Dover R, Patel K
Department of Anatomy, Queen Mary and Westfield College, University of London, UK.
Histochemistry. 1994 Nov;102(5):383-7. doi: 10.1007/BF00268909.
Detection of DNA synthesizing cells may often be achieved by immunocytochemical detection of bromodeoxyuridine (BrdU), which is rapid and appears to give similar results to those found using tritiated thymidine. However, the methodology for detection of BrdU involves a denaturation or digestion step to allow access of the antibody to BrdU incorporated into single- rather than double-stranded DNA. We wished to determine if microwave treatment could be used to enhance the detection of BrdU without the need for any other digestion/denaturation steps. An important consideration was to investigate whether such treatment produces a similar quantitative result, since BrdU detection is usually assessed on the basis of cell number rather than topographical distribution. We have found that microwave treatment can allow considerably lower antibody concentrations and eliminates the need for any other denaturation step. It also reduces the non-specific background staining found when using monoclonal antibodies on mouse tissue. We have performed cell counts and found that the number of BrdU positive cells remains constant for a range of different immunocytochemical parameters. We also report conditions where immunopositivity is adversely affected by changes in technique and describe the optimised conditions for obtaining reproducible results.
DNA合成细胞的检测通常可通过免疫细胞化学方法检测溴脱氧尿苷（BrdU）来实现，这种方法快速，且似乎能给出与使用氚标记胸腺嘧啶核苷所得到的结果相似的结果。然而，检测BrdU的方法涉及变性或消化步骤，以使抗体能够接触到掺入单链而非双链DNA中的BrdU。我们希望确定是否可以使用微波处理来增强BrdU的检测，而无需任何其他消化/变性步骤。一个重要的考虑因素是研究这种处理是否会产生相似的定量结果，因为BrdU检测通常是基于细胞数量而非拓扑分布进行评估的。我们发现，微波处理可以允许使用低得多的抗体浓度，并且无需任何其他变性步骤。它还减少了在小鼠组织上使用单克隆抗体时出现的非特异性背景染色。我们进行了细胞计数，发现在一系列不同的免疫细胞化学参数下，BrdU阳性细胞的数量保持恒定。我们还报告了技术变化对免疫阳性产生不利影响的情况，并描述了获得可重复结果的优化条件。
