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 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.

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 ...

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

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Developmental Biology

2.2K Views.  University of Pittsburgh School of Dental Medicine. 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. 

Video: Author Spotlight: Exploring Advanced Therapeutic Targets in Osteosarcoma Through Spatial 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 ...

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize mammalian adaptive immunity. Drosophila and mammalian hematopoiesis occur in spatially and temporally distinct phases to produce several blood cell lineages. Both systems maintain reservoirs of blood cell progenitors with which to expand or replace mature lineages. The hematopoietic system allows Drosophila and mammals to respond to and to adapt to immune challenges. Importantly, the transcriptional regulators and signaling pathways that control the generation, maintenance, and function of the hematopoietic system are conserved from flies to mammals. These similarities allow Drosophila to be used to genetically model hematopoietic development and disease.
Here we detail assays to examine the hematopoietic system of Drosophila larvae. In particular, we outline methods to measure blood cell numbers and concentration, visualize a specific mature lineage in vivo, and perform immunohistochemistry on blood cells in circulation and in the hematopoietic organ. These assays can reveal changes in gene expression and cellular processes including signaling, survival, proliferation, and differentiation and can be used to investigate a variety of questions concerning hematopoiesis. Combined with the genetic tools available in Drosophila, these assays can be used to evaluate the hematopoietic system upon defined genetic alterations. While not specifically outlined here, these assays can also be used to examine the effect of environmental alterations, such as infection or diet, on the hematopoietic system.

Methods to Examine the Lymph Gland and Hemocytes in Drosophila Larvae

Drosophila and mammalian hematopoietic systems share many common features, making Drosophila an attractive genetic model to study hematopoiesis. Here we demonstrate dissection and mounting of the major larval hematopoietic organ for immunohistochemistry. We also describe methods to assay various larval hematopoietic compartments including circulating hemocytes and sessile crystal cells.

Many parallels exist between the Drosophila and mammalian hematopoietic systems, even though Drosophila lack the lymphoid lineage that characterize ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart disease. When using prosthetic materials however, these grafts are susceptible to bacterial infections and various host responses.
Identification of bacterial and host factors that play a vital role in endovascular adherence of microorganisms is of importance to better understand the pathophysiology of the onset of infections such as infective endocarditis (IE) and to develop preventive strategies. Therefore, the development of competent models to investigate bacterial adhesion under physiological shear conditions is necessary. Here, we describe the use of a newly designed in vitro perfusion chamber based on parallel plates that allows the study of bacterial adherence to different components of graft tissues such as exposed extracellular matrix, endothelial cells and inert areas. This method combined with colony-forming unit (CFU) counting is adequate to evaluate the propensity of graft materials towards bacterial adhesion under flow. Further on, the flow chamber system might be used to investigate the role of blood components in bacterial adhesion under shear conditions. We demonstrated that the source of tissue, their surface morphology and bacterial species specificity are not the major determining factors in bacterial adherence to graft tissues by using our in-house designed in vitro perfusion model.

An In Vitro Model of a Parallel-Plate Perfusion System to Study Bacterial Adherence to Graft Tissues

We describe an in-house designed in vitro flow chamber model, which allows the investigation of bacterial adherence to graft tissues.

Various valved conduits and stent-mounted valves are used for right ventricular outflow tract (RVOT) valve replacement in patients with congenital heart ...

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...

Methods to Test Endocrine Disruption in Drosophila melanogaster

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor chemicals (EDCs). These chemicals may alter the normal balance of endocrine systems and lead to adverse effects, as well as an increasing number of hormonal disorders in the human population or disturbed growth and reduced reproduction in the wildlife species. For some EDCs, there are documented health effects and restrictions on their use. However, for most of them, there is still no scientific evidence in this sense. In order to verify potential endocrine effects of a chemical in the full organism, we need to test it in appropriate model systems, as well as in the fruit fly, Drosophila melanogaster. Here we report detailed in vivo protocols to study endocrine disruption in Drosophila, addressing EDC effects on the fecundity/fertility, developmental timing, and lifespan of the fly. In the last few years, we used these Drosophila life traits to investigate the effects of exposure to 17-&#945;-ethinylestradiol (EE2), bisphenol A (BPA), and bisphenol AF (BPA F). Altogether, these assays covered all Drosophila life stages and made it possible to evaluate endocrine disruption in all hormone-mediated processes. Fecundity/fertility and developmental timing assays were useful to measure the EDC impact on the fly reproductive performance and on developmental stages, respectively. Finally, the lifespan assay involved chronic EDC exposures to adults and measured their survivorship. However, these life traits can also be influenced by several experimental factors that had to be carefully controlled. So, in this work, we suggest a series of procedures we have optimized for the right outcome of these assays. These methods allow scientists to establish endocrine disruption for any EDC or for a mixture of different EDCs in Drosophila, although to identify the endocrine mechanism responsible for the effect, further essays could be needed.

Methods to Test Endocrine Disruption in Drosophila melanogaster

Endocrine disruptor chemicals (EDCs) represent a serious problem for organisms and for natural environments. Drosophila melanogaster represents an ideal model to study EDC effects in vivo. Here, we present methods to investigate endocrine disruption in Drosophila, addressing EDC effects on fecundity, fertility, developmental timing, and lifespan of the fly.

In recent years there has been growing evidence that all organisms and the environment are exposed to hormone-like chemicals, known as endocrine disruptor ...