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>

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

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

在使用定量免疫肿瘤的异质性蛋白表达的映射

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

科研

医学

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

使用荧光抗体对小鼠表皮石蜡切片的免疫组织化学

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

生物学

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 ...

癌症研究

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

使用反时相蛋白的阵列（RPPA）探索在个人的肾细胞癌蛋白表达的变化

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

Isolation of Stably Transfected Melanoma Cell Clones

Cell populations that have stable changes in their genomic information are widely used by scientists as a research model. They do not require repeated cell transfection as it can lead to a heterogeneous cell population and variable transfection efficiency, affecting reproducibility. Moreover, they are preferable for large-scale analyses. The generation of stable cell clones is useful for a wide range of applications, such as research on gene functions and recombinant protein production. There are a few methods to obtain a homogenous cell population upon initial transient transfection. Here, we describe the isolation of single cell clones with glass cylinders. Although this method has been known for some time, there are a few crucial steps, and neglecting them may lead to failure. We have successfully used this method to obtain clones stably overexpressing a protein of interest (POI) or with knockout of a gene of interest (GOI). We describe preparation steps such as the optimization of selecting drug concentrations, preparation of glass cylinders, and validation of whether the obtained clones have the desired change in the expression of the GOI by PCR, western blot analysis, immunostaining, or gDNA sequencing (depending on the type of derived clones). We also discuss the phenotypic heterogeneity of well-established cell lines as this might be an issue in obtaining stable cell clones.

<meta charset="utf8"></head><body>稳定转染黑色素瘤细胞克隆的分离

A protocol is described for isolating stably transfected melanoma cell clones using glass cylinders. We focus on practical advice that may be decisive for the success of the entire procedure.

稳定转染黑色素瘤细胞克隆的分离

Cell populations that have stable changes in their genomic information are widely used by scientists as a research model. They do not require repeated ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

定量免疫检测，以衡量在中心体的变异蛋白水平

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Quantitation of Protein Expression and Co-localization Using Multiplexed Immuno-histochemical Staining and Multispectral Imaging

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within tissues. Many semi-quantitative systems have been developed for scoring expression using immunohistochemistry, but inherent subjectivity limits reproducibility and accuracy of results. Furthermore, the investigation of spatially overlapping biomarkers such as nuclear transcription factors is difficult with current immunohistochemistry techniques. We have developed and optimized a system for simultaneous investigation of multiple proteins using high throughput methods of multiplexed immunohistochemistry and multispectral imaging. Multiplexed immunohistochemistry is performed by sequential application of primary antibodies with secondary antibodies conjugated to horseradish peroxidase or alkaline phosphatase. Different chromogens are used to detect each protein of interest. Stained slides are loaded into an automated slide scanner and a protocol is created for automated image acquisition. A spectral library is created by staining a set of slides with a single chromogen on each. A subset of representative stained images are imported into multispectral imaging software and an algorithm for distinguishing tissue type is created by defining tissue compartments on images. Subcellular compartments are segmented by using hematoxylin counterstain and adjusting the intrinsic algorithm. Thresholding is applied to determine positivity and protein co-localization. The final algorithm is then applied to the entire set of tissues. Resulting data allows the user to evaluate protein expression based on tissue type (ex. epithelia vs. stroma) and subcellular compartment (nucleus vs. cytoplasm vs. plasma membrane). Co-localization analysis allows for investigation of double-positive, double-negative, and single-positive cell types. Combining multispectral imaging with multiplexed immunohistochemistry and automated image acquisition is an objective, high-throughput method for investigation of biomarkers within tissues.

Immunohistochemistry is a powerful lab technique for evaluating protein localization and expression within tissues. Current semi-automated methods for quantitation introduce subjectivity and often create irreproducible results. Herein, we describe methods for multiplexed immunohistochemistry and objective quantitation of protein expression and co-localization using multispectral imaging.

蛋白表达和共定位使用复用免疫组织化学染色和多光谱成像定量

Immunohistochemistry is a commonly used clinical and research lab detection technique for investigating protein expression and localization within ...

Quantitative Immunoblotting of Cell Lines as a Standard to Validate Immunofluorescence for Quantifying Biomarker Proteins in Routine Tissue Samples

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An optimal method of quantification is accurate, has a broad linear dynamic range and maintains the structural integrity of the sample to allow for identification of individual cell types. Current methods such as immunohistochemistry (IHC), mass spectrometry, and immunoblotting each fail to meet these stipulations due to their categorical nature or need to homogenize the sample. As an alternative method, we propose the use of immunofluorescence (IF) and image analysis to determine the relative abundance of a protein of interest in FFPE tissues. Herein we demonstrate that this method is easily optimized, yields a wide dynamic range, and is linearly quantifiable as compared to the gold standard of quantitative immunoblotting. Furthermore, this method permits the maintenance of the structural integrity of the sample and allows for the distinction of various cell types, which may be crucial in diagnostic applications. Overall, this is a robust method for the relative quantification of proteins in FFPE samples and can be easily adapted to suit clinical or research needs.

We describe the use of quantitative immunoblotting to validate immunofluorescence histology coupled with image analysis as a means of quantifying a protein of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples. Our results demonstrate the utility of immunofluorescence histology for ascertaining the relative quantity of biomarker proteins in routine biopsy samples.

细胞系定量免疫印迹作为常规组织样本中生物标志物蛋白定量免疫荧光定量的标准

Quantification of proteins of interest in formalin-fixed, paraffin-embedded (FFPE) tissue samples is important in clinical and research applications. An ...

Standardized Processing for Formalin-Fixed, Paraffin-Embedded Cell Pellet Immunohistochemistry Controls

Positive and negative controls with known expression of target proteins are essential for the development of immunohistochemistry (IHC) assays. While tissue controls are beneficial for well-characterized proteins with defined tissue and cellular expression patterns, they are less suitable for the initial development of IHC assays for novel, poorly characterized, or ubiquitously expressed proteins. Alternatively, due to their standardized nature, cell pellets, including cancer cell lines with defined protein or transcript expression levels (e.g., high, medium, and low expression), transfected over-expressing cell lines, or cell lines with genes deleted through cell engineering technologies like CRISPR, can serve as valuable controls, especially for the initial antibody characterization and selection. In order for these cell pellets to be used in the development of IHC assays for formalin-fixed, paraffin-embedded tissues, they need to be processed and embedded in a manner that recapitulates the procedures used for tissue processing. This protocol describes a process for creating and processing formalin-fixed, paraffin-embedded cell pellet controls that can be used for IHC method developments.

Presented here is a protocol for generating formalin-fixed, paraffin-embedded cell pellet controls for immunohistochemistry.

福尔马林固定、石蜡包埋细胞沉淀免疫组化对照的标准化处理

Positive and negative controls with known expression of target proteins are essential for the development of immunohistochemistry (IHC) assays. While ...

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

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蛋白表达和共定位使用复用免疫组织化学染色和多光谱成像定量

细胞系定量免疫印迹作为常规组织样本中生物标志物蛋白定量免疫荧光定量的标准

福尔马林固定、石蜡包埋细胞沉淀免疫组化对照的标准化处理