanker die osteosarcoom wordt genoemd. Om dit te doen, willen we de biologische processen blootleggen die verantwoordelijk zijn voor het ontstaan van deze tumor en vooral voor de ontwikkeling van de metastase, die de doodsoorzaak van de patiënt verwijdert. Door ruimtelijke transcriptomics-analyse toe te passen op verschillende overeenkomende exemplaren van metastase van de menselijke primair...

Video: Auteur in de schijnwerpers: onderzoek naar geavanceerde therapeutische doelen bij osteosarcoom door middel van ruimtelijke transcriptomics

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4-/- fibroblast, compared to Mrp4+/+fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

MRP4 regulates various cyclic nucleotide-dependent signaling events including a recently elucidated role in cell migration. We describe a direct, but multifaceted approach to unravel the downstream molecular targets of MRP4 resulting in identification of a unique MRP4 interactome that plays key roles in the fine-tuned regulation of fibroblast migration.

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein and RNA multiplexing by leveraging photo-cleavable oligo-tagged antibodies and probes, respectively. Oligos are cleaved and quantified from specific regions across the tissue to elucidate the underlying biology. Here, the study demonstrates that automated custom antibody visualization protocols can be utilized to guide ROI selection in conjunction with spatial proteomics assays. This specific method did not show acceptable performance with spatial transcriptomics assays. The protocol describes the development of a 3-plex immunofluorescent (IF) assay for marker visualization on an automated platform, using tyramide signal amplification (TSA) to amplify the fluorescent signal from a given protein target and increase the antibody pool to choose from. The visualization protocol was automated using a thoroughly validated 3-plex assay to ensure quality and reproducibility. In addition, the exchange of DAPI for SYTO dyes was evaluated to allow imaging of TSA-based IF assays on the spatial profiling platform. Additionally, we tested the ability of selecting small ROIs using the spatial transcriptomics assay to allow the investigation of highly-specific areas of interest (e.g., areas enriched for a given cell type). ROIs of 50 &#181;m and 300 &#181;m diameter were collected, which corresponds to approximately 15 cells and 100 cells, respectively. Samples were made into libraries and sequenced to investigate the capability to detect signals from small ROIs and profile-specific regions of the tissue. We determined that spatial proteomics technologies highly benefit from automated, standardized protocols to guide ROI selection. While this automated visualization protocol was not compatible with spatial transcriptomics assays, we were able to test and confirm that specific cell populations can successfully be detected even in small ROIs with the standard manual visualization protocol.

Here, we describe a protocol for fine-tuning regions of interest (ROIs) for Spatial Omics technologies to better characterize the tumor microenvironment and identify specific cell populations. For proteomics assays, automated customized protocols can guide ROI selection, while transcriptomics assays can be fine-tuned utilizing ROIs as small as 50 &#181;m.

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein ...

Cancer Research

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 ...

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

Spatial High-resolution Analysis of Gene Expression Levels in Tendons

In recent years, many protocols have been developed for high-resolution transcriptomics in many different medical and biology fields. However, matrix-rich tissues, and specifically, tendons were left behind due to their low cell number, low RNA amount per cell, and high matrix content, which made them complicated to analyze. One of the recent and most important single-cell tools is the spatial analysis of gene expression levels in tendons. These RNA spatial tools have specifically high importance in tendons to locate specific cells of new and unknown populations, validate single-cell RNA-seq results, and add histological context to the single-cell RNA-seq data. These new methods will enable the analysis of RNA in cells with exceptional sensitivity and the detection of single-molecule RNA targets at the single-cell level, which will help to molecularly characterize tendons and promote tendon research. 
In this method paper, we will focus on the available methods to analyze spatial gene expression levels on histological sections by using novel in situ hybridization assays to detect target RNA within intact cells at single-cell levels. First, we will focus on how to prepare the tendon tissue for the different available assays and how to amplify target-specific signals without background noise but with high sensitivity and high specificity. Then, the paper will describe specific permeabilization methods, the different probe designs, and the signal amplification strategies currently available. These unique methods of analyzing transcription levels of different genes in single-cell resolution will enable the identification and characterization of the tendon tissue cells in young and aged populations of various animal models and human tendon tissues. This method will also help analyze gene expression levels in other matrix-rich tissues such as bones, cartilage, and ligaments.

This article describes how to perform an optimized in situ protocol for tendons. This method discusses tissue preparation, section permeabilization, probe design, and signal amplification methods.

In recent years, many protocols have been developed for high-resolution transcriptomics in many different medical and biology fields. However, matrix-rich ...

Principles and Protocols of Mechanical Testing for Bone Fracture Susceptibility

Practical Considerations for the Design, Execution, and Interpretation of Studies Involving Whole-Bone Bending Tests of Rodent Bones

Skeletal fragility leading to fracture is an American public health crisis resulting in 1.5 million fractures each year and $18 billion in direct care costs. The ability to understand the mechanisms underlying bone disease and the response to treatment is not only desired, but critical. Mechanical testing of bone serves as a valuable technique for understanding and quantifying a bone&#39;s susceptibility to fracture. While this method appears simple to perform, inappropriate and inaccurate conclusions may be reached if governing assumptions and key steps are disregarded by the user. This has been observed across disciplines as studies continue to be published with misuse of methods and incorrect interpretation of results. This protocol will serve as a primer for the principles associated with mechanical testing along with the application of these techniques-from considerations of sample size through tissue harvesting and storage, to data analysis and interpretation. With this in hand, valuable information regarding a bone&#39;s susceptibility to fracture may be obtained, furthering understanding for both academic research and clinical solutions.

Author Spotlight: Enhancing Accuracy and Reproducibility in Whole Bone Bending Tests 

Mechanical testing of rodent bones is a valuable method for extracting information regarding a bone's susceptibility to fracture. Lacking proper practical understanding, the results may be overinterpreted or lack validity. This protocol will serve as a guide to ensure mechanical tests are performed accurately to provide valid and functional data.

Skeletal fragility leading to fracture is an American public health crisis resulting in 1.5 million fractures each year and $18 billion in direct care ...

Bioengineering

HE Staining and Micro-CT to Evaluate Murine Trabecular Bone Microarchitecture

Trabecular Bone Microarchitecture Evaluation in an Osteoporosis Mouse Model

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton is crucial for gaining insight into the pathophysiology of osteoporosis and improving its treatment. However, handling bone samples can be complex due to their hard and dense properties. Secondly, specialized software makes image processing and analysis difficult. In this protocol, we present a cost-effective and easy-to-use solution for trabecular bone microstructure analysis. Detailed steps and precautions are provided. Micro-CT is a non-destructive three-dimensional (3D) imaging technique that provides high-resolution images of trabecular bone structure. It allows for the objective and quantitative evaluation of bone quality, which is why it is widely regarded as the gold standard method for bone quality assessment. However, histomorphometry remains indispensable as it offers crucial cellular-level parameters, bridging the gap between two-dimensional (2D) and 3D assessments of bone specimens. As for the histologic techniques, we chose to decalcify the bone tissue and then perform traditional paraffin embedding. In summary, combining these two methods can provide more comprehensive and accurate information on bone microstructure.

Author Spotlight: An Economic and Efficient Method for Quantitative Evaluation of Bone Microarchitecture in a Murine Osteoporosis Model 

This protocol presents an economical and efficient method for quantitative evaluation of bone microarchitecture in a mouse model of osteoporosis by combining Hematoxylin-Eosin (HE) staining and micro-computed tomography (Micro-CT) techniques.

Bone microstructure refers to the arrangement and quality of bone tissue at the microscopic level. Understanding the bone microstructure of the skeleton ...

Medicine

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

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

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

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

Methods to Enable Spatial Transcriptomics of Bone Tissues

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