我们感谢格拉茨医学院病理研究所分子病理学实验室的团队, 他们的专业知识和支持。此外, 我们感谢虹膜 Kufferath 和 Daniela Pabst 技术援助, 伯纳黛特丽格和西尔维亚 Eidenhammer 处理 HER2 病例以及坤 Szurian (病理学家) 和翳 Regényi (3DHISTEC)。我们感谢佩内洛普 Kungl 校对手稿。这项工作得到了基督教多普勒研究基金、奥地利联邦科学、研究和经济部以及国家研究、技术和发展基金会的资助。

这项研究由格拉茨医科大学伦理委员会批准 (参考数字 20-066), 奥地利。从两例经手术切除的原发性浸润性乳腺癌患者经书面知情同意后, 获得组织标本。
1. 组织固定
注: 用标准的缓冲福尔马林溶液 (SBFS) 进行组织固定, 定义为10% 福尔马林溶液, 其质量分数为 3.7% (对应于体积分数为 4%) 的甲醛缓冲到 ph 6.8 到 ph 7.2 根据岑/TS16827-1: 2015 或非连接的无福尔马林组织固定系统 (也称为下面的交替固定液), 由定影液和室温稳定剂溶液 (RT) 组成。
<ol>
	<li>将无菌手术刀切成两半, 每4毫米厚, 以确保足够的组织数量的乳腺癌组织样本。使用最大尺寸 4 x 15 x 15 毫米的 NCFPE 或 SBFS 组织固定。</li>
	<li>将肿瘤标本的每一半放置在一个标准的组织盒中, 并将其浸入 SBFS 或 24 h 的替代固定液中, 每体积比 (v/v) 的4份定影剂和一个部分组织 (4 mL/1 g 组织 (理想的 10 mL/1 g))。</li>
	<li>通过打开容器, 将纸巾盒按180°旋转, 并将其浸入水中, 将交替固定的样品转换为2-72 小时的稳定剂溶液。</li>
	<li>在 RT 中放置带 SBFS 固定样品的组织盒, 在250毫升70% 乙醇中进行30分钟的去除残余 SBFS。 注意: 这一步是重要的, 以避免 SBFS 污染的非连接固定处理的样品在自动组织处理器, 这将破坏非链固定的有益性质的生物大分子的保存。 注意: SBFS 是一种危险的溶液, 引起皮肤刺激性, 严重的眼部损伤, 可能引起过敏性皮肤反应, 被怀疑造成遗传缺陷, 可能导致癌症、呼吸道刺激、嗜睡并导致器官损害。该替代固色剂是有毒的吸入, 与皮肤接触, 如果吞食, 刺激眼睛和皮肤, 危险的非常严重的不可逆转的影响, 通过吸入, 与皮肤接触, 如果吞食。两种固定液都应避免吸入和身体接触。穿戴适当的防护服、手套、眼睛和脸部保护。在通风柜里工作。</li>
	<li>进行组织加工, 植入和组织切片 (2 和3节)。</li>
</ol>
2. 组织加工和植入
<ol>
	<li>在 70% (2 x 15 min)、80% (30 min)、90% (60 min) 和 99% (2 x 60 min) 乙醇, 其次是二甲苯 (2 x 60 分钟) 内, 对组织盒进行逐步脱水。然后在自动组织处理器中渗入石蜡 (4 x 45 min)。</li>
	<li>使用镊子, 将脱水和石蜡渗入的样品 (或者 SBFS 固定) 放置在金属模具中。</li>
	<li>将样品嵌入 5-10 毫升熔融石蜡 (凝固点56-58 ° c) 使用石蜡嵌入仪器。</li>
	<li>将模具放在-5 ° c 的冷却盘上30分钟。</li>
	<li>从模具中回收石蜡嵌入的组织块。</li>
	<li>存储结果块 (交替固定, 石蜡嵌入 (NCFPE) 和 SBFS 固定石蜡嵌入 (FFPE)) 在黑暗中在4° c 直到使用。</li>
</ol>
3. 鱼的组织切片和滑动准备
注: FFPE 剖面直接用于鱼, 而 NCFPE 切片的幻灯片在鱼术前24小时 post-fixed 在 SBFS。
<ol>
	<li>将一次性刀片插入切片, 并将切片调整到10µm。</li>
	<li>从冷却盘中取第一块, 并将其插入仪器中。</li>
	<li>修剪块, 直到达到一个均匀的表面。使用刷子清洗切片, 并将其调整为3µm. 丢弃前2-3 节。</li>
	<li>削减3µm 节的 NCFPE 和 FFPE 块, 并放置在冷水浴 (超纯水)。 注: 为了获得更好的照片没有重叠的细胞核, 这里, 图片2µm 的部分, 而不是3-5 µm 的建议, 在 manufacturer´s 指令显示。</li>
	<li>拿起每个浮动部分与罚款油漆刷和针到涂层显微镜幻灯片, 以粘合的组织部分。</li>
	<li>把这张幻灯片浸入一个温暖的 (40 ° c) 的水浴, 让切片完全伸展。</li>
	<li>通过小心地把滑梯从水中拉出, 避免皱纹, 把切片放回幻灯片上。</li>
	<li>让所有部分在室温下干燥1小时。</li>
	<li>在70° c 的温度下, 在恒温箱中加热30分钟以融化石蜡。</li>
	<li>将幻灯片水平放置在玻璃染色罐中。</li>
	<li>水化的部分逐步在 RT 中的染色罐子充满了250毫升以下液体: 100% 二甲苯 2 x 15 min, 96% 乙醇 2 x 15 min, 90% 乙醇 2 min, 80% 乙醇 2 min, 70% 乙醇 2 min, 50% 乙醇2分钟, 蒸馏水 2 x 10 分钟。 注意: 在通风柜中执行 deparaffinization 和补液步骤, 不要吸入有毒的挥发溶剂。 注意: 根据切片和组织, 可能需要预先测试不同的潜伏期。</li>
</ol>
4. NCFPE 标本的后固定
<ol>
	<li>执行 post-fixation 的 NCFPE 幻灯片, 把他们放入一个新的染色缸充满 SBFS 24 小时在 RT. 在24小时 post-fixation 步骤中将 jar 放置在通风罩中。</li>
	<li>用磷酸盐缓冲盐水 (3 x 10 分钟) 和蒸馏水 (2 x 10 分钟) 洗涤去除过量的 SBFS。</li>
</ol>
5. 荧光原位杂交 (fish)
注意: 这里的鱼是根据制造商的说明使用商用的 HER2/CEN17 双色探针套件进行的。在杂交和洗涤步骤中, 不允许组织切片干燥。不允许 dna 探针和 4 ", 6-diamidino-2-吲 (DAPI) dna counterstain 溶液暴露在较长的时间。使用透光容器在黑暗中执行这些步骤。根据样品材料的年龄和固定步骤, 增加或减少变性和洗涤温度以获得更好的杂交结果。
<ol>
	<li>在98° c 水浴中放置一个含有250毫升热预处理溶液的染色罐。</li>
	<li>将水化 FFPE 和 post-fixed NCFPE 滑入罐中, 孵育15分钟。</li></ol>每个着色罐不要使用超过六张幻灯片。
	<li>在新的染色罐中, 在 RT 中用蒸馏水冲洗3分钟。</li>
	<li>对于蛋白质水解, 将幻灯片放入温度控制杂交系统在37° c。<ol>
		<li>立即将滴 (2 毫克/毫升) 的胃蛋白酶溶液 (每滴30µl) 应用到组织切片, 直到完全覆盖。在杂交系统中孵育9分钟, 在37° c。 注: 建议5-15 分钟的潜伏期。蛋白质水解的最佳时间应提前确定。</li>
		<li>将幻灯片放入包含洗涤缓冲 SSC 的 jar 中, 并孵育5分钟。</li>
		<li>在 RT 中冲洗1分钟的蒸馏水中的幻灯片。</li>
		<li>在以下分级醇中执行脱水: 50%、70%、90% 和 100%, 每段1分钟空气干燥。</li>
		<li>对于杂交, 吸管10µL HER2/CEN17 探头到干燥组织切片, 覆盖每个样品与片和密封它与橡胶水泥。 注:10 µL 探头建议每 22 x 22 毫米组织面积。温和升温的探针使移过程更容易。避免气泡和长时间曝光探头的光线。</li>
		<li>共变性探针和标本 DNA 在杂交系统在75° c 为 10 min。</li>
		<li>将杂交系统设置为37° c 和杂交过夜。</li>
		<li>小心地将橡胶水泥用镊子 post-hybridization 和检测。</li>
		<li>在含有37° c 5ml 1 x 洗涤缓冲器 a 的染色罐中孵育幻灯片, 5 分钟取出片。</li>
		<li>使用 5ml 1 x 洗涤缓冲器两次在37° c 的5分钟洗涤幻灯片。</li>
		<li>脱水的幻灯片在 70%, 90% 和100% 乙醇, 每1分钟。</li>
		<li>在空气中干燥样品并保护光线。</li>
		<li>对于染色, 应用30µL DAPI-溶液到杂交区避免气泡。用片覆盖这些部分。</li>
		<li>孵育15分钟保护免受光。</li>
		<li>通过轻轻地按下过滤纸之间的滑动, 小心地去除多余的 DAPI 溶液。</li>
		<li>存储在 2-8 ° c 的幻灯片在黑暗中长达2周, 直到评价由共焦显微镜。</li>
	</ol>
	</li>

6. 共焦成像和扫描鱼幻灯片
<ol>
	<li>对装有 40 x/1.2 NA 目标的共聚焦数字扫描仪上的幻灯片进行共焦成像和扫描, 四波段滤波器 (DAPI/FITC/TRITC/Cy5) 和5.5Mpx、16位、冷却的科学互补金属氧化物半导体 (CMOS) 相机。</li>
	<li>获取图像与过滤器为绿色 (励磁: 503 毫微米, 放射: 528 毫微米) 和桔子 (励磁: 547 毫微米, 放射: 572 毫微米) 渠道。</li>
</ol>
7. 口译
注: HER2/CEN17 探针是一种红色荧光标记的 CEN17 探针的混合物, 特定于17号染色体 (D17Z1) 的α卫星丝区和荧光基因的绿色 HER2 标记的 HER2 探针 (探针位置 17q12)。肿瘤标本的 HER2:CEN17 比≤2.0 每核被评分为正常, 而那些与 HER2:CEN17 比率≥2.0 被评分为放大17。
<ol>
	<li>使用开放源码 CaseViewer 2.120对图像进行质量分析。<ol>
		<li>根据病理学家的定义, 评估至少20细胞位于肿瘤浸润部分的两个不同区域。在计数区包括清楚地可区分的分布的原子核。排除在边界和缩回或压缩区域中的组织区域计数。</li>
	</ol>
	</li>
</ol>
8. RNA 质量
<ol>
	<li>RNA 提取<ol>
		<li>确保所有仪器和工具核糖核酸免费。使用核糖核酸删除解决方案。</li>
		<li>使用切片后的 NCFPE 或 FFPE 样本, 将10到20节 (5 µm 厚) 剪切到步骤3.8。</li>
		<li>使用 rna 隔离套件从 NCFPE 样品中提取 rna (见材料表)。使用 FFPE rna 隔离试剂盒从 FFPE 样品中提取 rna。 注: 从非链固定中提取 RNA 需要一种与固定方法相适应的隔离方法。不要使用任何其他 RNA 隔离方法 (如 Trizol, RNeasy FFPE, 等 NCFPE 标本)。</li>
		<li>用分光光度计测定 RNA 的浓度和纯度。260 nm 和 280 nm (A260/280) 的吸光度比应为2.0。</li>
		<li>将分离的 RNA 储存在-70 ° c 直到进一步分析。</li>
	</ol>
	</li>
	<li>使用引物进行甘油3磷酸脱氢酶 (GAPDH) real-time 反转录 PCR, 生成不同长度的增8,9。<ol>
		<li>使用 cdna 反转转录试剂盒根据 manufacturer´s 指令的 cdna 合成。使用每个样本的 500 ng RNA。在-20 ° c 贮存 cDNA 直到进一步使用。</li>
		<li>根据 manufacturer´s 的指示准备 PCR 大师组合。</li>
		<li>将 PCR 主混合在 triplicates 的384井反应板中。添加4µL 1:16 稀释 cDNA (PCR 模板)。根据 manufacturer´s 指令进行 PCR。 注: 对于人类 GAPDH, 使用了以下引物: GAPDH 向前: 5´-CCACATCGCTCAGACACCAT-3´;GAPDH 反向:71 bp 5´-ACCAGGCGCCCAATACG-3´, 153 bp 5´-GTAAACCATGTAGTTGAGGTC-3´, 200 bp 5´-TTGACGGTGCCATGGAATTT-3´, 277 bp 5´-ACTTGATTTTGGAGGGATCT-3´, 323 bp 5´-AAGACGCCAGTGGACTCCA-3´, 530 bp 5´-ACGATACCAAAGTTGTCATG-3´。</li>
		<li>使用适用于 PCR 机的软件分析结果。非特异产品必须根据熔融曲线分析排除21。</li>
	</ol>
	</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HER2+amplification+ratios+by+fluorescence+in+situ+hybridization+and+correlation+with+immunohistochemistry+in+a+cohort+of+6556+breast+cancer+tissues.">HER2 amplification ratios by fluorescence in situ hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues.</a> Clin.Breast Cancer. 5 (1), 63-69 (2004).</li><li>Wolff, A. C., Hammond, M. E., Schwartz, J. N., Hagerty, K. L., Allred, D. C., Cote, R. J., 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=American+Society+of+Clinical+Oncology/College+of+American+Pathologists+guideline+recommendations+for+human+epidermal+growth+factor+receptor+2+testing+in+breast+cancer.">American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer.</a> Arch Pathol Lab Med. 131 (1), 18-43 (2007).</li><li>Press, M. F., Bernstein, L., Thomas, P. A., Meisner, L. F., Zhou, J. Y., Ma, 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=HER-2/neu+gene+amplification+characterized+by+fluorescence+in+situ+hybridization:+poor+prognosis+in+node-negative+breast+carcinomas.">HER-2/neu gene amplification characterized by fluorescence in situ hybridization: poor prognosis in node-negative breast carcinomas.</a> J. Clin. Oncol. 15 (8), 2894-2904 (1997).</li><li>Yamauchi, H., Stearns, V., Hayes, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+is+a+tumor+marker+ready+for+prime+time?+A+case+study+of+c-erbB-2+as+a+predictive+factor+in+breast+cancer.">When is a tumor marker ready for prime time? A case study of c-erbB-2 as a predictive factor in breast cancer.</a> J. Clin. Oncol. 19 (8), 2334-2356 (2001).</li><li>Paulik, R., Micsik, T., Kiszler, G., Kasz&#225;l, P., Sz&#233;kely, J., Paulik, N., V&#225;rhalmi, E., Pr&#233;musz, V., Kren&#225;cs, T., Moln&#225;r, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+image+analysis+algorithm+for+detecting+nuclear+signals+in+digital+whole+slides+for+histopathology.">An optimized image analysis algorithm for detecting nuclear signals in digital whole slides for histopathology.</a> Cytometry A. , (2017).</li><li>Applied Biosystems. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> QuantStudio 6 and 7 Flex Real-Time PCR System Software. , (2013).</li><li>NCCLS. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quality Assurance for Immunocytochemistry. , (1999).</li><li>Baker, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of Biological Microtechnique. , (1958).</li><li>Hewitt, S. M., Lewis, F., Cao, Y., Conrad, R. C., Cronin, M., Danenberg, K. D., 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=Tissue+handling+and+specimen+preparation+in+surgical+pathology:+issues+concerning+the+recovery+of+nucleic+acids+from+formalin-fixed,+paraffin-embedded+tissue.">Tissue handling and specimen preparation in surgical pathology: issues concerning the recovery of nucleic acids from formalin-fixed, paraffin-embedded tissue.</a> Arch Pathol. Lab Med. 132 (12), 1929-1935 (2008).</li></ol>

危险品受雇于 Qiagen, 并在公司的养老金计划中持有股票期权或债券。本手稿中的数据是指 PAXgene 组织系统, 它是由 Qiagen 和 PreAnalytiX 共同开发的。Qiagen 是公共资助的基督教多普勒实验室 Biospecimen 研究和 Biobanking 技术的工业伙伴。所有的作者都没有利益冲突, 没有竞争的金融利益。

结果显示了两个关键的发现。首先, 一个简单的 24 h SBFS post-fixation 步骤的切片从 NCFPE 组织是足以获得相同的结果, 与 FFPE 鱼分析使用的鱼化验批准 FFPE 组织 (也请参阅参考14)。本议定书的优势是使用最初开发和批准的鱼类化验 FFPE 不需要全面重新验证 (例如, 通过优化、条件非连接组织)。可以按照制造商的说明中所描述的那样使用 FISH 协议。
根据本协议中描述的 manufacturer´s 指令 (例如, deparaffinization 步骤中的组织切片直径和潜伏期) 进行的细微修改是由制造商明确推荐的。由于使用不同的组织类型和固定方法。
post-fixation 步骤非常关键, 因为它需要一个18-24 小时的化学反应时间, 它将分析时间延长一天, 但允许与 FFPE 组织相同的每日工作流 (图 1)。据报道, 根据组织类型23、24, SBFS 的平均每小时1毫米22到 5 mm 的穿透组织。观察到只有长时间的 post-fixation 期超过18小时, 才能改变 NCFPE 对 FFPE 节的性质, 表明不渗透的固定剂, 但化学反应时间是关键的达到预期的结果1 ,14。
其次, 剩余的 NCFPE 组织, 不用于 post-fixation 可以用于进一步的分子分析, 因为生物分子是保存完好。real-time 反向转录 PCR (图 3) 演示了这一点。该方法比从石蜡包埋组织中分离出的 rna 质量 (即化学修饰和分裂) 的电泳值 (rna 完整性数) 更敏感、更具体.8。本研究对 real-time PCR 的质量控制进行了限制, 因为独立组已表明, 除了 RNA 外, 从 NCFPE 中分离出的 DNA 和蛋白质的质量优于 FFPE 组织, 8,9,13。
所提出的协议如下, 在适用的情况下 (例如, SBFS 配方, 固定条件, RNA 质量控制, 验证), 要求的岑技术规范的分析程序的体外诊断 (IVD),这是最近由欧洲标准化委员会 (岑) (例如, 岑/TS 16827-1: 2015 的 RNA 分离的 FFPE 组织, 这是指 ISO 15189 标准)。
该方法仅限于此特定的定影液。虽然生物分子和形态学的良好保存使用非连接, 无福尔马林固定剂已提出了一些研究, 它主要用于研究, 但不是在常规医疗应用。其中一个原因是, 用另一个固定剂取代金本位 SBFS 固定, 需要进行各种验证研究, 因为并非所有为 FFPE 材料优化的协议都可以用于非连接固定的材料。其他的组织固定方法没有对此鱼议定书进行测试。
本手稿中描述的简单 post-fixation 步骤具有使用 IVD 认证的 FFPE 和 NCFPE 组织的方法的优势。

个性化医学越来越依赖于多参数测试, 包括形态学和分子组织分析。福尔马林固定组织作为金本位提供优异的形态学品质1,2。然而, 福尔马林诱导的蛋白质和核酸的化学修饰和交联3,4,5 , 对分子分析产生负面干扰6。这些分子修饰限制了核酸和蛋白质的质量, 并可能导致基因序列的人工制品7或降低了聚合酶链式反应 (PCR) 的灵敏度 (基于检测)8。尽管为优化 FFPE 组织的分子试验作出了重大努力, 但冷冻保存的组织总体上优于福尔马林固定, 因此有必要将组织标本分成不同的保存程序。为避免分子分析需要冷冻保存, 研制了一种非链、无福尔马林固定剂、PAXgene 组织。这种商用系统包括固定和含有不同醇、醋酸和可溶性有机化合物的稳定溶液。正确保存核酸, (phospo) 蛋白质和形态学显示在几个研究6,9,10,11,12.
癌症诊断的一个特殊应用是鱼类检测到一系列的染色体改变, 如易位、亚删除和放大14。例如, 20% 的浸润性乳腺癌肿瘤显示人表皮生长因子受体 2 (HER2) 基因的放大15,16,17, 这是与不良预后相关的18 ,19。HER2 在乳腺癌中的过度表达和扩增状态的测定需要选择患者进行 anti-HER2-directed 治疗, 并且是由联邦药物协会 (FDA) 列出的辅助诊断 (http://www.fda.gov/MedicalDevices/ProductsandMedicalProcedures/InVitroDiagnostics/ucm301431. htm)17。为了在医疗保健中广泛应用鱼伴诊断, 制定并批准了 FFPE 组织的化验。
在先前的研究中, 我们发现 NCFPE 组织不能用于已被批准用于 FFPE 组织的鱼类化验。然而, 4% 缓冲甲醛的 NCFPE 组织不同后固定术后的时间序列测试显示, NCFPE 组织切片的18小时或更长的后固定时间达到了与 FFPE 组织的等效结果 (14。
在这项研究中, 我们提供了一个详细的协议, 并证明了非的影响, 无福尔马林组织固定和24ĥ4% 缓冲甲醛 post-fixation 的部分用于 HER2 鱼和 RNA 质量从同一主要组织标本。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55885/55885fig1.jpg"> 
图 1: 显示荧光步骤的图表原位杂交 (fish) 和 RNA 分析福尔马林固定石蜡嵌入 (FFPE) 和非连接, 无福尔马林固定石蜡 (NCFPE) 组织.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55885/55885fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table>
	<tbody>
		<tr>
			<td>SAV LP GmbH, Flintsbach a. I., Germany</td>
			<td>www.sav-lp.de</td>
			<td>FN-200L-4-1</td>
			<td>Tissue fixative</td>
		</tr>
		<tr>
			<td>Simport Plastic Ltd., Beloeil, Canada</td>
			<td>www.simport.com</td>
			<td>M-498</td>
			<td>Safekeeping device providing labelling, fixation and paraffin embedding for tissue samples.</td>
		</tr>
		<tr>
			<td>Qiagen GmbH, Hilden Germany</td>
			<td>www.qiagen.com</td>
			<td>765112</td>
			<td>For collection, fixation, and stabilization of tissue samples: 10 Prefilled Reagent Containers, containing PAXgene Tissue FIX and PAXgene Tissue STABILIZER.</td>
		</tr>
		<tr>
			<td>ThermoFisher Scientific</td>
			<td>www.thermofisher.com</td>
			<td>813150</td>
			<td>Processing of biological specimens from chemical fixation to paraffin infiltration</td>
		</tr>
		<tr>
			<td>Merck Millipore, Munich, Germany</td>
			<td>www.analytics-shop.com/de/hersteller/millipore.html</td>
			<td>MC1009834000</td>
			<td>Dehydration of tissue samples</td>
		</tr>
		<tr>
			<td>VWR Chemicals, Darmstadt, Germany</td>
			<td>https://at.vwr.com</td>
			<td>10293EP</td>
			<td>Dehydration of tissue samples</td>
		</tr>
		<tr>
			<td>ACM Herba Chemosan Apotheker AG, Vienna, Austria</td>
			<td>www.herba-chemosan.at</td>
			<td>2549662 32</td>
			<td>Paraffin used in a tissue processing device</td>
		</tr>
		<tr>
			<td>Leica Mikrosysteme Handels GmbH, Vienna, Austria</td>
			<td>www.LeicaBiosystems.com</td>
			<td>39602004</td>
			<td>Paraffin embedding medium</td>
		</tr>
		<tr>
			<td>Sanova Diagnostik, Vienna, Austria</td>
			<td>www.sanova.at</td>
			<td>5229</td>
			<td>Paraffin embedding instrument for tissues for histology</td>
		</tr>
		<tr>
			<td>Histocom Medizintechnik Vertriebs GmbH, Wiener Neudorf, Austria</td>
			<td>www.histocom.info</td>
			<td>M 910010</td>
			<td>This device is used for especially sophisticated paraffin sectioning techniques in biology and medicine. Only skilled or specially trained personnel must operate the microtome, i.e. clamping the specimen, trimming, sectioning and taking off the sections from the instrument.</td>
		</tr>
		<tr>
			<td>Dako Denmark A/S, Glostrup, Denmark</td>
			<td>www.chem.agilent.com</td>
			<td>K802021-2</td>
			<td>Coated glass slides, intended for mounting formalin-fixed, paraffin-embedded tissue sections.</td>
		</tr>
		<tr>
			<td>Qiagen GmbH, Hilden Germany</td>
			<td>www.qiagen.com</td>
			<td>73504</td>
			<td>For purification of total RNA from formalin-fixed, paraffin-embedded tissue sections</td>
		</tr>
		<tr>
			<td>Qiagen GmbH, Hilden Germany</td>
			<td>www.qiagen.com</td>
			<td>765134</td>
			<td>For purification of total RNA from PAXgene-fixed, paraffin-embedded tissue sections</td>
		</tr>
		<tr>
			<td>ZytoVision GmbH, Bremerhaven, Germany</td>
			<td>www.zytovision.com</td>
			<td>Z-2015-200</td>
			<td>For the detection of ERBB2 (a.k.a HER2) gene amplification frequently observed in solid malignant neoplasms e.g. breast cancer samples.</td>
		</tr>
		<tr>
			<td>3DHISTEC, Budapest, Hungary</td>
			<td>www.3dhistech.com</td>
			<td></td>
			<td>Digital device for scanning tissue slides equipped with a 40x/1.2 NA objective, Quad band filters (DAPI/FITC/TRITC/Cy5) filters and a 5.5Mpx, 16 bit, cooled scientific CMOS (Complementary Metal Oxide Semiconductor) camera.</td>
		</tr>
		<tr>
			<td>ThermoFisher Scientific</td>
			<td>www.thermofisher.com</td>
			<td>4368814</td>
			<td>The High Capacity cDNA Reverse Transcription Kit contains all components necessary for the quantitative conversion of up to 2 &#181;g of total RNA to single-stranded cDNA in a single 20 &#181;L reaction.</td>
		</tr>
		<tr>
			<td>ThermoFisher Scientific</td>
			<td>www.thermofisher.com</td>
			<td>4367659</td>
			<td>PCR Master Mix containing polymerase, nucleotides and SYBR Green for PCR and quanitification of amplicons.</td>
		</tr>
		<tr>
			<td>ThermoFisher Scientific, Wilmington, DE</td>
			<td>www.thermofisher.com</td>
			<td>Ser. Nr. F239</td>
			<td>Spectrophotometer for nucleic acid quantification.</td>
		</tr>
		<tr>
			<td>QuantStudio 7 Flex Real-time PCR System</td>
			<td>www.thermofisher.com</td>
			<td>4485701</td>
			<td>PCR machine,</td>
		</tr>
		<tr>
			<td>Vysis/Abbott Laboratories. Abbott Park, Illinois, U.S.A</td>
			<td>www.molecular.abbott/us/en/products/instrumentation/thermobrite#order</td>
			<td>e.g. (ThermoBrite) 07J91-020</td>
			<td>Temperature controlled slide processing system for in-situ denaturation/hybridization procedures.</td>
		</tr>
	</tbody>
</table>

protocol for her2 fish using a non-cross-linking, formalin-free tissue fixative to combine advantages of cryo-preservation and formalin fixation

福尔马林固定、石蜡包埋 (FFPE) 组织标本的形态学评估是几十年来癌症诊断的黄金标准, 因为它具有优良的形态学保存。个性化药物越来越多地提供单独的适应和靶向治疗的特点个别疾病的组合形态和分子分析技术和诊断。由于化学修饰和核酸和蛋白质交联的影响, 甲醛对同一 FFPE 标本的形态学和分子检测的性能具有挑战性。最近开发了一种非连接的无福尔马林组织固定剂, 以满足这两种要求, 即保存像 FFPE 和生物分子的形态, 如冷冻保存。由于鱼类通常需要与组织病理学和分子诊断结合, 我们测试了鱼协议的适用性, 在这个新的定影剂治疗。我们发现, 福尔马林 post-fixation 的组织学切片的非连接, 无福尔马林和石蜡嵌入 (NCFPE) 乳腺癌组织产生的等效结果与 FFPE 组织的人表皮生长因子受体 2 (HER2)鱼分析。该协议描述了最初开发和验证的 FFPE 组织的鱼分析, 可以用简单的组织学切片 post-fixation 步骤用于 NCFPE 组织。

鱼 HER2 测定:
FFPE 和 NCFPE 的 HER2-FISH 信号强度相似 (图 2)。与红色信号 (CEN17 参考基因) 相比, 大多数细胞中的两种情况都显示出明显过量的绿色信号 (表明放大的 HER2 基因轨迹)。因此, 该病例显示了一个放大的 HER2 基因, 这意味着该病人有望回应曲妥珠, anti-HER2 靶向治疗。FFPE 标本充当阳性对照。在非细胞 (FFPE 和 NCFPE) 中观察到的信号是 HER2 基因放大的内部参考和负控制。对于每个分析系列, 至少有一个已知的基因扩增状态的部分应该被用作杂交反应的阳性控制。
RNA 质量:
应用 real-time 逆转录 PCR 技术对两种不同情况下的 FFPE 和 NCFPE 样本的 RNA 质量差异进行了分析。结果表明, 不同增长度 (即 71 bp、153 bp、200 bp、275 bp、323 bp) 对 GAPDH mRNA 的放大效率不同。从 FFPE 样品中获得的所有 Ct (周期阈值) 均高于 NCFPE。这种差异表明, FFPE 组织中分离出的 mRNA 的 PCR amplificability 更低, 这意味着更明显的化学修饰。此外, 较高的 Ct 值较短的增是一个指标的 mRNA 分裂。相比之下, 不同增长度的 Ct 值斜率在 FFPE 中显示出比 NCFPE 组织更明显的 mRNA 碎裂 (图 3)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55885/55885fig2v2.jpg"> 
图 2: HER2 鱼的代表性结果为相应的福尔马林固定石蜡镶嵌 (FFPE) 和非链, 无福尔马林固定石蜡嵌入 (NCFPE) 组织切片.相应的样品从相同的 FFPE (a) 和 NCFPE (B) 组织显示。与正常的相间细胞 (C) 相比, 两个绿色 (HER2) 和两个红色 (CEN17) 信号的预期, 细胞与放大 HER2 基因所在地代表多个副本的绿色信号 (HER2) (D)。HER2 的放大状态是相同的 FFPE 和 post-fixed NCFPE 节 (a, B)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55885/55885fig2v2large.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/55885/55885fig3.jpg"> 
图 3: 用于 RNA 质量控制的 real-time 反转录 PCR 检测结果.Y 轴: Ct (周期阈值) 的 GAPDH 基因显示不同的增长度 (71 bp, 153 bp, 200 bp, 275 bp, 323 bp) 在 X 轴。对两种不同的乳腺癌组织标本的 FFPE 和 NCFPE 进行了分离, 并转录成 cDNA 进行 PCR 处理。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55885/55885fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation at JoVE.com

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

荧光原位杂交 (fish) 通常需要结合组织病理学和分子诊断技术来选择个性化药物治疗。一种新的非连接, 无福尔马林组织固定剂, 允许高品质的形态, 分子和鱼类分析从同一标本, 加入了 post-fixation 步骤之前, 鱼提出。

非交联无甲醛组织固定剂 HER2 鱼的协议, 结合低温保存和福尔马林固色的优点

Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its ...

protocol-for-her2-fish-using-non-cross-linking-formalin-free-tissue

Nanyang Technological University

Research

JoVE Journal

Cancer Research

13.6K 次观看.  Medical University Graz. 荧光原位杂交 (fish) 通常需要结合组织病理学和分子诊断技术来选择个性化药物治疗。一种新的非连接, 无福尔马林组织固定剂, 允许高品质的形态, 分子和鱼类分析从同一标本, 加入了 post-fixation 步骤之前, 鱼提出。

视频: Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a viable treatment strategy and biopsy is not routinely performed, the availability of patient samples for research is limited. Consequently, efforts to study this disease have been challenged by a paucity of faithful disease models. To address this need, we describe here a protocol for the rapid processing of post-mortem autopsy tissue samples in order to generate durable patient-derived cell culture models that can be used in in vitro assays or in vivo orthotopic xenograft experiments. These models can be used to screen for potential drug targets and to study fundamental pathobiological processes within DIPG. This protocol can further be extended to analyze and isolate tumor and microenvironmental cells using Fluorescence-activated Cell Sorting (FACS), which enables subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA at the bulk cell or single cell level. Finally, this protocol can also be adapted to generate patient-derived cultures for other central nervous system tumors.

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

This protocol describes a method for the rapid processing of post-mortem diffuse intrinsic pontine glioma samples for the establishment of patient-derived cell culture models or direct characterization of tumor and microenvironmental cells.

一种内在的弥漫性脑胶质瘤脑桥快速验尸细胞培养协议（DIPG）

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

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 ...

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

测定急性髓系白血病可测残留病和白血病干细胞的骨髓标本和过程综合协议

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

向上移动: 一个简单而灵活的体外三维入侵检测协议

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

用医用金属丝捕获循环肿瘤细胞的肿瘤细胞: 基于免疫荧光和 DNA 的3D 方法

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

Imaging Approaches to Assessments of Toxicological Oxidative Stress Using Genetically-encoded Fluorogenic Sensors

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including destruction of the sample, introduction of potential artifacts, and a lack of specificity for the reactive species involved. Thus, there is a current need in the field of toxicology for non-destructive, sensitive, and specific methods that can be used to observe and quantify intracellular redox perturbations, more commonly referred to as oxidative stress. Here, we present a method for the use of two genetically-encoded fluorogenic sensors, roGFP2 and HyPer, to be used in live-cell imaging studies to observe xenobiotic-induced oxidative responses. roGFP2 equilibrates with the glutathione redox potential (EGSH), while HyPer directly detects hydrogen peroxide (H2O2). Both sensors can be expressed into various cell types via transfection or transduction, and can be targeted to specific cellular compartments. Most importantly, live-cell microscopy using these sensors offers high spatial and temporal resolution that is not possible using conventional methods. Changes in the fluorescence intensity monitored at 510 nm serves as the readout for both genetically-encoded fluorogenic sensors when sequentially excited by 404 nm and 488 nm light. This property makes both sensors ratiometric, eliminating common microscopy artifacts and correcting for differences in sensor expression between cells. This methodology can be applied across a variety of fluorometric platforms capable of exciting and collecting emissions at the prescribed wavelengths, making it suitable for use with confocal imaging systems, conventional wide-field microscopy, and plate readers. Both genetically-encoded fluorogenic sensors have been used in a variety of cell types and toxicological studies to monitor cellular EGSH and H2O2 generation in real-time. Outlined here is a standardized method that is widely adaptable across cell types and fluorometric platforms for the application of roGFP2 and HyPer in live-cell toxicological assessments of oxidative stress.

This manuscript describes the use of genetically-encoded fluorogenic reporters in an application of live-cell imaging for the examination of xenobiotic-induced oxidative stress. This experimental approach offers unparalleled spatiotemporal resolution, sensitivity, and specificity while avoiding many of the shortcomings of conventional methods used for the detection of toxicological oxidative stress.

用基因编码荧光传感器评估毒理学氧化应激的成像方法

While oxidative stress is a commonly cited toxicological mechanism, conventional methods to study it suffer from a number of shortcomings, including ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

超声引导组织定向细胞植入术在生物学相关转移性肿瘤移植中的应用

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

Automated Multiplex Immunofluorescence Panel for Immuno-oncology Studies on Formalin-fixed Carcinoma Tissue Specimens

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4&#8242;,6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.

A detailed protocol for a six-marker multiplex immunofluorescence panel is optimized and performed, using an automated stainer for more consistent results and a shorter procedure time. This approach can be directly adapted by any laboratory for immuno-oncology studies.

福尔马林固定癌组织标本免疫肿瘤学自动复合免疫荧光小组研究

Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of ...

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

使用微生理系统模拟人体乳房组织中的乳腺癌

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&#38;E) annotations are used to target small tumor areas using a blade that is 250 &#181;m2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 &#181;m in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

Digital annotation with automated tissue dissection provides an innovative approach to enriching tumor in low tumor content cases and is adaptable to both paraffin and frozen tissue types. The described workflow improves accuracy, reproducibility and throughput and could be applied to both research and clinical settings.

用于低肿瘤含量组织中肿瘤富集的自动解剖方案

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly ...