The Opal multiplex technique is an established methodology for the detection of multiple biomarkers in one section. The protocol encompasses iterative single stainings and heating-mediated removal of the primary and secondary antibodies after each staining round, leaving untouched the Opal fluorophores which are deposited onto the antigen of interest. According to our experience, repetitive heating of skin sections often results in tissue damage, indicating an urgent need for milder alternatives to strip immunoglobulins. In this study, we demonstrate that considerable heating-related damage was found not only in skin but also in tissues of different origin, mostly characterized by low cell density. Importantly, the morphology remained fully intact when sections were repetitively exposed to β-mercaptoethanol-containing stripping buffer instead of multiple heating cycles. However, target epitopes appeared sensitive at a differential degree to multiple treatments with stripping buffer, as shown by loss in staining intensity, but in all cases, the staining intensity could be restored by increment of the primary antibody concentrations. Application of β-mercaptoethanol-containing stripping buffer instead of heating for antibody removal markedly improved the quality of the Opal multiplex technique, as a substantial higher number of differently colored cells could be visualized within a well-conserved morphological context:

蛋白石多重技术是一种用于在一个区域中检测多种生物标志物的既定方法。该方案包括迭代的单次染色，以及在每个染色回合后加热介导的一级抗体和二级抗体的去除，使蛋白石荧光蛋白原样保留在感兴趣的抗原上。根据我们的经验，皮肤切片的反复加热通常会导致组织损伤，这表明迫切需要更温和的替代方法来剥离免疫球蛋白。在这项研究中，我们证明不仅在皮肤中而且在不同来源的组织中都发现了与加热相关的相当大的损害，其主要特征是细胞密度低。重要的，当切片重复暴露于含β-巯基乙醇的剥离缓冲液中而不是多次加热循环时，形态仍保持完整。但是，目标表位似乎对脱膜缓冲液的多次处理有不同程度的敏感性，如染色强度的损失所示，但在所有情况下，可以通过增加一抗浓度来恢复染色强度。应用含β-巯基乙醇的剥离缓冲液代替加热来去除抗体可显着提高Opal多重技术的质量，因为在良好保存的形态学背景下可以看到大量不同颜色的细胞：如染色强度的损失所示，但是在所有情况下，可以通过增加一抗浓度来恢复染色强度。应用含β-巯基乙醇的剥离缓冲液代替加热来去除抗体可显着提高Opal多重技术的质量，因为在良好保存的形态学背景下可以看到大量不同颜色的细胞：如染色强度的损失所示，但是在所有情况下，可以通过增加一抗浓度来恢复染色强度。应用含β-巯基乙醇的剥离缓冲液代替加热来去除抗体可显着提高Opal多重技术的质量，因为在良好保存的形态学背景下可以看到大量不同颜色的细胞：