Shi S R, Imam S A, Young L, Cote R J, Taylor C R
Department of Pathology, University of Southern California School of Medicine, Los Angeles 90033.
J Histochem Cytochem. 1995 Feb;43(2):193-201. doi: 10.1177/43.2.7822775.
Antigen retrieval (AR) incorporating high-temperature microwave (MW) heating of tissue sections before immunostaining is a revolutionary technique that can unmask the antigens in formalin-fixed tissue sections, thus making them available for immunohistochemical staining. Although high temperature is believed to be the primary mechanism in retrieval of antigens, a variety of chemical solutions have been tested to define an optimal AR solution. We tested the hypothesis that pH of the AR solution may influence the quality of immunostaining by using seven different AR buffer solutions at a series of different pH values ranging from 1 to 10. We evaluated the staining of monoclonal antibodies to cytoplasmic antigens (AE1, HMB45, NSE), nuclear antigens (MIB-1, PCNA, ER), and cell surface antigens (MT1, L26, EMA) on routinely formalin-fixed, paraffin-embedded sections under different pH conditions with MW heating for 10 min. The intensity of immunostaining was graded in a blinded fashion. The pH value of the AR buffer solution was carefully measured before, immediately after, and 15 min after the AR procedure. The influence of pH on AR immunohistochemical staining can be summarized into three patterns. Some antigens (L26, PCNA, AE1, EMA, and NSE) showed excellent retrieval throughout the pH range. Other antigens (MIB1 and ER) showed strong intensity of immunohistochemical staining at very low pH and at neutral to high pH, but a dramatic decrease in the intensity of the AR immunostaining at moderately acidic pH (pH 3-6). Still others (MT1 and HMB45) showed increasing intensity of the AR immunostaining with increasing pH, but only weak immunostaining at low pH. Among the seven buffer solutions at any given pH value, the intensity of AR immunostaining was very similar. However, Tris-HCl buffer tended to produce better results at higher pH, compared with other buffers. Although high-temperature heating is believed to be the most important factor for the AR technique, the pH value of the AR solution is an important co-factor for some antigens. Optimization of the AR system should therefore include optimization of the pH of the AR solution. Our results indicate that AR immunostaining of Tris-HCl or sodium acetate buffer at pH 8-9 may be suitable for most antigens, although certain nuclear antigens show optimal staining at low pH.
抗原修复（AR）是一种革命性技术，即在免疫染色前对组织切片进行高温微波（MW）加热，它可以使福尔马林固定的组织切片中的抗原暴露出来，从而使其可用于免疫组织化学染色。尽管高温被认为是抗原修复的主要机制，但人们已经测试了多种化学溶液以确定最佳的AR溶液。我们通过使用七种不同的AR缓冲溶液，在一系列从1到10的不同pH值下，检验了AR溶液的pH值可能影响免疫染色质量这一假设。我们评估了针对细胞质抗原（AE1、HMB45、NSE）、核抗原（MIB-1、PCNA、ER）和细胞表面抗原（MT1、L26、EMA）的单克隆抗体在常规福尔马林固定、石蜡包埋切片上，在不同pH条件下经MW加热10分钟后的染色情况。免疫染色强度采用盲法分级。在AR程序进行前、刚结束后以及结束15分钟后，仔细测量AR缓冲溶液的pH值。pH值对AR免疫组织化学染色的影响可归纳为三种模式。一些抗原（L26、PCNA、AE1、EMA和NSE）在整个pH范围内都显示出良好的修复效果。其他抗原（MIB1和ER）在极低pH值以及中性至高pH值时显示出强烈的免疫组织化学染色强度，但在中等酸性pH值（pH 3 - 6）时，AR免疫染色强度急剧下降。还有一些抗原（MT1和HMB45）显示出随着pH值升高，AR免疫染色强度增加，但在低pH值时只有微弱的免疫染色。在任何给定pH值的七种缓冲溶液中，AR免疫染色强度非常相似。然而，与其他缓冲液相比，Tris - HCl缓冲液在较高pH值时往往能产生更好的结果。尽管高温加热被认为是AR技术最重要的因素，但AR溶液的pH值对于某些抗原来说是一个重要的辅助因素。因此，AR系统的优化应包括AR溶液pH值的优化。我们的结果表明，pH 8 - 9的Tris - HCl或醋酸钠缓冲液的AR免疫染色可能适用于大多数抗原，尽管某些核抗原在低pH值时显示出最佳染色效果。
