Name: elastic staining on paraffin-embedded slides of pt3n0m0 gastric cancer tissue
Uploaded: 2019-05-01T12:30:00
Description: Watch this Scientific Journal Video about Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue at JoVE.com

作者部分感谢国家自然科学基金(NO.81401902和NO.81501992)和湖南省自然科学基金(NO.2017SK2134,NO.2018JJ2238)的支持。手稿由广磊撰写。整个实验由杨光磊和杨海燕进行。作者感谢病理学家和跟踪患者数据的研究员的帮助。

1994年至2005年间,中南大学湘雅医学院附属肿瘤医院的两名经验丰富的胃肠道病理学家从胃肿瘤胃切除术患者中抽取了患者样本,pT3N0M0阶段胃癌术后病理诊断(根据第七期TNM分期)。这项研究得到了道德审查委员会的批准。
1. 分段
<ol><li>将石蜡块放入雪橇微体固定支架中,该支架可在刀片上前后移动。调整块和微托刀之间的最佳角度。 注:最佳角度不仅取决于刀片的几何形状,还?...

<ol>
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这里提出的该方法为识别亚血清弹性层压提供了一种准确的方法。该方法可用于评估肿瘤细胞是否侵入了pT3N0M0胃癌12中的弹性层。弹性层压在H&E染色部分是红色的,很难与胶原纤维区分,其他现有的辅助诊断方法,如细胞学和免疫组织化学,也具有有限的诊断价值。在围肠灌溉液中发现肿瘤细胞并不一定表明原发性肿瘤存在血清入侵,这可能是由于淋巴结转移或其他遥远的转移5。<sup cl...

根据肿瘤、节点和转移(TNM)癌症分期系统,病理阶段pT4的胃癌预后明显比pT3(第8次UICC/AJCC)1的晚期差。与胃癌,原发性肿瘤(pT)的分类通常取决于肿瘤入侵的深度2。病理阶段 pT3 胃癌被定义为通过肌肉体侵入到亚血清组织中的癌症,而 pT4 胃癌被定义为渗透到内脏围气体的表面的癌症。围层表面侵入性的存在是区分两个阶段2的关键。然而,由于围肠间细胞层非常薄,在手术、术后治疗和固定3过程中可能会受损。此外,肿瘤相关的炎症变化和纤维化会损害围肠的正常解剖结构,因此很难仅使用H&E染色4准确判断围肠表面入侵。目前的辅助诊断方法,如细胞学和免疫组织化学,由于是假阳性或成本效益低,诊断价值有限。
血清膜包括围肠、胸膜和心包膜,由中皮膜、基底膜和亚中上层7组成。血清膜的一个常见组织学特征是,在亚中皮层中存在弹性层,与中上层细胞8、9相邻。弹性拉米纳具有很强的抗损伤能力,如果间皮细胞被严重的肿瘤相关炎症或纤维化3破坏,可以作为替代预后标记。弹性层压片主要由弹性蛋白和微纤维10组成,可以通过弹性染色进行可视化。弹性染色使用的主要原因是弹性素与弹性素溶液中的红素酚组形成氢键,导致弹性纤维被染成蓝黑色。在范吉森(VG)对比染色后,胶原纤维可以染成红色,肌肉纤维和红血球可以染黄色8,11,12,13。
肺癌的TNM分期第八版将内脏胸膜入侵(T2)定义为肿瘤入侵,超出弹性层膜或内脏胸膜2表面的入侵。对结肠直肠癌的研究表明,pT3结肠直肠癌的弹性层压瘤入侵可能是预后不佳的原因。大肠癌的5年无病和整体存活率也被证明像pT4a13,14,15。根据我们以前对弹性染色的研究,弹性拉米纳入侵对pT3胃癌的预后有显著的负面影响,应像pT4a胃癌12一样对待。因此,这种简单且经济高效的方法可以帮助消除通过H&E染色获得的结果的不确定性,以及与细胞学和免疫性化学在内的其他辅助诊断方法相关的其他常见限制。别人。该方法可有效地诊断胃肠癌的围膜表面入侵,特别是在一些模棱两可的情况下。它很方便,因为H&E污渍和其他污渍通常很难识别这种入侵。这种方法还确保了结果的可靠性,因为它非常有选择性,尤其是弹性纤维。在这里,我们进行弹性染色,以确定肿瘤细胞是否侵入了pT3N0M0胃癌12的弹性层压。
该方法用于识别形式固定、石蜡嵌入部分的组织中的弹性纤维,也可用于冷冻部分。在这里,我们选择石蜡嵌入部分,以最佳维护细胞和组织形态。此协议中的所有步骤都在室温下执行。一种商业染色试剂盒,包括高锰酸钾溶液、草酸溶液、艾莱他溶液和Van Gieson溶液。

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	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:192px;">Elastin-Van Gieson staining kit</td>
			<td style="width:108px;">Baso</td>
			<td style="width:99px;">BA4083B</td>
			<td style="width:221px;">Used for staining of elastic fibers, collagen fibers and myofibers&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:192px;">Permount TM Mounting Medium</td>
			<td style="width:108px;">MXB Biotechnologies</td>
			<td style="width:99px;">DAB-0033</td>
			<td>Used for mounting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Ethanol</td>
			<td style="width:108px;">LMAI Bio&#160;</td>
			<td style="width:99px;">LM64-17-5&#160;</td>
			<td align="right">100%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:192px;">Xylene</td>
			<td style="width:108px;">LMAI Bio&#160;</td>
			<td style="width:99px;">LX820585</td>
			<td></td>
		</tr>
	</tbody>
</table>

elastic staining on paraffin-embedded slides of pt3n0m0 gastric cancer tissue

弹性层状通常位于亚中上层,毗邻围肠中皮细胞,在血氧素和欧辛(H&E)染色上是两年期的,但可以通过弹性染色可视化。弹性染色的一个重要好处是,它很容易确定肿瘤细胞是否侵入了弹性层压。这有助于检查围膜表面入侵的程度,从而区分胃肠道癌的pT3和pT4阶段。在这项研究中,我们提出了一个协议,以识别在形式固定,石蜡嵌入pT3N0M0胃癌组织部分的弹性纤维。我们准备固定在幻灯片上的5μm石蜡部分,然后在染色过程中对所有幻灯片进行脱蜡和补水。随后,所有部分均被高锰酸钾氧化,由草酸漂白,被弹性染色染色,并反染范吉森。最后,我们考察了染色的效果;弹性层压是彩色蓝黑色和胶原纤维被染成红色,而癌细胞被染色在不同色调的黄色。我们还详细介绍了用于确定癌细胞和弹性层压作用关系的方法。该方法简单、成本低,因其对弹性纤维的选择性和真实效果,在胃肠癌的围膜表面入侵的鉴定中应用广泛。

成功的弹性染色清楚地揭示了pT3胃癌中的弹性拉米纳和癌细胞。图1 - 低功率场,在弹性蛋白溶液染色后进行显微检查,在范吉森的逆色之前,根据上述协议 - 显示弹性层状接近血清中质细胞,并在长丝的形式,并且肿瘤细胞和弹性拉比纳之间仍有一定的距离。图2是范吉森的反面染色的典型弹性染色,其中弹性层片被染成蓝黑色,胶原纤?...

Watch this Scientific Journal Video about Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue at JoVE.com

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

在这里,我们提出了弹性染色方案,以识别pT3N0M0胃癌组织在形式固定,石蜡嵌入部分的弹性纤维。随后,我们描述了一种确定肿瘤细胞是否侵入弹性层压外的方法。

pT3N0M0 胃癌组织石蜡嵌入幻灯片上的弹性染色

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

此方法有助于确定肿瘤细胞是否侵入在 pT3 胃癌中超过弹性层。pT3 胃癌预后图中的弹性层层侵蚀。该技术的主要优点是，它方便，准确的方法，以确定弹性层，这使得它更精确的病理学家评估肿瘤细胞是否侵入超过弹性层在pT3胃癌。该技术可用于肺癌和癌症的病理测试阶段诊断。该方法也适用于肺癌和结肠癌的TNM阶段系统。首先，将幻灯片浸入二甲苯中，在科普林罐中浸泡 10 分钟。从这个罐子里取下幻灯片，将它们浸入另一个Coplin罐子中的新鲜二甲苯中再浸泡10分钟。将幻灯片浸入 100%乙醇中 5 分钟，然后将它们转移到新鲜 100%乙醇中再浸泡 5 分钟。接下来，将幻灯片浸入95%乙醇中两分钟。将幻灯片转移到装有 70% 乙醇的罐子中两分钟。在此之后，用蒸馏水在一个新的Coplin罐中短暂冲洗幻灯片。首先，使用纸巾擦去幻灯片上或组织周围的任何多余的溶液。将幻灯片放在与室温和湿度相匹配的条件下。在每张幻灯片中加入几滴高锰酸钾以氧化它们，确保添加的体积足以覆盖每张幻灯片上的组织部分。五分钟后，用科普林罐中的蒸馏水冲洗氧化滑梯，重复冲洗两到三次，每次冲洗使用一罐新水。组织应具有可观察到的棕色。现在，在每张幻灯片中加入几滴草酸来漂白它们，确保添加的体积足以覆盖每张幻灯片上的组织部分。五分钟后，用蒸馏水冲洗漂白的滑梯，重复冲洗两到三次，每次冲洗使用一罐新水。用 95% 的酒精短暂清洗滑梯。将洗涤的幻灯片浸入弹性液中8至24小时。然后直接将幻灯片浸入 95%乙醇中一到两分钟，使每个组织部分都分好。用蒸馏水完全冲洗幻灯片。从微观上检查蓝黑色弹性纤维染色和灰色背景。将幻灯片在 van Gieson 溶液中对进行一分钟对，确保使用的体积足以完全覆盖每张幻灯片上的组织部分。接下来，迅速将95%乙醇滴到组织切片上几秒钟，使每个组织部分都分好。首先，将幻灯片浸入 95% 酒精中五分钟，快速脱水。将幻灯片转移到 100%酒精五分钟，以继续脱水。然后，将幻灯片转移到新鲜 100% 酒精中，再多喝五分钟。将幻灯片浸入两种不同的二甲苯变化中，每次浸入持续三分钟。在此之后，在幻灯片中加入一滴树脂安装介质，并在顶部放置盖滑。使用显微镜观察文本协议中概述的组织部分。在这项研究中，弹性染色被用于视觉识别亚性弹性层。一个具有代表性的成功染色表明，在弹性蛋白溶液染色后，但在范吉森的反染色之前，弹性层可以清楚地看到以丝片的形式染色蓝黑色。它们靠近血清间血细胞，弹性层和肿瘤细胞之间仍有一定距离。典型的弹性染色与范吉森的反面表明，弹性层是染色的蓝黑色。胶原纤维染成红色，肌肉纤维和癌细胞染成黄色。穿透弹性层的癌细胞被认为是弹性层侵入。而癌细胞接近，但没有入侵弹性层被认为是弹性层非入侵。如果染色显示多层弹性层重复结构，且肿瘤不侵入最外层，则应诊断样品为弹性层非侵入。弹性蛋白溶液是一个，染色时间相对长，所以最好将弹性蛋白染色的所有幻灯片浸入密封容器中。弹性蛋白溶液染色后，应用蒸馏水冲走。

Research

JoVE Journal

Cancer Research

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

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 ...

科研

癌症研究

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

基于芯片的数字 PCR 检测新鲜冷冻胃癌组织中的一个CDH1罕见转录变体

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

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

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

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array (HMCA)-based Plates

Analysis of Cancer Cell Invasion and Anti-metastatic Drug Screening Using Hydrogel Micro-chamber Array HMCA-based Plates

用水凝胶微室阵列 (HMCA) 板进行肿瘤细胞侵袭和抗转移药物筛选的分析

Cancer metastasis is known to cause 90% of cancer lethality. Metastasis is a multistage process which initiates with the penetration/invasion of tumor ...

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

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 ...

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

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 ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

建立胃癌患者衍生异种移植模型和原细胞系

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Expanding the Comprehension of the Tumor Microenvironment using Mass Spectrometry Imaging of Formalin-Fixed and Paraffin-Embedded Tissue Samples

使用福尔马林固定和石蜡包埋组织样品的质谱成像扩展肿瘤微环境的理解

Advances in immune-based therapies have revolutionized cancer treatment and research. This has triggered growing demand for the characterization of the ...

Deciphering Tumor Microenvironment Using mIHC in Lung Squamous Carcinoma

Multiplex Immunohistochemistry Staining for Paraffin-embedded Lung Cancer Tissue

Author Spotlight: Enhancing Multicolor Fluorescence Localization in Lung Carcinoma Sample

Lung cancer is the leading cause of malignant tumor-related morbidity and mortality all over the world, and the complex tumor microenvironment has been ...