Kann M L, Fouquet J P
Groupe d'Etude de la Formation et de la Maturation du Gamète Mâle, Laboratoire d'Histologie, Paris, France.
Histochemistry. 1989;91(3):221-6. doi: 10.1007/BF00490136.
The efficiency of various postembedding procedures for actin immunogold detection was compared using testicular tissue as a model. Whatever the fixative, testes embedded in LR White resin or in Lowicryl K4M showed few differences as regard ultrastructural preservation and gave similar actin antigenicity preservation. A purified polyclonal antibody (IgG) and a monoclonal antibody (IgM) visualized with gold secondary antibody yielded high labeling intensity whereas the IgG-protein-A gold association was less efficient. Crude antisera gave a low specific staining/background ratio. Samples of testes, fixed in different conditions, were also embedded in Epon, omitting propylene oxide and lowering polymerization temperature to 40 degrees-50 degrees C. This slight modification improved ultrastructural preservation which was better than with hydrophilic resins, as well as made possible immunogold detection of actin though antigenicity preservation was lesser than with these resins. Thus, in Epon embedded samples actin labeling, using IgG antiactin-gold secondary antibody, was similar to that observed after hydrophilic resin-protein-A gold procedures. In addition to actin labeling of various somatic cells it was confirmed that actin is a consistent component of the subacrosomal space of spermatids during the greater part of spermiogenesis in rat.
以睾丸组织为模型，比较了各种用于肌动蛋白免疫金检测的包埋后程序的效率。无论使用何种固定剂，包埋在LR White树脂或Lowicryl K4M中的睾丸在超微结构保存方面几乎没有差异，并且肌动蛋白抗原性保存情况相似。用金标二抗显色的纯化多克隆抗体（IgG）和单克隆抗体（IgM）产生了高标记强度，而IgG-蛋白A金复合物的效率较低。粗抗血清的特异性染色/背景比值较低。将在不同条件下固定的睾丸样本也包埋在Epon中，省略环氧丙烷并将聚合温度降至40℃-50℃。这种微小的改进提高了超微结构的保存效果，比亲水性树脂更好，并且尽管抗原性保存不如这些树脂，但仍能进行肌动蛋白的免疫金检测。因此，在Epon包埋的样本中，使用IgG抗肌动蛋白-金标二抗进行的肌动蛋白标记与亲水性树脂-蛋白A金程序后观察到的标记相似。除了各种体细胞的肌动蛋白标记外，还证实了在大鼠精子发生的大部分过程中，肌动蛋白是精子细胞顶体下间隙的一个恒定成分。
