Department of Pathology.
Department of Biochemistry.
J Histochem Cytochem. 2022 Feb;70(2):181-187. doi: 10.1369/00221554211055240. Epub 2021 Oct 29.
The tight junction membrane protein claudin 1 and the adherens junction protein E-cadherin play critical roles in cell-cell communication and in cell signaling. As a result, their protein levels and distribution in cancer have been a focus of cancer researchers in recent years. The loss of sensitivity to contact inhibition and the establishment of invasive properties in cancer are thought to be a result of the mislocalization of these membrane proteins to the cytoplasm. However, reports on their distribution and levels have been inconsistent. It is therefore critical that the techniques used to determine the cellular localization of these proteins be both consistent and reliable. This study was undertaken to determine the optimal fixation method, methanol or formalin, for the detection of claudin 1 and E-cadherin by immunofluorescence in five different human breast cancer cell lines. Both methods exhibited staining of the cell membrane and cytoplasm, but the strongest and most distinct signals were obtained using methanol fixation. Interestingly, cell-specific differences were also observed that appeared to be associated with levels of claudin 1 and E-cadherin as seen by Western blotting. Therefore, when evaluating cellular localization of the junction proteins claudin 1 and E-cadherin, expression level and cell type differences must be considered.
紧密连接膜蛋白 Claudin 1 和黏着连接蛋白 E-cadherin 在细胞间通讯和细胞信号转导中发挥着关键作用。因此，近年来癌症研究人员一直关注这些蛋白在癌症中的蛋白水平和分布情况。癌细胞对接触抑制的敏感性丧失和侵袭性的建立被认为是这些膜蛋白错误定位于细胞质的结果。然而，关于它们分布和水平的报道一直不一致。因此，确定这些蛋白细胞定位的技术必须既一致又可靠。本研究旨在确定 5 种不同的人乳腺癌细胞系中，免疫荧光法检测 Claudin 1 和 E-cadherin 的最佳固定方法，甲醇或福尔马林。这两种方法都显示出细胞膜和细胞质的染色，但甲醇固定得到的信号最强、最清晰。有趣的是，还观察到了细胞特异性差异，这些差异似乎与 Western blot 检测到的 Claudin 1 和 E-cadherin 的水平有关。因此，在评估连接蛋白 Claudin 1 和 E-cadherin 的细胞定位时，必须考虑表达水平和细胞类型的差异。
