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 br...

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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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.&#160;</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&#160;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&#160; 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&#160;</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&#160;</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&#160;</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.

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

Methods to Enable Spatial Transcriptomics of Bone Tissues

Our goal is to find better therapeutic approaches to treat a human bone cancer called osteosarcoma. In order to do so, we want to uncover the biological processes responsible for the onset of this tumor and above all for the development of its metastasis, which is deleting cause of patient's death. By employing spatial transcriptomics analysis on several matching specimens of human primary osteosarcoma line metastasis, we located in multiple patient common cell populations residing within the tumoral areas, and by comparing them, we identified several key genes which may represent valuable therapeutic targets to prevent lung metastasis.Special transcriptomics analysis of heart tissue, such as bone have been very challenging because of the inability to preserve good quality RNA and tissue morphology, while at the same time processing the heart tissue for sectioning. Our protocol addresses this gap, allowing researchers to successfully apply special transcriptomics analysis also to heart tissues.

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

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 ...

methods-to-enable-spatial-transcriptomics-of-bone-tissues