Beier K
Institut für Anatomie und Zellbiologie II, Universität Heidelberg, Federal Republic of Germany.
J Histochem Cytochem. 1992 Jan;40(1):115-21. doi: 10.1177/40.1.1370307.
The feasibility of light microscopic post-embedding immunocytochemistry for morphometry of peroxisomes using automatic image analysis was investigated and compared with the classical alkaline DAB method. Perfusion-fixed rat liver tissue was either embedded in LR White or incubated in the alkaline diaminobenzidine (DAB) medium for cytochemical visualization of catalase. Sections from the LR White-embedded material were incubated with a monospecific antibody against catalase, followed by protein A-gold and silver intensification. Determination of peroxisomal volume density in sections of different thickness revealed that the values increased with section thickness in DAB-stained sections but were unaffected in immunostained preparations. Moreover, the absolute value for volume density of peroxisomes, as determined by light microscopy in immunostained sections, was quite close to the value obtained by analysis of electron microscopic preparations. Finally, morphometric analysis of bezafibrate-induced peroxisome proliferation revealed that the ratio of proliferation obtained by light microscopy in immunostained sections was very close to the results obtained by electron microscopic morphometry. The main advantage of post-embedding immunostaining for light microscopic morphometry is that it restricts the immunocytochemical reaction product to the surface of the section, thus making it independent of section thickness.
研究了采用自动图像分析技术对过氧化物酶体进行形态计量的光镜包埋后免疫细胞化学方法的可行性，并与经典的碱性二氨基联苯胺（DAB）方法进行了比较。将灌注固定的大鼠肝脏组织包埋于LR White中，或置于碱性二氨基联苯胺（DAB）培养基中孵育，以进行过氧化氢酶的细胞化学可视化。将LR White包埋材料制成的切片与抗过氧化氢酶单特异性抗体孵育，随后进行蛋白A-金和银增强。对不同厚度切片中过氧化物酶体体积密度的测定表明，在DAB染色切片中，该值随切片厚度增加而升高，但在免疫染色标本中不受影响。此外，通过光镜在免疫染色切片中测定的过氧化物酶体体积密度绝对值，与通过电子显微镜标本分析获得的值非常接近。最后，对苯扎贝特诱导的过氧化物酶体增殖的形态计量分析表明，通过光镜在免疫染色切片中获得的增殖率与通过电子显微镜形态计量获得的结果非常接近。包埋后免疫染色用于光镜形态计量的主要优点是，它将免疫细胞化学反应产物限制在切片表面，从而使其与切片厚度无关。
