An antigen retrieval method using an alkaline solution allows immunoelectron microscopic identification of secretory granules in conventional epoxy-embedded tissue sections
An antigen retrieval method using an alkaline solution allows immunoelectron microscopic identification of secretory granules in conventional epoxy-embedded tissue sections
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DOI：
10.1177/002215540305100208
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发表时间：
2003-02-01
影响因子：
3.2
通讯作者：
Yokoyama, S
中科院分区：
文献类型：
--
作者：
Yano, S;Kashima, K;Yokoyama, S
免疫电子显微镜使用嗜铬粒蛋白A特异性抗体已被提出作为一种有效的技术，用于识别分泌颗粒（SGs）在肿瘤细胞的证据明显的神经内分泌分化。使用抗原修复（AR）的方法，我们成功地免疫标记SG与抗体在常规处理的环氧包埋块的组织切片。将胰岛素瘤样本固定在2%戊二醛中，后固定在1%OSO4中，并包埋在环氧树脂中。超薄切片用嗜铬粒蛋白A特异性抗体和金标记的第二抗体进行免疫染色。在不存在AR的情况下没有显著标记。无论是蚀刻与偏高碘酸钠，也没有微波照射的柠檬酸盐缓冲液（pH 6.0）或EDTA缓冲液（pH 8.0）中的免疫标记的效率是有效的提高。然而，在碱性溶液（pH 10）中微波处理的环氧树脂包埋切片被充分标记（5.2 +/- 0.34个颗粒/SG）。此外，通过在碱性溶液（pH 10）中微波切片，随后在60 ℃下进行免疫染色（12.2 +/-0.51个颗粒/SG），实现了免疫染色效率的显著提高。这种方法也可以应用于从福尔马林固定的石蜡包埋的组织块获得的环氧包埋切片，甚至对15年前制备的旧环氧包埋的组织块也有效。
Immunoelectron microscopy using chromogranin A-specific antibodies has been proposed as an efficient technique for identification of secretory granules (SGs) in tumor cells with evidence of apparent neuroendocrine differentiation. Using an antigen retrieval (AR) method, we succeeded in immunolabeling SGs with antibodies in ultrathin sections of routinely processed epoxy-embedded blocks of tissue. Samples of an insulinoma were fixed in 2% glutaraldehyde, postfixed in 1% OSO4, and embedded in epoxy resin. Ultrathin sections were immunostained with chromogranin A-specific antibodies and gold-conjugated second antibodies. There was no significant labeling in the absence of AR. Neither etching with sodium metaperiodate nor microwave irradiation of ultrathin sections in citrate buffer (pH 6.0) or in EDTA buffer (pH 8.0) was effective in improving the efficiency of immunolabeling. However, ultrathin epoxy-embedded sections that were microwaved in alkaline solution (pH 10) were adequately labeled (5.2 +/- 0.34 particles per SG). Moreover, considerably improved efficiency of immunostaining was achieved by microwaving sections in alkaline solution (pH 10) with subsequent immunostaining at 60C (12.2 +/- 0.51 particles per SG). This method can also be applied to epoxy-embedded sections obtained from formalin-fixed, paraffin-embedded blocks of tissue and was even valid for an old epoxy-embedded block of tissue prepared 15 years previously.