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>

<ol>
	<li>Fox, C. H., Johnson, F. B., Whiting, J., Roller, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formaldehyde+fixation.">Formaldehyde fixation.</a> J. Histochem.Cytochem. 33 (8), 845-853 (1985).</li><li>Oosterhuis, J. W., Coebergh, J. W., van Veen, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour+banks:+well-guarded+treasures+in+the+interest+of+patients.">Tumour banks: well-guarded treasures in the interest of patients.</a> Nat. Rev. Cancer. 3 (1), 73-77 (2003).</li><li>Masuda, N., Ohnishi, T., Kawamoto, S., Monden, M., Okubo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+chemical+modification+of+RNA+from+formalin-fixed+samples+and+optimization+of+molecular+biology+applications+for+such+samples.">Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples.</a> Nucleic Acids Res. 27 (22), 4436-4443 (1999).</li><li>Metz, B., Kersten, G. F., Hoogerhout, P., Brugghe, H. F., Timmermans, H. A., de Jong, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+formaldehyde-induced+modifications+in+proteins:+reactionswith+model+peptides.">Identification of formaldehyde-induced modifications in proteins: reactionswith model peptides.</a> J. Biol. Chem. 279 (8), 6235-6243 (2003).</li><li>Evers, D. L., Fowler, C. B., Cunningham, B. R., Mason, J. T., O'Leary, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+formaldehyde+fixation+on+RNA:+optimization+of+formaldehyde+adduct+removal.">The effect of formaldehyde fixation on RNA: optimization of formaldehyde adduct removal.</a> J.Mol. Diagn. 13 (3), 282-288 (2011).</li><li>Groelz, D., Sobin, L., Branton, P., Compton, C., Wyrich, R., Rainen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-formalin+fixative+versus+formalin-fixed+tissue:+A+comparison+of+histology+and+RNA+quality.">Non-formalin fixative versus formalin-fixed tissue: A comparison of histology and RNA quality.</a> Exp. Mol. Pathol. 94 (1), 188-194 (2013).</li><li>Do, H., Dobrovic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+artifacts+in+DNA+from+formalin-fixed+tissues:+causes+and+strategies+for+minimization.">Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization.</a> Clin. Chem. 61, 64-71 (2015).</li><li>Kashofer, K., Viertler, C., Pichler, M., Zatloukal, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quality+Control+of+RNA+Preservation+and+Extraction+from+Paraffin-Embedded+Tissue:+Implications+for+RT-PCR+and+Microarray+Analysis.">Quality Control of RNA Preservation and Extraction from Paraffin-Embedded Tissue: Implications for RT-PCR and Microarray Analysis.</a> PloS One. 8 (7), e70714 (2013).</li><li>Viertler, C., Groelz, D., G&#252;ndisch, S., Kashofer, K., Reischauer, B., Riegman, P. H., 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=A+new+technology+for+stabilization+of+biomolecules+in+tissues+for+combined	histological+and+molecular+analyses.">A new technology for stabilization of biomolecules in tissues for combined	histological and molecular analyses.</a> J. Mol. Diagn. 14 (5), 458-466 (2012).</li><li>Gundisch, S., Slotta-Huspenina, J., Verderio, P., Maura, C., Ciniselli, S. P., Schott, 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=Evaluation+of+colon+cancer+histomorphology:+A+comparison+between+formalin+and+PAXgene+tissue+fixation+by+an+international+ring+trial.">Evaluation of colon cancer histomorphology: A comparison between formalin and PAXgene tissue fixation by an international ring trial.</a> Virchows Arch. 465 (5), 509-519 (2014).</li><li>Ergin, B., Meding, S., Langer, R., Kap, M., Viertler, C., Schott, C., 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=Proteomic+analysis+of+PAXgene-fixed+tissues.">Proteomic analysis of PAXgene-fixed tissues.</a> J. Proteome Res. 9 (10), 5188-5196 (2010).</li><li>Kap, M., Smedts, F., Oosterhuis, W., Winther, R., Christensen, N., Reischauer, B., 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=Histological+Assessment+of+PAXgene+Tissue+Fixation+and+Stabilization+Reagents.">Histological Assessment of PAXgene Tissue Fixation and Stabilization Reagents.</a> PLoS ONE. 6 (11), e27704 (2011).</li><li>Mathieson, W., Marcon, N., Antunes, L., Ashford, D. A., Betsou, F., Frasquilho, S. G., 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=A+Critical+Evaluation+of+the+PAXgene+Tissue+Fixation+System:+Morphology,+Immunohistochemistry,+Molecular+Biology,+and+Proteomics.">A Critical Evaluation of the PAXgene Tissue Fixation System: Morphology, Immunohistochemistry, Molecular Biology, and Proteomics.</a> Am J Clin Pathol. 146, 25-40 (2016).</li><li>Oberauner-Wappis, L., Loibner, M., Viertler, C., Groelz, D., Wyrich, R., Zatloukal, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+for+HER2+FISH+determination+on+PAXgene-fixed+and+paraffin-embedded+tissue+in+breast+cancer.">Protocol for HER2 FISH determination on PAXgene-fixed and paraffin-embedded tissue in breast cancer.</a> Int J. Exp. Path. 97 (2), 202-206 (2016).</li><li>Pauletti, G., Godolphin, W., Press, M. F., Slamon, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantitation+ofHER-2/neu+gene+amplification+in+human+breast+cancer+archival+material+using+fluorescence+in+situ+hybridization.">Detection and quantitation ofHER-2/neu gene amplification in human breast cancer archival material using fluorescence in situ hybridization.</a> Oncogene. 13 (1), 63-72 (1996).</li><li>Owens, M. A., Horten, B. C., Da Silva, M. 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.bdigitaluss.remotexs.co/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 &amp;micro;g of total RNA to single-stranded cDNA in a single 20 &amp;micro;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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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

Universidad San Sebastian

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

Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based multicolor labeling is challenging, especially when utilized for assessing interrelations between target proteins in the tissue with a high fat content such as the central nervous system (CNS).
Our protocol represents a refinement of the standard immunolabeling technique particularly adjusted for detection of both structural and soluble proteins in the rat CNS and peripheral lymph nodes (LN) affected by neuroinflammation. Nonetheless, with or without further modifications, our protocol could likely be used for detection of other related protein targets, even in other organs and species than here presented.

We here present an optimized, detailed protocol for double immunostaining in formalin-fixed, paraffin-embedded rat central nervous system (CNS) and peripheral lymph node (LN) tissue sections.

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

Immunohistochemistry (IHC) provides highly specific, reliable and attractive protein visualization. Correct performance and interpretation of an IHC-based ...

科研

神经科学

Do's and Don'ts in the Preparation of Muscle Cryosections for Histological Analysis

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of neuromuscular disorders. However, in order to do so, proper care needs to be taken when excising and preserving tissues to achieve optimal staining. One method of tissue preservation involves fixing tissues in formaldehyde and then embedding them with paraffin wax. This method preserves morphology well and allows for long-term storage at RT but is cumbersome and requires handling of toxic chemicals. Further, formaldehyde fixation results in antigen cross-linking, which necessitates antigen retrieval protocols for effective immunostaining. On the contrary, frozen sectioning does not require fixation and thus retains biological antigen conformation. This method also provides a distinct advantage in quick turn around time, making it especially useful in situations needing quick histological evaluation like intraoperative surgical biopsies. Here we describe the most effective method of preparing muscle biopsies for visualization with different histological and immunological stains.

Here we demonstrate the most efficient methods for freezing, embedding, cryosectioning, and staining of muscle biopsies to avoid freezing artifacts.

该做什么和不该做什么的肌肉冰冻切片的准备组织学分析

Histological evaluation of muscle biopsies has served as an indispensable tool in the understanding of the development and progression of pathology of ...

医学

Fixation of Embryonic Mouse Tissue for Cytoneme Analysis

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens act in a concentration- and time-dependent manner to specify distinct transcriptional programs that instruct and reinforce cell fate. One mechanism by which appropriate morphogen signaling thresholds are ensured is through delivery of the signaling proteins by specialized filopodia called cytonemes. Cytonemes are very thin (&#8804;200 nm in diameter) and can grow to lengths of several hundred microns, which makes their preservation for fixed-image analysis challenging. This paper describes a refined method for delicate handling of mouse embryos for fixation, immunostaining, and thick sectioning to allow for visualization of cytonemes using standard confocal microscopy. This protocol has been successfully used to visualize cytonemes that connect distinct cellular signaling compartments during mouse neural tube development. The technique can also be adapted to detect cytonemes across tissue types to facilitate the interrogation of developmental signaling at unprecedented resolution.

Here, an optimized step-by-step protocol is provided for fixation, immunostaining, and sectioning of embryos to detect specialized signaling filopodia called cytonemes in developing mouse tissues.

固定胚胎小鼠组织以进行细胞内膜分析

Developmental tissue patterning and postdevelopmental tissue homeostasis depend upon controlled delivery of cellular signals called morphogens. Morphogens ...

发育生物学

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

非人类灵长类动物脑组织的制备预嵌入免疫组化和电子显微镜

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

小鼠视网膜冷冻节的制备,用于免疫性植物化学

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

视网膜平片准备成像和免疫组化优化方案

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

生物学

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

将多重荧光 原 位杂交与荧光免疫组化相结合，对新鲜冷冻或固定的小鼠脑切片进行研究

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

Tandem High-pressure Freezing and Quick Freeze Substitution of Plant Tissues for Transmission Electron Microscopy

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, because of laborious and time-consuming protocols that also demand experience in preparation of artifact-free samples, TEM is not considered a user-friendly technique. Traditional sample preparation for TEM used chemical fixatives to preserve cellular structures. High-pressure freezing is the cryofixation of biological samples under high pressures to produce very fast cooling rates, thereby restricting ice formation, which is detrimental to the integrity of cellular ultrastructure. High-pressure freezing and freeze substitution are currently the methods of choice for producing the highest quality morphology in resin sections for TEM. These methods minimize the artifacts normally associated with conventional processing for TEM of thin sections. After cryofixation the frozen water in the sample is replaced with liquid organic solvent at low temperatures, a process called freeze substitution. Freeze substitution is typically carried out over several days in dedicated, costly equipment. A recent innovation allows the process to be completed in three hours, instead of the usual two days. This is typically followed by several more days of sample preparation that includes infiltration and embedding in epoxy resins before sectioning. Here we present a protocol combining high-pressure freezing and quick freeze substitution that enables plant sample fixation to be accomplished within hours. The protocol can readily be adapted for working with other tissues or organisms. Plant tissues are of special concern because of the presence of aerated spaces and water-filled vacuoles that impede ice-free freezing of water. In addition, the process of chemical fixation is especially long in plants due to cell walls impeding the penetration of the chemicals to deep within the tissues. Plant tissues are therefore particularly challenging, but this protocol is reliable and produces samples of the highest quality.

Obtaining high-quality transmission electron microscopy images is challenging, especially in the case of plant cells, which have abundant large water-filled vacuoles and aerated spaces. Tandem high-pressure freezing and quick freeze substitution greatly reduce preparation time of plant samples for TEM while producing samples with excellent ultrastructural preservation.

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

Since the 1940s transmission electron microscopy (TEM) has been providing biologists with ultra-high resolution images of biological materials. Yet, ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

对小鼠乳腺冰冻切片间接免疫荧光

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

High-Throughput, Multi-Image Cryohistology of Mineralized Tissues

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious projects to disrupt every gene in the mouse genome. The research community needs rapid and inexpensive means to phenotype tissues via histology. Histological analyses of skeletal tissues are often time consuming and semi-quantitative at best, regularly requiring subjective interpretation of slides from trained individuals. Here, we present a cryohistological paradigm for efficient and inexpensive phenotyping of mineralized tissues. First, we present a novel method of tape-stabilized cryosectioning that preserves the morphology of mineralized tissues. These sections are then adhered rigidly to glass slides and imaged repeatedly over several rounds of staining. The resultant images are then aligned either manually or via computer software to yield composite stacks of several layered images. The protocol allows for co-localization of numerous molecular signals to specific cells within a given section. In addition, these fluorescent signals can be quantified objectively via computer software. This protocol overcomes many of the shortcomings associated with histology of mineralized tissues and can serve as a platform for high-throughput, high-content phenotyping of musculoskeletal tissues moving forward.

In this manuscript, we present a high-throughput, semi-automated cryohistology platform to produce aligned composite images of multiple response measures from several rounds of fluorescent imaging on frozen sections of mineralized tissues.

高通量，矿化组织的多图像Cryohistology

There is an increasing need for efficient phenotyping and histopathology of a variety of tissues. This phenotyping need is evident with the ambitious ...

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

摘要

探索更多视频

此视频中的章节

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

该做什么和不该做什么的肌肉冰冻切片的准备组织学分析

固定胚胎小鼠组织以进行细胞内膜分析

非人类灵长类动物脑组织的制备预嵌入免疫组化和电子显微镜

小鼠视网膜冷冻节的制备,用于免疫性植物化学

视网膜平片准备成像和免疫组化优化方案

将多重荧光原位杂交与荧光免疫组化相结合，对新鲜冷冻或固定的小鼠脑切片进行研究

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

对小鼠乳腺冰冻切片间接免疫荧光

高通量，矿化组织的多图像Cryohistology

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

摘要

探索更多视频

此视频中的章节

免疫组化分析大鼠中枢神经系统和周围淋巴结组织切片

该做什么和不该做什么的肌肉冰冻切片的准备组织学分析

固定胚胎小鼠组织以进行细胞内膜分析

非人类灵长类动物脑组织的制备预嵌入免疫组化和电子显微镜

小鼠视网膜冷冻节的制备,用于免疫性植物化学

视网膜平片准备成像和免疫组化优化方案

将多重荧光 原 位杂交与荧光免疫组化相结合，对新鲜冷冻或固定的小鼠脑切片进行研究

串联高压冷冻和植物组织中快速冷冻替代透射电子显微镜

对小鼠乳腺冰冻切片间接免疫荧光

高通量，矿化组织的多图像Cryohistology

将多重荧光原位杂交与荧光免疫组化相结合，对新鲜冷冻或固定的小鼠脑切片进行研究