Mrini A, Moukhles H, Jacomy H, Bosler O, Doucet G
Département de Pathologie, Université de Montréal, Québec, Canada.
J Histochem Cytochem. 1995 Dec;43(12):1285-91. doi: 10.1177/43.12.8537644.
Optimal ultrastructural preservation of brain tissue for electron microscopy is best achieved with fixatives containing high concentrations of glutaraldehyde, which is generally considered detrimental to the immunogenicity of most protein antigens. We tested seventeen mono- or polyclonal antibodies against peptide or protein antigens, including a majority for which immunoreactivity had previously been reported to be sensitive to glutaraldehyde fixation. Forebrain sections of rats or mice fixed by perfusion with 3.5% glutaraldehyde were processed for pre-embedding immunocytochemistry by the avidin-biotin method. The resulting immunostaining was in most cases at least similar to that obtained in sections fixed with paraformaldehyde. Immunoreactivity against the mouse or human neurofilament protein NF-L was even improved, being similar to that previously reported for unfixed brain tissue. Of all antigens tested, only choline acetyltransferase, phenylethanolamine-N-methyl transferase, and neuropeptide Y were detected with lower sensitivity than after paraformaldehyde fixation, which was attributed to a rather restricted penetration of the primary antibody into glutaraldehyde-fixed tissue sections. These results indicate that glutaraldehyde may be envisaged as a possible fixative for optimal immunocytochemical detection of any tissue antigen at the electron microscopic level, including antigens which, on the basis of results obtained after fixation with paraformaldehyde-glutaraldehyde mixtures, were considered highly sensitive to glutaraldehyde fixation.
对于电子显微镜检查而言，使用含有高浓度戊二醛的固定剂能最佳地实现脑组织的超微结构保存，而戊二醛通常被认为对大多数蛋白质抗原的免疫原性有害。我们测试了针对肽或蛋白质抗原的17种单克隆或多克隆抗体，其中大多数抗体的免疫反应性此前据报道对戊二醛固定敏感。用3.5%戊二醛灌注固定的大鼠或小鼠前脑切片，采用抗生物素蛋白-生物素方法进行包埋前免疫细胞化学处理。在大多数情况下，所得免疫染色至少与用多聚甲醛固定的切片中获得的免疫染色相似。针对小鼠或人类神经丝蛋白NF-L的免疫反应性甚至有所改善，与先前报道的未固定脑组织的免疫反应性相似。在所有测试的抗原中，只有胆碱乙酰转移酶、苯乙醇胺-N-甲基转移酶和神经肽Y的检测灵敏度低于多聚甲醛固定后，这归因于一抗对戊二醛固定的组织切片的穿透相当有限。这些结果表明，戊二醛可被设想为在电子显微镜水平对任何组织抗原进行最佳免疫细胞化学检测的一种可能的固定剂，包括那些基于多聚甲醛-戊二醛混合物固定后获得的结果而被认为对戊二醛固定高度敏感的抗原。
