Heat-induced antigen retrieval (HIAR) treatment improves the antigen immunodetection in formalin-fixed, paraffin-embedded tissue samples and it can also improve the detection of intracellular antigens in alcohol-fixed cytological samples, although it could deleteriously impact immunodetection, particularly that of membranous antigens. We examined the differences in cell surface topography on MCF7 cells fixed in methanol/acetone (M/A) or 4% paraformaldehyde (4% PFA), as well as the changes caused by HIAR treatment at three different temperatures (60, 90, and 120°C), using atomic force microscopy. Furthermore, the consequences for immunostaining of five membranous antigens [epidermal growth factor receptor (EGFR), E-cadherin, CD9, CD24, and CD44] were examined. Our results illustrate that while there was no one single optimal immunostaining condition for the tested antibodies, the surface topography could be an important factor in successful staining. Generally, the best conditions for successful immunostaining were M/A fixation with no HIAR treatment, whereas in 4% PFA-fixed cells, HIAR treatment at 120°C was optimal. These conditions showed similarity in cell surface skewness. A correlation factor between successful immunocytochemical staining and the skewness parameter was 0.8000. Our results indicate that the presence of valleys, depressions, scratches, and pits on the cell surface is unfavorable for the successful immunodetection of cell surface antigens.

中文翻译：

---

细胞膜纳米结构被热诱导的抗原检索改变：膜抗原的免疫细胞化学检测的可能后果。

热诱导抗原取回（HIAR）处理可改善福尔马林固定，石蜡包埋的组织样品中的抗原免疫检测，还可以改善酒精固定细胞学样品中细胞内抗原的检测，尽管它可能对免疫检测产生不利影响，尤其是对膜抗原。我们检查了固定在甲醇/丙酮（M / A）或4％多聚甲醛（4％PFA）中的MCF7细胞在细胞表面形貌上的差异，以及在三种不同温度（60、90和90°C）下HIAR处理引起的变化120°C），使用原子力显微镜。此外，检查了五个膜抗原[表皮生长因子受体（EGFR），E-钙粘蛋白，CD9，CD24和CD44]的免疫染色结果。我们的结果说明，尽管没有一种针对测试抗体的最佳免疫染色条件，但表面形貌可能是成功染色的重要因素。通常，成功进行免疫染色的最佳条件是不进行HIAR处理的M / A固定，而在4％PFA固定的细胞中，120°C的HIAR处理是最佳的。这些条件显示出细胞表面偏度的相似性。免疫细胞化学染色成功与偏度参数之间的相关因子为0.8000。我们的结果表明，在细胞表面存在谷，凹陷，划痕和凹坑不利于细胞表面抗原的成功免疫检测。成功进行免疫染色的最佳条件是不进行HIAR处理的M / A固定，而在4％PFA固定的细胞中，120°C的HIAR处理是最佳的。这些条件显示出细胞表面偏度的相似性。免疫细胞化学染色成功与偏度参数之间的相关因子为0.8000。我们的结果表明，在细胞表面存在谷，凹陷，划痕和凹坑不利于细胞表面抗原的成功免疫检测。成功进行免疫染色的最佳条件是不进行HIAR处理的M / A固定，而在4％PFA固定的细胞中，120°C的HIAR处理是最佳的。这些条件显示出细胞表面偏度的相似性。免疫细胞化学染色成功与偏度参数之间的相关因子为0.8000。我们的结果表明，在细胞表面存在谷，凹陷，划痕和凹坑不利于细胞表面抗原的成功免疫检测。