Brandon C, Criswell M H
Department of Cell Biology and Anatomy, Chicago Medical School, Illinois 60064.
J Histochem Cytochem. 1991 Nov;39(11):1547-53. doi: 10.1177/39.11.1918929.
We present a new method for the preparation of antisera to Lucifer Yellow, and these antisera are here shown to be particularly suitable for immunocytochemical localization of multiple dye-injected cells in large pieces of vertebrate retina. The method involves the preparation of covalent conjugates of the VS isomer of Lucifer Yellow with keyhole limpet hemocyanin (KLH) or rabbit serum albumin (RSA), and their use as immunogens in rabbits. Both carrier protein conjugates yielded robust antibody responses. Antiserum to the KLH-LY conjugate contained precipitating antibodies against LY and KLH, although activity to the latter did not interfere with immunocytochemical staining. Rabbit antiserum to the RSA-LY conjugate contained precipitating antibody only against LY. When used for immunocytochemical staining of large retinal pieces containing many LY-filled cells, both antisera yielded well-stained, darkly filled cells similar to those seen with the Golgi technique; even very fine dendritic processes of retinal ganglion cells could be followed for long distances. LY immunocytochemistry provides a useful alternative to photooxidation for the analysis of multiple dye injected cells, especially in whole mounts. This approach may also be useful for immunocytochemical identification of cells filled with LY after tissue fixation.
我们提出了一种制备抗路西法黄抗血清的新方法，并且在此表明这些抗血清特别适用于对大片脊椎动物视网膜中多个注射了染料的细胞进行免疫细胞化学定位。该方法包括制备路西法黄的VS异构体与钥孔血蓝蛋白（KLH）或兔血清白蛋白（RSA）的共价缀合物，并将它们用作兔的免疫原。两种载体蛋白缀合物均产生了强烈的抗体反应。针对KLH-LY缀合物的抗血清含有针对路西法黄和KLH的沉淀抗体，尽管对后者的活性并不干扰免疫细胞化学染色。针对RSA-LY缀合物的兔抗血清仅含有针对路西法黄的沉淀抗体。当用于对含有许多充满路西法黄的细胞的大片视网膜进行免疫细胞化学染色时，两种抗血清都能产生染色良好、充满深色的细胞，类似于用高尔基技术观察到的细胞；甚至视网膜神经节细胞非常细小的树突状突起也能被追踪很长距离。路西法黄免疫细胞化学为分析多个注射了染料的细胞提供了一种有用的替代光氧化的方法，特别是在整装标本中。这种方法对于组织固定后对充满路西法黄的细胞进行免疫细胞化学鉴定也可能有用。
