这项工作得到了匹兹堡治愈肉瘤（PCS）和骨肉瘤研究所（OSI）的资金支持。

下面描述的所有动物程序均已根据匹兹堡大学牙科医学院的《实验动物护理和使用指南》获得批准。
1.一种需要脱矿质的骨组织样品FFPE块的制备方法
<ol><li>试剂和材料的制备<ol><li>制备EDTA 20%pH 8.0。对于1L，将200g EDTA溶解在800mL超纯水中，用氢氧化钠10N调节pH至8.0，最后用超纯水使体积达到1L。在室温下储存。 注意：始终使用新鲜制备的 EDTA 20% pH 8.0。氢氧化钠 （NaOH） 10 N 是通过将 400 g NaOH 溶解在 1 L 超纯水中来制备的。</li>
		<li>在通风橱中使用 1x PBS 制备 4% 多聚甲醛 （PFA） 不含钙和镁。 注意：在通风橱中指定一个区域用于处理浓缩多聚甲醛溶液或多聚甲醛固体，并贴上标签。为避免暴露，请尽可能保持容器密闭。以正在进行的实验所需的最小实际量使用。如果称量多聚甲醛粉末和称重秤不能在通风橱中使用，请先拿一个容器，然后在通风橱和盖子中加入PFA粉，然后再返回秤称量粉末。始终使用新鲜制备的 4% PFA。</li>
		<li>为每个标本使用合适的容器。 注意：例如，对于 1 mg 骨组织，使用允许至少 1 mL 任何溶液与骨接触的容器。</li>
		<li>对解剖所需的所有手术设备进行消毒。</li>
		<li>用去污溶液清洁所有区域以去除 RNase。</li>
		<li>收集剩余的实验材料，包括冰容器，70%乙醇喷雾剂，并可以使用轨道振荡器。</li>
	</ol></li>
	<li>从小鼠中分离骨组织样本<ol><li>通过CO2 暴露对小鼠实施安乐死，然后进行颈椎脱位并将其放在背上。用70%乙醇喷洒小鼠皮肤。</li>
		<li>使用无菌解剖剪刀，打开皮肤，露出腿部肌肉。切开腿部旁边的肌肉，以获得清晰的骨头位置视图，然后轻轻地分离骨头，以将其移除而不会损坏。 注意：没有必要完全切除附着在骨头上的所有肌肉。尝试尽可能快地限制 RNA 降解。</li>
		<li>立即将骨样本放入含有 4% PFA 的试管中，然后将试管放在冰上。</li>
		<li>根据需要重复该过程以收集其他骨骼样本。</li>
		<li>将含有4%PFA的样品在轨道振荡器上以140rpm，在4°C下搅拌至少24小时，以便正确固定。</li>
	</ol></li>
	<li>骨组织样品的脱钙<ol><li>固定后从眼眶振荡器中取出骨组织。</li>
		<li>在轨道振荡器上以140rpm的速度用1x PBS洗涤骨组织2次3分钟，以除去任何剩余的PFA。</li>
		<li>使用无菌解剖剪刀，去除仍附着在骨头上的任何剩余肌肉。</li>
		<li>用1x PBS再次洗涤骨组织3分钟。</li>
		<li>将骨组织放入含有 20% EDTA pH 8.0 的新容器中。然后，将容器在室温（RT）下以140rpm的速度放在轨道振荡器上30分钟。</li>
		<li>弃去溶液，在容器中加入新鲜的20%EDTA pH 8.0，并将其置于轨道振荡器上，在室温下以140rpm放置30分钟。</li>
		<li>重复步骤 1.3.6 10 次。</li>
		<li>弃去溶液，在容器中加入新鲜的20%EDTA pH 8.0，并将其置于4°C的冷藏室中，在轨道振荡器上以140rpm过夜。</li>
	</ol></li>
	<li>骨组织样本脱水<ol><li>从容器中取出骨组织样本。</li>
		<li>检查组织并通过轻轻转动和扭曲骨头来验证脱钙的效率。或者，使用 X 光机对骨骼进行 X 光检查。 注意：如果试样容易弯曲，则已达到良好的脱钙等级。如果没有，应增加脱钙参数（时间、搅拌速度、20%EDTA pH 8.0的体积等）。</li>
		<li>用 1x PBS 洗涤骨组织 2 次 3 分钟以去除 EDTA 残留物。</li>
		<li>将骨组织放入新容器中，并用70%乙醇开始脱水。以 140 rpm 孵育 15 分钟。</li>
		<li>弃去70%乙醇溶液，将样品放入90%乙醇溶液中，以140rpm孵育15分钟。</li>
		<li>弃去90%乙醇溶液，将样品放入100%乙醇中，并以140rpm孵育15分钟。</li>
		<li>丢弃100%乙醇，将样品放入新的100%乙醇中，并以140rpm孵育15分钟。</li>
		<li>丢弃100%乙醇，将样品放入新的100%乙醇中，并以140rpm孵育15分钟。</li>
		<li>丢弃100%乙醇，将样品放入新的100%乙醇中，并以140rpm孵育30分钟。</li>
		<li>弃去100%乙醇，将样品放入新的100%乙醇中，并以140rpm孵育45分钟。 注意：也可以使用自动处理器进行脱水。在这种情况下，请使用为手动脱水报告的相同条件设置程序。报告的条件适用于厚度不超过 4 毫米的标本（即从小鼠获得的骨组织样本）。对于厚度大于 4 mm 的试样，必须通过实验确定脱水时间。</li>
	</ol></li>
	<li>清除骨组织样本<ol><li>将骨组织样本放入新容器中，并使用二甲苯开始透明化过程。以 140 rpm 孵育 20 分钟。</li>
		<li>丢弃用过的二甲苯，加入新的二甲苯，并以 140 rpm 的速度再孵育 20 分钟。</li>
		<li>丢弃用过的二甲苯，加入新的二甲苯，并以140rpm孵育45分钟。 注意：也可以使用自动处理器执行清算。在这种情况下，请使用为手动清除报告的相同条件设置程序。报告的条件适用于厚度不超过 4 毫米的标本（即从小鼠获得的骨组织样本）。对于厚度大于 4 mm 的试样，必须通过实验确定时间。</li>
	</ol></li>
	<li>骨组织样本的浸润和包埋<ol><li>清除后从轨道振荡器/自动处理器中取出骨组织样本。</li>
		<li>将骨组织样品放入包埋盒中，并开始用蜡浸润（参见 材料表）。在60°C下孵育30分钟。</li>
		<li>丢弃用过的蜡，加入新蜡，并在60°C下孵育30分钟。</li>
		<li>丢弃用过的蜡，加入新蜡，并在60°C下孵育45分钟。</li>
		<li>小心地将骨组织样本放入具有所需方向的模具中。</li>
		<li>让试样块在冷表面上凝固。</li>
		<li>将获得的FFPE储存在4°C，直到准备开始切片。 注意：在进行切片之前，FFPE块可以存放多年。</li>
	</ol></li>
</ol>2. 未脱矿质FFPE样品切片指南
注意：以下指南提供了宝贵的提示和说明，有助于大大提高组织切片的质量，同时避免 RNA 降解，尤其是在未脱钙的小骨标本的情况下。如果可用，这些指南也适用于脱钙骨标本。对于较大的骨组织样本，应进行脱矿质以获得更高质量的切片;在这种情况下，应遵循上述方法，并应相应调整参数（时间、溶液体积等）。当FFPE骨组织已经处理过（例如，从组织库或其他实验室收集的FFPE块）时，或者当收集的切片将用于执行需要高质量RNA、高质量组织切片或两者兼而有之的任何类型的分析时，以下指南都适用。
<ol><li>设备准备<ol><li>用去污溶液喷洒所有工作表面和仪器，以去除 RNase。</li>
		<li>用双蒸水准备一个水浴，并将温度设置为42°C。</li>
		<li>取切片机刀片，喷洒 70% 乙醇以去除油残留物，然后喷洒去污溶液以去除 RNase。</li>
		<li>将刀片固定在切片机上。确保间隙角设置为 10°。 注意： 始终使用新刀片进行切片。</li>
		<li>准备两桶冰块。</li>
		<li>拿来镊子、刷子和探针，用去污溶液喷洒它们以去除 RNase，然后将它们放入两个冰桶中的一个中。</li>
		<li>通过向另一个冰桶中加入双蒸水来准备冰浴。 注意： FFPE 块不应漂浮在添加的水中;因此，向冰桶中添加的水量必须刚好足以使样品湿润而不漂浮。</li>
	</ol></li>
	<li>切片<ol><li>从4°C的储存中取出FFPE块，并将其放在切片机上。将命令设置为 修剪 或 14 μm 的涡旋厚度，然后开始修剪样品，直到组织暴露出来。 注意：如果 FFPE 块已经被修剪并且组织已经暴露，请跳过步骤 2.2.1。确认块没有孔和裂纹。如果是这样，请直接执行步骤 2.2.2。如果没有，请切几段以平坦表面并避免孔洞和裂纹，然后继续执行步骤 2.2.2。</li>
		<li>将FFPE块放在冰浴上以水合样品。水合作用的时间必须通过实验确定。<ol><li>从 2 分钟开始，如果水合作用不足，则增加时间。不要超过 10 分钟。水合作用会扩大组织并减少“威尼斯百叶窗”和线条的形成。较长的水化时间可能会导致石蜡块出现裂纹，并可能导致组织脱落。</li>
			<li>如果 10 分钟不足以水合样品，则执行不超过 10 分钟的水合循环并再次尝试切片。</li>
			<li>如果块的表面由于裂纹或割伤而不光滑和清洁，请考虑重新嵌入样品。RNA质量不会受到重新包埋过程的影响。</li>
		</ol></li>
		<li>将样品放入切片机的标本夹中，将其与刀片对齐，然后开始切片。将涡旋厚度设置为 5 μm。</li>
		<li>收集切片并使用冷镊子和刷子将其置于42°C的水浴中。 注：或者，如果要进行组织切片的 RNA 分离，请将切片收集在离心管中，并遵循 RNA 制备的生产规格（参见 材料表）。</li>
		<li>将切片在水浴中孵育，以拉伸组织并消除任何残留的皱纹和褶皱。 注意：每个部分在水浴中漂浮的时间必须通过实验确定。如果出现组织脱离问题，请考虑将骨骼部分多留一分钟。不要让该部分漂浮太久，因为长时间漂浮可能会损坏组织。</li>
		<li>将切片放在载玻片上，然后在42°C下孵育3小时。</li>
		<li>将载玻片放入干燥器或烤箱中，在室温下过夜以适当干燥。</li>
		<li>在室温下将带有连接部分的载玻片存放在载玻片盒中。 注意：载玻片在室温下可以存放多年。如果要进行空间转录组学，而不是载玻片，请将该部分放在制造商提供的空间基因表达载玻片上，并按照报告的建议进行操作（参见 材料表）。</li>
	</ol></li>
</ol>

制备具有 RNA 完整性的脱矿质 FFPE 骨组织样品的方案 

<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>

作者没有要披露的利益冲突。

本文提供了一种详细的方法来制备脱钙骨的FFPE块，并保持RNA的完整性，用于测序（即下一代测序（NGS））或其他RNA相关技术（即 原位 杂交、定量逆转录聚合酶链反应（qRT-PCR）等）。
该方法利用EDTA对骨组织样品进行脱钙;EDTA孵育允许样品进行缓慢而精细的脱矿质，从而保持组织的最佳组织学特征。通常，使用 10% EDTA pH 7.0 进行骨脱钙至少 2 周，每隔一天刷新一次溶液<s...

骨骼是一种特殊的结缔组织，主要由 1 型胶原纤维和1 型无机盐组成。因此，骨骼非常坚固和坚硬，同时又具有轻便性和抗创伤性。骨骼的巨大强度源于其矿物质含量。事实上，矿物质含量百分比每增加一个百分点，刚度就会增加五倍2。因此，研究人员在通过组织学切片分析骨标本的生物学时面临重大问题。
未脱钙的骨组织学检查是可行的，有时是必需的，具体取决于检查的类型（例如，研究骨骼的微观结构）;然而，这是非常具有挑战性的，特别是如果标本很大。在这些情况下，用于组织学目的的组织处理需要对标准协议和技术进行多次修改3.一般来说，为了进行常见的组织学评估，骨组织在固定后立即脱钙，这一过程可能需要几天到几周的时间，具体取决于组织的大小和所使用的脱钙剂4.脱钙切片常用于骨髓的检查、肿瘤的诊断等。脱钙剂主要有三种类型：强酸（如硝酸、盐酸）、弱酸（如甲酸）和螯合剂（如乙二胺三链酸或EDTA）5。强酸可以非常迅速地使骨骼脱钙，但它们会损害组织;弱酸很常见，适用于诊断程序;螯合剂是迄今为止使用最多且适用于研究应用的，因为在这种情况下，脱矿质过程缓慢而温和，可以保留高质量的形态并保留基因和蛋白质信息，这是许多程序（例如， 原位 杂交、免疫染色）所要求的。然而，当需要保留整个转录组时，例如用于基因表达分析，即使是缓慢而温和的去矿质化也可能是有害的。因此，当组织的形态学分析需要与细胞的基因表达分析配对时，需要更好的方法和方法。
由于单细胞 RNA 测序 （scRNA-seq） 和空间转录组学的最新改进，现在甚至可以使用福尔马林固定石蜡包埋 （FFPE） 来储存组织样本 6,7,8。这个机会开启了对更多样本的访问，例如储存在全球组织库中的样本。如果要采用scRNA-seq，RNA完整性是最重要的要求;然而，在FFPE样品的空间转录组学中，高质量的组织切片和高质量的RNA对于在每个组织切片的组织学背景下可视化基因表达都是必需的。虽然这在软组织中很容易实现，但对于骨骼等硬组织来说却不能这样。事实上，据我们所知，从未对 FFPE 骨样本进行过使用空间转录组学的研究。这是因为缺乏能够有效处理FFPE骨组织同时保留其RNA含量的方案。本文首先提供了一种在避免RNA降解的同时对新鲜获得的骨组织样品进行处理和脱钙的方法。然后，认识到需要对全球组织库中收集的FFPE样本进行转录组学分析，并提出了制定的指南，以正确处理非脱矿质骨骼的FFPE样本。

<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

了解细胞之间的关系及其在每个组织中的位置对于揭示与正常发育和疾病病理学相关的生物学过程至关重要。空间转录组学是一种强大的方法，可以分析组织样本中的整个转录组，从而提供有关细胞基因表达和细胞所在的组织学背景的信息。虽然这种方法已被广泛用于许多软组织，但其在骨骼等硬组织分析中的应用一直具有挑战性。主要挑战在于在处理硬组织样品进行切片时无法保留高质量的 RNA 和组织形态。因此，本文描述了一种处理新鲜获得的骨组织样本以有效生成空间转录组学数据的方法。该方法允许对样品进行脱钙，从而成功获得组织切片，保留形态细节，同时避免 RNA 降解。此外，还为以前未进行脱矿质的石蜡包埋样品（例如从组织库收集的样品）提供了详细的指南。使用这些指南，显示了从原发肿瘤和骨肉瘤肺转移的组织库样本生成的高质量空间转录组学数据。

本文介绍的方法描述了如何处理新鲜分离的骨头以获得脱矿质的FFPE样品，这些样品可以很容易地用切片机切片，同时保持RNA的完整性（图1）。该方法已成功用于小鼠股骨，但可以用于其他类似尺寸的骨组织样本，或者可以通过增加所有参数（时间、溶液体积等）来适应更大的骨样本（例如，人类样本）。
<img alt="Figure 1" ...

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

作者聚焦：通过空间转录组学探索骨肉瘤的先进治疗靶点

在这里，我们描述了一种方法，该方法允许对新鲜获得的骨组织进行脱钙并保留高质量的RNA。还展示了一种方法，用于对非脱矿质骨骼的福尔马林固定石蜡包埋 （FFPE） 样品进行切片，以便在无法获得或无法收集新鲜组织的情况下获得高质量的结果。

实现骨组织空间转录组学的方法

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

我们的目标是找到更好的治疗方法来治疗称为骨肉瘤的人类骨癌。为了做到这一点，我们希望揭示导致这种肿瘤发病的生物学过程，尤其是导致其转移发展的生物学过程，这就是患者死亡的原因。通过对人类原发性骨肉瘤系转移的几个匹配标本进行空间转录组学分析，我们定位了位于肿瘤区域内的多个患者常见细胞群，并通过比较它们，我们确定了几个关键基因，这些基因可能代表预防肺转移的有价值的治疗靶点。心脏组织（如骨骼）的特殊转录组学分析非常具有挑战性，因为无法保留高质量的 RNA 和组织形态，同时处理心脏组织进行切片。我们的协议填补了这一空白，使研究人员能够成功地将特殊的转录组学分析应用于心脏组织。

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

Research

JoVE Journal

Developmental Biology

Methods to Enable Spatial Transcriptomics of Bone Tissues

2.0K 次观看.  University of Pittsburgh School of Dental Medicine. 我们的目标是找到更好的治疗方法来治疗称为骨肉瘤的人类骨癌。为了做到这一点，我们希望揭示导致这种肿瘤发病的生物学过程，尤其是导致其转移发展的生物学过程，这就是患者死亡的原因。通过对人类原发性骨肉瘤系转移的几个匹配标本进行空间转录组学分析，我们定位了位于肿瘤区域内的多个患者常见细胞群，并通过比较它们，我们确定了几个关键基因，这些基因?...

视频: 作者聚焦：通过空间转录组学探索骨肉瘤的先进治疗靶点

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.

方法在成纤维细胞迁移的规律研究大分子复合物含MRP4，

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

科研

发育生物学

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.

方法来检查淋巴腺和血细胞中<em&gt;果蝇</em&gt;幼虫

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.

在Active ERK的时空分析<em&gt;℃。线虫</em&gt;生殖系

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.

从详细的空间表达图谱的动态表达数据的时间排序

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.

脊髓培养法研究胚胎鸡组织的发展

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.

成人斑马鱼损伤模型研究泼尼松对再生骨组织的影响

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.

平行板灌注系统的体外模型研究了细菌对移植组织的粘附性

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.

小鼠颅面组织和未切除骨骼的组织制备及免疫染色

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.

检测果蝇黑色素的内分泌干扰的方法

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

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its longevity can be a powerful way to reveal mechanisms of tissue development and maintenance. One of the most important tools currently available to monitor cells in vivo is the Confetti mouse model. The Confetti model can be used to genetically label individual cells in living mice with various fluorescent proteins in a cell type-specific manner and monitor their fate, as well as the fate of their progeny over time, in a process called clonal genetic tracing or clonal lineage tracing. This model was generated almost a decade ago and has contributed to an improved understanding of many biological processes, particularly related to stem cell biology, development, and renewal of adult tissues. However, preserving the fluorescent signal until image collection and simultaneous capturing of various fluorescent signals is technically challenging, particularly for mineralized tissue. This publication describes a step-by-step protocol for using the Confetti model to analyze growth plate cartilage that can be applied to any mineralized or nonmineralized tissue.

This method describes the use of the R26R-Confetti (Confetti) mouse model to study mineralized tissues, covering all steps from the breeding strategy to the image acquirements. Included is a general protocol that can be applied to all soft tissues and a modified protocol that can be applied to mineralized tissues.

使用纸屑小鼠研究矿化组织的克隆遗传追踪

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...