Herman I M, Pollard T D
J Cell Biol. 1979 Mar;80(3):509-20. doi: 10.1083/jcb.80.3.509.
We purified actin antibodies by affinity chromatography from the serum of rabbits immunized with glutaraldehyde-fixed chicken gizzard actin filaments and used this anti-actin to localize actin in myofibrils and fixed cultured cells at each stage of the cell cycle. By double immunodiffusion the anti-actin reacted with both smooth and skeletal muscle actin. Several blocking and absorption experiments demonstrated that the antibodies also bound specifically to actin in nonmuscle cells. The same structures stained using either the direct or the indirect fluorescent antibody technique; and, while the indirect method was more sensitive, the direct method was superior because there was no detectable nonspecific staining. As expected, anti-actin stained the I-band of myofibrils. It also stained stress fibers and membrane ruffles in HeLa cells. Some PtK-2 cells have straight stress fibers which stained with anti-actin, but in confluent cultures all PtK-2 cells have, instead, sinuous phase-dense fibers which stained with antibody. At prophase the whole cytoplasm stained uniformly with anti-actin. During metaphase and anaphase, anti-actin staining was concentrated diffusely in the mitotic spindle. In contrast, fluorescent heavy meromyosin stained discrete fine spindle fibers in these fixed cells. During cytokinesis, anti-actin stained the whole cytoplasm uniformly and was not concentrated in the cleavage furrow.
我们通过亲和层析从用戊二醛固定的鸡砂囊肌动蛋白丝免疫的兔血清中纯化肌动蛋白抗体，并使用这种抗肌动蛋白来定位细胞周期各阶段肌原纤维和固定培养细胞中的肌动蛋白。通过双向免疫扩散，抗肌动蛋白与平滑肌和骨骼肌肌动蛋白均发生反应。多项封闭和吸收实验表明，这些抗体也能特异性结合非肌肉细胞中的肌动蛋白。使用直接或间接荧光抗体技术均可对相同结构进行染色；虽然间接方法更灵敏，但直接方法更优，因为未检测到非特异性染色。正如预期的那样，抗肌动蛋白对肌原纤维的I带进行了染色。它还对HeLa细胞中的应力纤维和膜皱褶进行了染色。一些PtK - 2细胞有可被抗肌动蛋白染色的直应力纤维，但在汇合培养物中，所有PtK - 2细胞取而代之的是有可被抗体染色的蜿蜒的相致密纤维。在前期，整个细胞质被抗肌动蛋白均匀染色。在中期和后期，抗肌动蛋白染色分散地集中在有丝分裂纺锤体中。相比之下，荧光重酶解肌球蛋白对这些固定细胞中的离散细纺锤体纤维进行了染色。在胞质分裂期间，抗肌动蛋白均匀地对整个细胞质进行染色，且未集中在分裂沟中。
