Decker B, Sender S, Gros G
Abteilung Zellbiologie und Elektronenmikroskopie, Medizinische Hochschule Hannover, Germany.
Histochem Cell Biol. 1996 Oct;106(4):405-11. doi: 10.1007/BF02473299.
Carbonic anhydrase IV (CA IV) was examined by light microscopy and electron microscopy in rat soleus muscle. Semithin sections of aldehyde-fixed Epon-embedded muscle were stained with rabbit anti-rat lung CA IV and the avidin-biotin-peroxidase complex. With this technique, capillaries and sarcolemma showed positive CA IV staining. For electron microscopy, rat soleus specimens were aldehyde-fixed, with or without subsequent osmication, and embedded in Epon. Ultrathin sections were immunostained with anti-rat lung CA IV/immunogold. Omitting osmium allowed ample antigen-antibody reactions but could not prevent the release of glycosylphosphatidylinositol-anchored CA IV from the membranes, which led to apparent background staining. Postosmication significantly reduced tissue antigenicity but kept the antigen bound to the membranes and thus allowed a very precise localization of CA IV. By electron microscopy, membrane-bound CA IV is found to be associated with capillary endothelium, sarcolemma, and sarcoplasmic reticulum (SR). Conceivably, the presence of SR staining in ultrathin sections and its absence in semithin sections reflect a problem of accessibility of the antigenic sites.
运用光学显微镜和电子显微镜对大鼠比目鱼肌中的碳酸酐酶IV（CA IV）进行了检测。用兔抗大鼠肺CA IV和抗生物素蛋白-生物素-过氧化物酶复合物对醛固定、环氧树脂包埋的肌肉半薄切片进行染色。采用该技术，毛细血管和肌膜显示出CA IV阳性染色。对于电子显微镜检查，大鼠比目鱼肌标本经醛固定，有或没有随后的锇酸处理，然后包埋在环氧树脂中。超薄切片用抗大鼠肺CA IV/免疫金进行免疫染色。省略锇酸处理可使抗原-抗体充分反应，但无法阻止糖基磷脂酰肌醇锚定的CA IV从膜上释放，这导致了明显的背景染色。后锇酸处理显著降低了组织抗原性，但使抗原与膜结合，从而实现了CA IV的精确定位。通过电子显微镜观察，发现膜结合的CA IV与毛细血管内皮、肌膜和肌浆网（SR）相关。可以想象，超薄切片中存在SR染色而半薄切片中不存在，这反映了抗原位点可及性的问题。
