Sender S, Gros G, Waheed A, Hageman G S, Sly W S
Zentrum Physiologie, Medizinische Hochschule Hannover, Germany.
J Histochem Cytochem. 1994 Sep;42(9):1229-36. doi: 10.1177/42.9.8064130.
We used polyclonal antisera raised in rabbits against membrane-bound rat lung and human lung carbonic anhydrase (CA) IV in immunofluorescence studies to stain cryosections of rat soleus and extensor digitorum longus (EDL) and several human skeletal muscles. There was strong specific staining of capillaries in all muscles investigated. Several techniques were applied to verify this result. (a) Serial sections were either incubated with anti-CA IV/FITC or processed for endothelial ATPase reaction. There was precise co-localization of antibody marked structures and ATPase stained capillaries. (b) Human muscle sections were double stained with anti-CA IV/TRITC and anti-von Willebrand factor (vWF)/FITC. vWF, a capillary marker, and CA IV were localized at identical sites. (c) The CAIV was released from capillaries by treatment with phosphatidylinositol specific phospholipase C, suggesting that the enzyme is anchored to the endothelial cell membrane via a phosphatidylinositolglycan anchor. (d) A rat hindlimb was perfused with diluted antiserum. Cryosections of perfused soleus and EDL processed for anti-rabbit IgG/FITC staining showed clear fluorescence associated with capillaries, indicating that the antigen was accessible from the capillary lumen. (e) Immune complexes formed during antiserum perfusion as described in d were precipitated from muscle homogenates. SDS-PAGE followed by immunoblotting showed that the predominant portion of total muscle CA IV was bound in these complexes and therefore must be located intravascularly.
在免疫荧光研究中，我们使用了针对膜结合型大鼠肺和人肺碳酸酐酶（CA）IV免疫家兔制备的多克隆抗血清，对大鼠比目鱼肌、趾长伸肌（EDL）以及几块人类骨骼肌的冰冻切片进行染色。在所研究的所有肌肉中，毛细血管均呈现出强烈的特异性染色。我们应用了多种技术来验证这一结果。（a）连续切片要么用抗CA IV/FITC孵育，要么进行内皮ATP酶反应处理。抗体标记结构与ATP酶染色的毛细血管精确共定位。（b）人类肌肉切片用抗CA IV/TRITC和抗血管性血友病因子（vWF）/FITC进行双重染色。vWF作为一种毛细血管标记物，与CA IV定位在相同位点。（c）通过用磷脂酰肌醇特异性磷脂酶C处理，CAIV从毛细血管中释放出来，这表明该酶通过磷脂酰肌醇聚糖锚定在内皮细胞膜上。（d）用稀释的抗血清灌注大鼠后肢。灌注后的比目鱼肌和EDL的冰冻切片进行抗兔IgG/FITC染色，显示与毛细血管相关的清晰荧光，表明抗原可从毛细血管腔进入。（e）如d中所述，在抗血清灌注过程中形成的免疫复合物从肌肉匀浆中沉淀出来。SDS - PAGE随后进行免疫印迹分析表明，总肌肉CA IV的主要部分结合在这些复合物中，因此必定位于血管内。
