A METHOD FOR EXPOSING HIDDEN ANTIGENIC SITES IN PARAFORMALDEHYDE-FIXED CULTURED-CELLS, APPLIED TO INITIALLY UNREACTIVE ANTIBODIES
A METHOD FOR EXPOSING HIDDEN ANTIGENIC SITES IN PARAFORMALDEHYDE-FIXED CULTURED-CELLS, APPLIED TO INITIALLY UNREACTIVE ANTIBODIES
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DOI：
10.1177/41.3.8429208
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发表时间：
1993-03-01
影响因子：
3.2
通讯作者：
KAARIAINEN, L
中科院分区：
文献类型：
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作者：
PERANEN, J;RIKKONEN, M;KAARIAINEN, L
我们描述了一种简单的方案，该方案能够获取培养细胞中被掩盖或隐藏的细胞内抗原。该方案基于将多聚甲醛固定且用Triton X - 100透化的细胞暴露于盐酸胍（GdnHCl）。我们通过免疫荧光显微镜将其用于BHK - 21细胞中不同抗原的定位。我们的结果表明，六种抗体中有五种最初无反应，但当与GdnHCl联合使用时，成为了出色的定位工具。当用不同的细胞器特异性抗体监测时，用GdnHCl使固定细胞变性并不影响细胞的整体结构。在17种情况中有2种，抗原位点在变性后丢失。然而，通过在固定剂中加入少量戊二醛，这个问题可以部分得到解决。我们认为这种方法在免疫荧光定位研究中可能普遍有用，特别是在已知抗体仅与变性抗原反应的情况下。
We describe a simple protocol that allows the retrieval of masked or hidden intracellular antigens in cultured cells. The protocol is based on the exposure of paraformaldehyde-fixed and Triton X-100-permeabilized cells to guanidine hydrochloride (GdnHCl). We used it for localization of different antigens in BHK-21 cells by immunofluorescence microscopy. Our results showed that five out of six antibodies, initially unreactive, became excellent localization tools when used in conjunction with GdnHCl. Denaturation of fixed cells with GdnHCl did not affect the overall cell architecture, when monitored with different organelle-specific antibodies. In two cases out of 17 the antigenic sites were lost after denaturation. However, this problem could be partially overcome by including low amounts of glutaraldehyde in the fixative. We think that this method could be generally useful in immunofluorescence localization studies, particularly in cases where the antibodies are known to react only with denatured antigens.