Riederer B M
Department of Physiology, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033.
J Histochem Cytochem. 1989 May;37(5):675-81. doi: 10.1177/37.5.2495321.
The effects of aldehyde fixatives on immunochemical detection of cytoskeletal proteins were demonstrated by applying several quantitative assays to evaluate antigen conservation. Immunologically detectable brain spectrin (240/235) was measured by dot-immunobinding and quantitative immunodot assay using a polyclonal antibody. Paraformaldehyde fixation led to a 43-66% reduction in brain spectrin (240/235) immunodetection, and increasing glutaraldehyde concentrations decreased the immunological detection even more. Quantitative cryosection immunoassay and immunocytochemical localization confirmed the aldehyde sensitivity of brain spectrin (240/235). Brain spectrin (240/235) immunoreactivity decreased with increasing protein crosslinking and was dependent on glutaraldehyde concentration and post-fixation period. The assays were also used to test for conservation of antigenicity of neurofilament proteins by two monoclonal antibodies. Neurofilament detection was abolished in brain tissue after aldehyde fixation. The described methods allow screening within 24 hr of many fixation conditions by use of purified proteins as well as brain tissue samples, and allow an estimate of fixative influence on the conservation of protein antigenicity.
通过应用几种定量测定方法来评估抗原保存情况，证明了醛类固定剂对细胞骨架蛋白免疫化学检测的影响。使用多克隆抗体，通过斑点免疫结合和定量免疫斑点测定法测量免疫可检测的脑血影蛋白（240/235）。多聚甲醛固定导致脑血影蛋白（240/235）免疫检测减少43%-66%，并且戊二醛浓度增加会使免疫检测减少得更多。定量冷冻切片免疫测定和免疫细胞化学定位证实了脑血影蛋白（240/235）对醛类的敏感性。随着蛋白质交联增加，脑血影蛋白（240/235）的免疫反应性降低，并且依赖于戊二醛浓度和后固定时间。这些测定方法还用于通过两种单克隆抗体测试神经丝蛋白抗原性的保存情况。醛类固定后，脑组织中的神经丝检测被消除。所述方法允许在24小时内通过使用纯化蛋白以及脑组织样本对许多固定条件进行筛选，并允许估计固定剂对蛋白质抗原性保存的影响。
