Galand P, Degraef C
Laboratory of Cytology and Experimental Cancerology, School of Medicine, Free University of Brussels, Belgium.
Cell Tissue Kinet. 1989 Sep;22(5):383-92. doi: 10.1111/j.1365-2184.1989.tb00223.x.
Paraffin sections from animal or human tissues fixed in different fixatives were submitted to immunostaining with the mouse monoclonal antibody 19A2, developed by Ogata et al. (1987a) against cyclin/PCNA. Detection of the bound antibody was performed by the indirect method with biotinylated sheep antibody and streptavidin-biotin-peroxidase complexes. No, or faint, nuclear staining was seen in material fixed in ethanol, Bouin, Bouin-Hollande, Carnoy or formaldehyde, whereas readily detectable immunocytochemical reaction was constantly observed over nuclei of methanol-fixed tissues. Hydrolysis with 2 N HCl prior to immunocytochemistry (as currently performed to render incorporated BrdU accessible to antibodies) somewhat improved the results with Bouin or Carnoy and markedly augmented the intensity of the peroxidase reactions in formaldehyde and in methanol-fixed tissues. The distribution of the positive nuclei in the two latter cases coincided with the proliferative compartment. On the other hand, double labelling with [3H]-thymidine and with the cyclin/PCNA antibody revealed that in methanol-fixed tissues the cyclin/PCNA labelling index did not differ by more than 6% from the [3H]-thymidine index. Besides the two labels overlapped in a proportion of labelled cells that was in reasonable agreement with expectation considering cells flow in and out of S phase since the time of [3H]-thymidine injection. This indicates that both labels recognize the same cells in this material. In contrast, in formaldehyde-fixed tissues, the cyclin/PCNA labelling index markedly exceeded the [3H]-thymidine labelling index. From this it is concluded that cyclin/PCNA immunostaining can be used: (1) In formaldehyde-fixed tissues (including existing material stored as paraffin blocks): for defining and mapping the proliferative (or germinative) compartment. (2) In methanol-fixed tissues as a substitute to the [3H]-thymidine autoradiographic labelling index. From this, a method is proposed (derived from classical 'double-labelling' technique) for measuring S phase duration in tissues fixed at a known interval time after a single labelling with [3H]-thymidine (or BrdU) and submitted to cyclin/PCNA immunocytochemical detection and to autoradiography (or to BrdU immunostaining).
将用不同固定剂固定的动物或人体组织的石蜡切片，用绪方等人（1987年a）研制的抗细胞周期蛋白/增殖细胞核抗原的小鼠单克隆抗体19A2进行免疫染色。结合抗体的检测采用间接法，使用生物素化羊抗体和链霉亲和素-生物素-过氧化物酶复合物。在乙醇、布安氏液、布安-奥兰德液、卡诺氏液或甲醛固定的材料中，未观察到或仅观察到微弱的核染色，而在甲醇固定的组织细胞核上，始终能观察到易于检测的免疫细胞化学反应。在免疫细胞化学之前用2N盐酸水解（目前用于使掺入的溴脱氧尿苷能被抗体识别），在一定程度上改善了布安氏液或卡诺氏液固定材料的结果，并显著增强了甲醛和甲醇固定组织中过氧化物酶反应的强度。后两种情况下阳性细胞核的分布与增殖区一致。另一方面，用[3H] - 胸腺嘧啶核苷和细胞周期蛋白/增殖细胞核抗原抗体进行双重标记显示，在甲醇固定的组织中，细胞周期蛋白/增殖细胞核抗原标记指数与[3H] - 胸腺嘧啶核苷指数相差不超过6%。此外，考虑到自注射[3H] - 胸腺嘧啶核苷以来细胞进出S期的情况，两种标记在一定比例的标记细胞中重叠，这与预期合理相符。这表明在这种材料中两种标记识别的是相同的细胞。相反，在甲醛固定的组织中，细胞周期蛋白/增殖细胞核抗原标记指数明显超过[3H] - 胸腺嘧啶核苷标记指数。由此得出结论，细胞周期蛋白/增殖细胞核抗原免疫染色可用于：（1）在甲醛固定的组织（包括作为石蜡块保存的现有材料）中：用于定义和绘制增殖（或生发）区。（2）在甲醇固定的组织中作为[3H] - 胸腺嘧啶核苷放射自显影标记指数的替代方法。据此，提出了一种（源自经典的“双重标记”技术）用于测量在单次用[3H] - 胸腺嘧啶核苷（或溴脱氧尿苷）标记并在已知间隔时间固定后，经细胞周期蛋白/增殖细胞核抗原免疫细胞化学检测和放射自显影（或溴脱氧尿苷免疫染色）的组织中S期持续时间的方法。
