Comparison of the effects of microwave heating and high pressure cooking for antigen retrieval of human and rat Bcl-2 protein in formaldehyde-fixed, paraffin-embedded sections
Comparison of the effects of microwave heating and high pressure cooking for antigen retrieval of human and rat Bcl-2 protein in formaldehyde-fixed, paraffin-embedded sections
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DOI：
10.1080/714028191
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发表时间：
2002-05-01
影响因子：
1.6
通讯作者：
van Dierendonck, JH
中科院分区：
文献类型：
--
作者：
Hoetelmans, RWM;van Slooten, HJ;van Dierendonck, JH
抗凋亡Bcl-2蛋白表达的免疫组织化学检测作为各种类型癌症的推定预后和预测因子被广泛研究。为此目的，在免疫染色之前，在pH 6.0的柠檬酸盐缓冲液中通过微波（MW）加热10分钟至100 ℃，通常用于在存档的福尔马林固定的石蜡包埋组织中回收Bcl-2抗原。我们最近报道，Bcl-2不仅是一种细胞质蛋白，但它也存在于间期核，它与有丝分裂染色体密切相关。此外，我们发现，单克隆抗体（MAb）#124与核/染色体表位的结合被基于甲酰胺的固定剂减弱，并且不能通过MW处理10分钟恢复。在这里，我们报告了延长MW加热或在高压锅（HPC）中加热至130 ℃，尽管改善了细胞质免疫染色，不能在人组织中检索MAb#124识别的核/染色体Bcl-2表位。相比之下，这些程序可以在大鼠组织中检索由多克隆#15616E抗体检测到的核/染色体Bcl-2表位。这些表位的特异性通过MW加热或HPC处理的组织的Western印迹分析来证实。
Immunohistochemical detection of expression of the anti-apoptotic Bcl-2 protein is widely studied as a putative prognostic and predictive factor in various types of cancer. For that purpose, heating for 10 min by microwave (MW) up to 100 C in citrate buffer, pH 6.0, prior to immunostaining is often used to retrieve Bcl-2 antigens in archival formalin-fixed, paraffin-embedded tissue. We recently reported that Bcl-2 is not only a cytoplasmic protein, but that it is present also in interphase nuclei and that it strongly associates with mitotic chromosomes. Furthermore, we showed that binding of the monoclonal antibody (MAb) #124 with nuclear/chromosomal epitopes is diminished by formaldehyde-based fixatives and cannot be restored by MW treatment for 10 min. Here we report that prolonged MW heating or heating up to 130 C in a high pressure cooker (HPC), despite improved cytoplasmic immunostaining, fails to retrieve nuclear/chromosomal Bcl-2 epitopes recognized by the MAb #124 in human tissues. In contrast, these procedures can retrieve nuclear/chromosomal Bcl-2 epitopes detected by polyclonal #15616E antibodies in rat tissues. The specificity of these epitopes was confirmed by Western blot analysis of tissues treated by MW heating or HPC.