这项工作得到了研究补助金的支持, M.H. 来自国家癌症中心, 韩国 (1510121) 和国家研究基金会, 韩国 (不。NRF-2015R1A2A2A04007432)。

该研究议定书获得韩国国家癌症中心 (NCCNCS-12-630) 机构审查委员会的批准。
1. 样品准备 (30 分钟)
<ol><li>对于 HeLa 细胞 (CCL-2, ATCC) 的培养, 在100毫米培养皿中使用10毫升完整的 DMEM 培养基 (10% 胎牛血清, 1% 枝链球菌)。如前一项研究11所述, 制备血清饥饿的 hela 细胞, 在无胎牛血清的 DMEM 中孵育汇合的 hela 细胞48小时。</li>
	<li>要分离细胞, 用2毫升磷酸盐缓冲盐水 (PBS) 冲洗, 并添加2毫升 0.25% EDTA 胰蛋白酶每100毫米培养皿。然后, 在37摄氏度的 CO2孵化器中孵育 2-3 分钟。</li>
	<li>添加5毫升完整的 DMEM 培养基到培养皿停用胰蛋白酶, 并将分离的细胞转移到15毫升管。然后, 离心细胞在 300 x g 10 分钟形成一个小球。</li>
	<li>取出上清液, 用2毫升冷 PBS 将颗粒清洗两次, 离心 300 x g 10 分钟。</li>
	<li>清除清液后, 将1毫升95% 乙醇添加到细胞颗粒中, 用涡流将细胞颗粒与乙醇混合。把固定的细胞放在冰上直到细胞块准备。</li>
</ol>2. 细胞块制备 (1 小时30分钟)
<ol><li>为制备冷冻血浆整除数, 从健康献血者血液和离心机中收集 1.3万 x g 的 EDTA 血浆, 10 分钟. 收集上清血浆, 并整除 200-400 µL 成离心管。将整除数保存在-80 摄氏度, 直到使用。当冷冻血浆整除数准备就绪时, 通过孵化37摄氏度以5分钟的时间提取并解冻血浆。</li>
	<li>将固定细胞离心 700 x 克, 10 分钟, 然后丢弃上清。</li>
	<li>将细胞颗粒与2滴 (大约200µL) 的血浆、2滴凝血酶和2滴0.025 米氯化钙混合在一起。然后, 混合他们的吹打。</li>
	<li>允许混合物在室温下形成细胞血栓10分钟, 如图 2A所示。然后, 通过用吸管浇和去除 pbs 两次冲洗细胞血栓。代表的白细胞血栓显示在图 2B中。</li>
	<li>用福尔马林滋润一张滤纸, 用预湿滤纸包裹细胞凝结。然后, 将凝结的混合物与 pincette 放在纸巾盒中, 如图 2C所示。</li>
	<li>将纸巾盒放在装有50毫升缓冲福尔马林 (PBS 10% 福尔马林) 的玻璃瓶中, 隔夜4摄氏度用于福尔马林固定。</li>
</ol>3. 组织加工和石蜡嵌入 (隔夜)
<ol><li>将含有福尔马林固定细胞凝块的组织盒装入一夜之间, 如前所述11。组织处理是在石蜡嵌入前对固定细胞进行除水和调理。对于没有组织处理的研究, 执行组织处理过程, 如表 1所示。</li>
	<li>打开加热的嵌入站, 60 分钟后, 检查其中的金属模具中是否有熔融石蜡 (图 2D)。</li>
	<li>从纸巾盒中取出形成的细胞血栓, 将其放入金属模内的熔融石蜡中。</li>
	<li>将新的纸巾盒放在不带盖子的金属模内, 放入熔融石蜡中间的细胞血栓, 然后将更多的熔融石蜡倒入纸巾盒中, 放入金属模上。然后, 让石蜡凝固在冷板 30-60 秒。</li>
	<li>将纸巾盒与金属模具分开。然后, 石蜡细胞块已经准备好用于细胞化学。在石蜡嵌入的单元格块中的嵌入细胞血栓由图 2E中的箭头指示。</li>
</ol>4. 制备免疫细胞化学 (1 h) 的幻灯片
<ol><li>检查石蜡细胞块中细胞血栓的面积, 用切片将细胞块切成 3-4 µm 的切片。将石蜡切片放在硅烷涂层的玻璃幻灯片上, 如图 2F所示。</li>
	<li>将截面幻灯片放在37°c 烤箱中30分钟, 以使各部分符合幻灯片。</li>
</ol>5. 细胞化学 (6 小时)
注: 对于单元格块部分的免疫细胞化学染色, 可以使用各种套件 (参见材料表)。所有这些套件都有不同的灵敏度和特殊性, 这取决于它们的修改。
<ol><li>将50毫升二甲苯中的剖面孵育4分钟, paraffinize。</li>
	<li>在100% 乙醇中孵化2分钟脱水, 两次95% 乙醇, 80% 乙醇为2分钟。然后, 在自来水中取出乙醇10分钟。</li>
	<li>对于抗原检索, 将幻灯片放在含有40毫升三 EDTA 检索缓冲器的罐子中, pH 值为 9.0, 然后在炊具中煮沸30分钟。</li>
	<li>在流水下冲洗滑梯, 在4摄氏度的95% 乙醇中孵化10分钟。然后, 用 Pap 笔标记幻灯片上的细胞染色区域, 以便于识别。</li>
	<li>将含有0.2% 吐温 20 (tb T) 的三缓冲盐水中的幻灯片洗净, 并在室温下在过氧化氢块中孵化 (参见材料表), 以除去残余过氧化物酶活性。然后, 每三次用2分钟的时间来冲洗幻灯片。</li>
	<li>通过稀释蛋白块中的原抗体来制备稀释的抗体溶液 (参见材料表)。例如, CKAP2 原抗体可以稀释 1:100, Ki-67 抗体1:500。然后, 在100µL 的稀释抗体溶液中孵化出1小时的幻灯片。</li>
	<li>在每2分钟洗五次后, 在黑暗中室温下, 在试剂盒中的主要抗体增强剂中孵化出15分钟的幻灯片。</li>
	<li>在四次洗涤后, 每2分钟, 添加2滴的 HRP 聚合物 (一次抗体标记为辣根过氧化物酶), 并孵化在室温下的幻灯片30分钟。</li>
	<li>在每五次冲洗2分钟后, 添加100µL (diaminobenzidine) 解决方案 (参见材料表), 并将幻灯片孵化3分钟。</li>
	<li>冲洗后两次, 添加100µL 的苏木精溶液 (100 µL 的苏木精和600µL 的蒸馏水), 并孵化的幻灯片1分钟。</li>
	<li>在冲洗过一次的幻灯片后, 脱水通过孵化95% 乙醇2分钟, 浸泡在95% 乙醇一次, 并浸泡在100% 乙醇两次。然后, 在一个玻璃罐子里孵育40毫升二甲苯中的滑梯5分钟。</li>
	<li>当从二甲苯干燥的幻灯片, 装载各自的盖玻片。</li>
	<li>使用光镜观察染色图案。</li>
</ol>

<ol>
	<li>Bhattacharyya, D., Hammond, A. T., Glick, B. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-quality+immunofluorescence+of+cultured+cells.">High-quality immunofluorescence of cultured cells.</a> Methods Mol Biol. 619, 403-410 (2010).</li><li>Brown, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+microscopy+-+Avoiding+the+pit+falls.">Fluorescence microscopy - Avoiding the pit falls.</a> J Cell Sci. 120, 1703-1705 (2007).</li><li>Odell, I. D., Cook, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescence+Techniques.">Immunofluorescence Techniques.</a> J Invest Dermatol. 133, e4 (2013).</li><li>Kulkarni, M. B., Desai, S. B., Ajit, D., Chinoy, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+utility+of+the+thromboplastin-plasma+cell-block+technique+for+fine-needle+aspiration+and+serous+effusions.">The utility of the thromboplastin-plasma cell-block technique for fine-needle aspiration and serous effusions.</a> Diagn Cytopathol. 37 (2), 86-90 (2009).</li><li>Shivakumarswamy, U., Arakeri, S. U., Karigowdar, M. H., Yelikar, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+utility+of+the+cell+block+method+versus+the+conventional+smear+study+in+pleural+fluid+cytology.">Diagnostic utility of the cell block method versus the conventional smear study in pleural fluid cytology.</a> J Cytol. 29 (1), 11-15 (2012).</li><li>Nathan, N. A., Narayan, E., Smith, M. M., Horn, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+block+cytology:+Improved+preparation+and+its+efficacy+in+diagnostic+cytology.">Cell block cytology: Improved preparation and its efficacy in diagnostic cytology.</a> Am J Clin Pathol. 114 (4), 599-606 (2000).</li><li>Keyhani-Rofagha, S., Vesey-Shecket, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+value,+feasibility,+and+validity+of+preparing+cell+blocks+from+fluid-based+gynecologic+cytology+specimens.">Diagnostic value, feasibility, and validity of preparing cell blocks from fluid-based gynecologic cytology specimens.</a> Cancer. 96, 204-209 (2002).</li><li>Young, N. A., Naryshkin, S., Katz, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+value+of+electron+microscopy+on+paraffin-embedded+cytologic+material.">Diagnostic value of electron microscopy on paraffin-embedded cytologic material.</a> Diagn Cytopathol. 9, 282-290 (1993).</li><li>Jin, Y., Murakumo, Y., Ueno, K., Hashimoto, M., Watanabe, T., Shimoyama, Y., 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=Identification+of+a+mouse+cytoskeleton-associated+protein,+CKAP2,+with+microtubule-stabilizing+properties.">Identification of a mouse cytoskeleton-associated protein, CKAP2, with microtubule-stabilizing properties.</a> Cancer Sci. 95 (10), 815-821 (2004).</li><li>Hong, K. U., Choi, Y. -. B., Lee, J. -. H., Kim, H. -. J., Kwon, H. -. R., Seong, Y. -. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+phosphorylation+of+tumor+associated+microtubule+associated+protein+(TMAP)/cytoskeleton+associated+protein+2+(CKAP2)+at+Thr-596+during+early+phases+of+mitosis.">Transient phosphorylation of tumor associated microtubule associated protein (TMAP)/cytoskeleton associated protein 2 (CKAP2) at Thr-596 during early phases of mitosis.</a> Exp Mole Med. 40, 377-386 (2008).</li><li>Sim, S. H., Bae, C. D., Kwon, Y., Hwang, H. -. L., Poojan, S., Hong, H. -. I., 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=CKAP2+(cytoskeleton-associated+protein2)+is+a+new+prognostic+marker+in+HER2-negative+luminal+type+breast+cancer.">CKAP2 (cytoskeleton-associated protein2) is a new prognostic marker in HER2-negative luminal type breast cancer.</a> PLoS ONE. 12 (8), e0182107 (2017).</li><li>Inwald, E. C., Klinkhammer-Schalke, M., Hofstadter, F., Zeman, F., Koller, M., Gerstenhauer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ki-67+is+a+prognostic+parameter+in+breast+cancer+patients:+results+of+a+large+population-based+cohort+of+a+cancer+registry.">Ki-67 is a prognostic parameter in breast cancer patients: results of a large population-based cohort of a cancer registry.</a> Breast Cancer Res Treat. 139 (2), 539-552 (2013).</li><li>Katikireddy, K. R., O'Sullivan, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+and+immunofluorescence+procedures+for+protein+analysis.">Immunohistochemical and immunofluorescence procedures for protein analysis.</a> Methods Mol Biol. 784, 155-167 (2011).</li><li>Beutner, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunofluorescent+staining:+The+fluorescent+antibody+method.">Immunofluorescent staining: The fluorescent antibody method.</a> Microbiol Mol Biol Rev. 25 (1), 49-76 (1961).</li></ol>

所有作者都宣布没有利益冲突。

固定培养细胞的免疫荧光染色目前是测定细胞中蛋白质表达水平的首选方法, 同时保留形态学信息1。然而, 在石蜡嵌入细胞块中免疫化学可以是一个很好的选择。本议定书介绍了石蜡嵌段和免疫细胞化学制备的详细程序, 希望能促进免疫细胞化学的应用。
免疫细胞化学在免疫荧光染色方面有优势。细胞免疫荧光染色通常需要新鲜培养的细胞, 但石蜡细胞块可以保持在室温下的13年。此外, 细胞免疫组化可以通过使用与人体组织的常规免疫化学方法相同的抗体4来探索胞内表达模式。此外, 它可以探索蛋白质水平或翻译后修改的变化, 无论是预先孵化细胞与各种药物或在不同的文化条件下 11.
与免疫细胞化学染色的优点相比, 石蜡嵌块的制备需要时间, 成本很高14。此外, 大多数研究实验室缺乏这种技术的经验, 在这种情况下技术错误是常见的。最常见的错误是细胞形态的保存不畅, 石蜡细胞块切片上的免疫化学染色不良或不规则。这些和大多数其他可以避免通过制造细胞块在最佳的细胞条件下, 并使用足够的解决方案形成细胞血栓。
作为本议定书的示范, 为 HeLa 细胞准备了细胞块, 并对两种增殖标记、CKAP2 和 Ki-67 进行了免疫组织化学染色, 如先前报告的11。对于免疫细胞化学, 通过在无胎牛血清的培养基中孵化, 可以观察到血清饥饿的影响。这些制备的石蜡嵌入细胞块可以用于大量的抗体, 因为许多幻灯片可以从一个细胞块, 只使用一个 4-5 µm 厚的细胞块部分, 每张幻灯片。因此, 可以用几种不同的抗体对两种不同条件下的表达模式进行评价。癌症组织中 CKAP2 和 Ki-67 的染色模式已经报告了9,10,11,12, 和免疫细胞化学染色结果可以很容易地评估, 因为染色模式与免疫组化法相当相似。
结论: 石蜡细胞化学染色可作为免疫荧光染色的最佳替代品;此外, 在保留形态学信息的同时, 也可以方便可靠地应用于细胞系表达谱的基础研究中。

在大多数实验室, 石蜡嵌入细胞块是不常用的。相反, 固定培养细胞, 而不是石蜡嵌入细胞, 被用于亚细胞定位研究。对于那些固定培养细胞, 使用荧光代替色原。因此, 免疫荧光染色是目前的选择方法, 以确定蛋白质表达水平的研究使用细胞培养1。然而, 为免疫荧光染色而准备的幻灯片只能在免疫荧光显微镜下观察, 这可能显示出与平面显微镜2下所示的图像完全不同的图片。此外, 保存的幻灯片的免疫荧光染色需要保护, 从明亮的光, 和荧光信号变得较弱的重复曝光的成像由于荧光信号的损失3。石蜡嵌段细胞免疫组织化学的结果与石蜡嵌段的免疫组化有很大的相似之处在4, 可以很容易地转化为临床信息。因此, 免疫细胞化学可以是一个很好的选择。然而, 细胞块的制备在基础研究实验室中并不流行。在该协议中, 为促进在细胞培养研究领域中的应用, 提供细胞块制备和免疫化学染色。
细胞块制备和免疫化学不是唯一的方法, 它们已经从临床诊断应用到基础研究4,5。虽然已报告了各种单元格块准备方法4,6凝血酶等离子方法简单、经济高效且易于适应。因此, 在本文所提出的协议中, 凝血酶等离子体方法4,5,6,7,8用于制备石蜡嵌入的细胞块。
在本研究中, 凝血酶血浆细胞块制备的详细程序和使用两种增殖标记的免疫化学方法进行了论证。一个标记是细胞骨架相关蛋白 2 (CKAP2), 它最近被报告为有丝分裂标记9,10,11;另一个是 Ki-67, 它是最著名的扩散标记12。代表性方案显示在图 1中。

<table border="0" cellpadding="0" cellspacing="0" style="width:844px;" width="844">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">HeLa Cells</td>
			<td style="width:221px;">ATCC</td>
			<td style="width:291px;">HeLa (ATCC&#160;CCL-2)</td>
			<td style="width:71px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Ki-67 Antibody</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td>RM-9106-S1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Paraffin</td>
			<td style="width:221px;">Leica</td>
			<td style="width:291px;">39601006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Xylene</td>
			<td style="width:221px;">Fisher SCientific</td>
			<td>1330-20-7,100-41-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Ethenol</td>
			<td style="width:221px;">GD Chem</td>
			<td style="width:291px;">DJ16016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Hematoxylin</td>
			<td style="width:221px;">Agilent</td>
			<td style="width:291px;">10118581</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">DAB Quanto Kit</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">TA-125-QHDX, QHCX 170405</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Ultravision LP detection Kit</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">PBQ141209, LPB141209, LPH141210</td>
			<td>Kit for immunocytochemistry; contains protein block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">TintoRetriever Pressure Cooker</td>
			<td style="width:221px;">Bio SB Corporation</td>
			<td>BSB 7008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Tris-buffered saline</td>
			<td style="width:221px;">iNtRON Biotechnology</td>
			<td style="width:291px;">IBS-BT008</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Tween 20</td>
			<td style="width:221px;">USB Corporation</td>
			<td style="width:291px;">115106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Thromboplastin</td>
			<td>Neoplastin Cl Plus</td>
			<td>NC0591432</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Tris-EDTA retrival Buffer</td>
			<td style="width:221px;">Dignostic Biosystem</td>
			<td style="width:291px;">E625-A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Trypsin EDTA</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal&#160;bovine serum (FBS)</td>
			<td>Hyclone Laboratories Inc</td>
			<td>SH30910.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM</td>
			<td>Hyclone Laboratories Inc</td>
			<td style="width:291px;">SH30243.01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Antibiotic-Antimycotic, 100X</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">15240062</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:261px;">Ultra vision peroxide block H2O2</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">02Q141212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Mounting Medium</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">363313</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Pap-Pen</td>
			<td>Vector Laboratories</td>
			<td style="width:291px;">H-4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Filter Paper</td>
			<td style="width:221px;">GE Healthcare Life Sciences</td>
			<td style="width:291px;">1001-0155</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Calcium Chloride</td>
			<td style="width:221px;">Sigma-Aldrich</td>
			<td style="width:291px;">21115-100ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Phosphate-buffered saline</td>
			<td style="width:221px;">PAA Laboratories</td>
			<td style="width:291px;">H21-002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Formalin</td>
			<td style="width:221px;">Daejung</td>
			<td style="width:291px;">50-00-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">15 ml tube</td>
			<td style="width:221px;">SPL Life Sciences</td>
			<td style="width:291px;">50015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">tissue processing cassette</td>
			<td style="width:221px;">Simport</td>
			<td style="width:291px;">M492-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100 mm culture dishes</td>
			<td style="width:221px;">BD Biosciences</td>
			<td style="width:291px;">08-772E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Glass Slide</td>
			<td style="width:221px;">Muto Pure Chemicals</td>
			<td style="width:291px;">140712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Cover Glass</td>
			<td style="width:221px;">Marienfeld</td>
			<td style="width:291px;">2262817</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Glass Zar</td>
			<td style="width:221px;">Hyunil Lab-Mate</td>
			<td style="width:291px;">HIP-1027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Centrifuge</td>
			<td style="width:221px;">Eppendorf</td>
			<td>5810R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Class II Laminar Flow Hood</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">1300 series A2</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:261px;">CO2 Incubator</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">Series A2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Tissue Processor</td>
			<td style="width:221px;">Leica BioSystem</td>
			<td style="width:291px;">TP1020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Microtome</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">HM340E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Microscope</td>
			<td style="width:221px;">Olympus</td>
			<td style="width:291px;">CX-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Paraffin embedding station</td>
			<td style="width:221px;">Thermo Fisher Scientific</td>
			<td style="width:291px;">EC 350-1, EC 350-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Tissue Section Bath (Round)</td>
			<td style="width:221px;">CellPath</td>
			<td style="width:291px;">HCP-JAW-0100-00AEU</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:261px;">Fume Hood</td>
			<td style="width:221px;">Hanyang Scientific Equipment Co. Ltd.</td>
			<td style="width:291px;">FH-150</td>
			<td></td>
		</tr>
	</tbody>
</table>

determination of protein expression level in cultured cells by immunocytochemistry on paraffin-embedded cell blocks

免疫荧光染色是目前测定细胞培养系统中蛋白质表达水平的首选方法, 当形态学信息也是必要的时候。本文介绍了石蜡嵌段细胞免疫组织化学染色的方法, 是非石蜡嵌固细胞免疫荧光染色的最佳替代方案。本协议采用凝血酶-血浆法制备了 HeLa 细胞的石蜡细胞块, 并对两种增殖标记、CKAP2 和 Ki-67 进行了免疫组织化学分析。细胞块切片中, HeLa 细胞的细胞核和细胞质形态学保存完好。同时, 免疫细胞化学中的 CKAP2 和 Ki-67 染色模式与石蜡癌组织免疫组化染色的方式相当相似。经改良的细胞培养条件, 包括在无血清条件下 HeLa 细胞的预培养, 可以在保存建筑信息的同时对其效果进行评价。总之, 石蜡嵌段细胞免疫化学是一种很好的替代免疫荧光染色。

在由石蜡嵌入的细胞块 (图 3A, B) 中的苏木精和-红染色的幻灯片中, 细胞的大部分细胞核和细胞质都完好无损, 这表明形态学保存与当前的协议是很好的 (图 3A)。在免疫细胞化学染色中, 在凝染色质、有丝分裂纺锤体和细胞质 (图 4) 中观察到阳性 CKAP2 染色, 如先前报告的10。Ki-67 染色是在细胞核中观察到的 (如预期的那样) (图 4)。只有在凝聚染色质中 CKAP2 染色的细胞 (见图 4A, B中的箭头) 是有丝分裂细胞。许多 CKAP2-positive 细胞被显示在高度有丝分裂的 HeLa 细胞, 已准备在完全培养基的孵化 (图 4A)。相比之下, 血清饥饿的 HeLa 细胞中几乎没有 CKAP2-positive 细胞 (图 4B)。大多数高度有丝分裂的 HeLa 细胞 Ki-67 阳性 (图 4C)。Contrastingly, 血清饥饿的 HeLa 细胞的 Ki-67-positive 率保持高达 50% (图 4D)。这些结果与以前报告11中的数据相当, 这表明 CKAP2 是癌细胞中比 Ki-67 更可靠的增殖标记.
在制备不良的细胞块中, 细胞核与细胞质分离, 也有 resultantly, 形态保存不良。冰箱中固定细胞的长期孵化可能会造成如此糟糕的结果。另外一个重要的问题是, 尽管细胞的形态学很好, 但它并没有被免疫组织化学染色。当细胞血栓小的时候, 这个问题就会出现。通常, 染色强度不规则, 如图 3B所示;但当细胞血栓增大时, 不规则染色的几率就会大大降低。因此, 在本议定书中, 我们增加了血浆, 凝血酶和氯化钙的体积, 以形成一个大的细胞血栓。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57369/57369fig1.jpg"> 图 1: 单元格块准备方案.<a href="/files/ftp_upload/57369/57369fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57369/57369fig2.jpg"> 图 2: 石蜡电池块准备的插图.(A) 凝血酶血浆细胞血栓。(B) PBS 洗涤后 TP 细胞血栓。(C) 湿滤纸上的细胞血栓。(D) 带有熔融蜡的组织嵌入站。金属模具 (箭) 持有熔融蜡固化。(E) 石蜡嵌入的细胞血栓 (箭头) 嵌入石蜡或石蜡嵌入细胞块。(F) 在涂层的滑动的中间部分薄石蜡切片。<a href="/files/ftp_upload/57369/57369fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57369/57369fig3.jpg"> 图 3: 细胞块的制备和质量的确认, 苏木精和染色.(a) 在石蜡嵌入的细胞块切片中的 HeLa 细胞的苏木精和-伊红染色图像。(B) 在制备不良的石蜡嵌入细胞块切片中, 对免疫组化 Ki-67 的不规则染色。刻度条为 (A) 100 和 (B) 200 微米。<a href="/files/ftp_upload/57369/57369fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57369/57369fig4.jpg"> 图 4: 用于 HeLa 细胞的石蜡嵌段细胞免疫组织化学染色.(A) 在高度有丝分裂条件下 CKAP2 染色。(B) 在血清饥饿条件下 CKAP2 染色。(C) 在高度有丝分裂条件下 Ki-67 染色。(D) 在血清饥饿条件下 Ki-67 染色。显示100微米刻度条。箭头表示 CKAP2-positive 细胞。<a href="/files/ftp_upload/57369/57369fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>程序</td>
			<td>步骤</td>
			<td>解决 方案</td>
			<td>时间/温度</td>
		</tr>
<tr>
<td rowspan="4">脱水</td>
			<td>1</td>
			<td>70% 酒精</td>
			<td>15分钟/RT</td>
		</tr>
<tr>
<td>2</td>
			<td>80% 酒精</td>
			<td>15分钟/RT</td>
		</tr>
<tr>
<td>3</td>
			<td>95% 酒精</td>
			<td>15分钟/RT</td>
		</tr>
<tr>
<td>4</td>
			<td>100% 酒精</td>
			<td>15分钟/RT</td>
		</tr>
<tr>
<td rowspan="4">结算</td>
			<td>1</td>
			<td>二甲苯</td>
			<td>60 min/4 °c</td>
		</tr>
<tr>
<td>2</td>
			<td>二甲苯</td>
			<td>10分钟/RT</td>
		</tr>
<tr>
<td>3</td>
			<td>二甲苯</td>
			<td>10分钟/RT</td>
		</tr>
<tr>
<td>4</td>
			<td>二甲苯</td>
			<td>10分钟/RT</td>
		</tr>
<tr>
<td>* 室温</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
</tbody></table>表1。组织加工程序。

Watch this Scientific Journal Video about Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks at JoVE.com

Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks

石蜡嵌段细胞免疫细胞化学法测定培养体细胞蛋白质表达水平

目前, 固定细胞免疫荧光染色是确定形态学信息时蛋白质表达水平的选择方法。本协议提供了一种替代方法的免疫细胞化学在石蜡嵌入单元块。

Immunofluorescent staining is currently the method of choice for determination of protein expression levels in cell-culture systems when morphological ...

determination-protein-expression-level-cultured-cells

Research

JoVE Journal

Cancer Research

11.4K 次观看.  National Cancer Center. 目前, 固定细胞免疫荧光染色是确定形态学信息时蛋白质表达水平的选择方法。本协议提供了一种替代方法的免疫细胞化学在石蜡嵌入单元块。

视频: Determination of Protein Expression Level in Cultured Cells by Immunocytochemistry on Paraffin-embedded Cell Blocks

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

该<em&gt;果蝇</em&gt;成虫盘瘤模型:基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

科研

癌症研究

Four-color Fluorescence Immunohistochemistry of T-cell Subpopulations in Archival Formalin-fixed, Paraffin-embedded Human Oropharyngeal Squamous Cell Carcinoma Samples

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3+), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3+), and T helper 17 (Th17) cells (i.e., IL-17+CD3+) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17+ non-T cells. This suggests that IL-17+ non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.

Multiparameter fluorescence immunohistochemistry can be used to assess the number, relative distribution, and localization of immune cell populations in the tumor microenvironment. This manuscript describes the use of this technique to analyze T-cell subpopulations in oropharyngeal cancer.

档案福尔马林固定，石蜡包埋的人类口咽鳞状细胞癌样品中T细胞亚群的四色荧光免疫组织化学

The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their ...

Preparation of Primary Acute Lymphoblastic Leukemia Cells in Different Cell Cycle Phases by Centrifugal Elutriation

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.

This protocol describes the use of centrifugal elutriation to separate primary acute lymphoblastic leukemia cells into different cell cycle phases.

离心淘洗制备不同细胞周期的原发性急性淋巴细胞白血病细胞

The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum ...

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (&#8805;1%, &#8805;50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

To expand the ability of laboratories worldwide to assess the eligibility of patients with lung cancer for treatment with pembrolizumab, in a reliable and reproducible manner, we developed an assay that uses the 22C3 antibody concentrate on a widely available immunohistochemical autostainer, for both biopsy and cytology specimens.

22C3 Anti-PD-L1 抗体精矿在非小细胞肺癌活检及细胞学标本中的应用

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and eosin (H&amp;E) staining but can be visualized through elastic staining. One of the important benefits of elastic staining is that it makes it easy to determine whether tumor cells have invaded beyond the elastic lamina. This helps in examining the extent of peritoneal surface invasion, thereby distinguishing the pT3 and pT4 stages in gastrointestinal cancer. In this study, we present a protocol to identify elastic fibers in the formalin-fixed, paraffin-embedded pT3N0M0 gastric cancer tissue sections. We prepare 5-&#181;m paraffin sections fixed on slides, and then all the slides are deparaffinated and rehydrated during the staining procedure. Subsequently, all the sections are oxidized by potassium permanganate, bleached by oxalic acid, stained by elastic staining, and counterstained by Van Gieson's. Finally, we examine the effect of staining; elastic lamina is stained blue-black and collagen fibers are stained red, while the cancer cells are stained in varying shades of yellow. We also describe in detail the methods used for determining the positional relationship between cancer cells and elastic lamina. This method is simple, low-cost, and widely applicable in the identification of peritoneal surface invasion in gastrointestinal cancer because of its outstanding selectivity for elastic fibers and authentic results.

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

pT3N0M0 胃癌组织石蜡嵌入幻灯片上的弹性染色

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and ...

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

石蜡包埋三维胶质瘤神经球培养的免疫组织化学评价胶质瘤生物标志物

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

毛细管电泳质谱法提取培养细胞中的水代谢物进行代谢组学分析

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

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

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

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

组织中蛋白质和RNA表达的空间分析:一种微调虚拟显微切割的方法

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

Live-3D-Cell Immunocytochemistry Assays of Pediatric Diffuse Midline Glioma

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using three-dimensional (3D) cell-based invasion and migration assays. In this study, we have optimized these 3D assays for live-cell immunocytochemistry. An Antibody Labeling Reagent was used to detect in real-time the expression of the adhesion molecule CD44, on the plasma membrane of migrating and invading cells of a DMG H3K27M primary patient-derived cell line. CD44 is associated with cancer stem cell phenotype and tumor cell migration and invasion and is involved in the direct interactions with the central nervous system (CNS) extracellular matrix. Neurospheres (NS) from the DMG H3K27M cell line were embedded into the basal membrane matrix (BMM) or placed onto a thin coating layer of BMM, in the presence of an anti-CD44 antibody in conjunction with the antibody labeling reagent (ALR). The live-3D-cell immunocytochemistry image analysis was performed on a live-cell analysis instrument to quantitatively measure the overall CD44 expression, specifically on the migrating and invading cells. The method also allows visualizing in real-time the intermittent expression of CD44 on the plasma membrane of migrating and invading cells. Moreover, the assay also provided new insights into the potential role of CD44 in the mesenchymal to amoeboid transition in DMG H3K27M cells.

This study presents a protocol of live-3D-cell immunocytochemistry applied to a pediatric diffuse midline glioma cell line, useful to study in real-time the expression of proteins on the plasma membrane during dynamic processes like 3D cell invasion and migration.

小儿弥漫性中线胶质瘤的活-3D细胞免疫细胞化学测定

Cell migration and invasion are specific hallmarks of Diffuse Midline Glioma (DMG) H3K27M-mutant tumors. We have already modeled these features using ...