Antoine J C, Avrameas S, Gonatas N K, Stieber A, Gonatas J O
J Cell Biol. 1974 Oct;63(1):12-23. doi: 10.1083/jcb.63.1.12.
Normal rat and mouse lymphoid cells were incubated at 0 degrees -4 degrees C for 1 h with purified rabbit or sheep antirat (mouse) immunoglobulin (Ig)-horseradish peroxidase (PO) conjugates or with Fab fragments of antibody coupled with peroxidase. Cells were subsequently washed and incubated in fresh medium, without labeled antibody or Fab fragments for 5-30 min at 20 degrees or 37 degrees C. With the use of the diaminobenzidine (DAB) method, distribution of peroxidase was studied in the light and electron microscopes. Fab fragments of antirat Ig antibody were iodinated with (125)I and subsequently coupled with horseradish PO. Plasma membrane and internalized immunoglobulins were detected by electron microscope autoradiography and peroxidase cytochemistry. Single- (Fab-PO), and double- ([(125)I]Fab-PO) labeled lymphoid cells showed identical patterns of surface or internal distribution of immunoglobulins. In the electron microscope, Fab-PO conjugates at 0 degrees -4 degrees C resulted in a diffuse specific staining of the plasmalemma of lymphocytes and plasma cells. Most of the small dark lymphocytes (T cells?) did not show plasma membrane Ig. Macrophages did not show plasmalemma staining, but displayed nonspecific cytoplasmic staining after incubation at 20 degrees or 37 degrees C with antibody or Fab-PO conjugates. Lymphocytes and plasma cells, after incubation with antibody-PO conjugates at 0 degrees -4 degrees C, had patchy deposits of oxidized DAB on their plasma membranes. Macrophages, similarly treated, had no plasmalemmal staining. Patch and cap formation on the plasma membrane of lymphocytes and plasma cells was seen regularly after antibody-PO incubation at 37 degrees C. Internalization patterns were different in lymphocytes and plasma cells. In lymphocytes, peroxidase staining was observed in small round or oval vesicles clustered at one pole of the cell (30 min at 37 degrees C). In plasma cells, peroxidase staining was seen in clusters of tubules resembling the Golgi apparatus. Internalization of plasma membrane IgG was less pronounced after antibody-PO labeling as compared to Fab-PO labeling.
将正常大鼠和小鼠的淋巴细胞在0℃-4℃下与纯化的兔或羊抗大鼠（小鼠）免疫球蛋白（Ig）-辣根过氧化物酶（PO）偶联物或与与过氧化物酶偶联的抗体Fab片段一起孵育1小时。随后洗涤细胞，并在新鲜培养基中于20℃或37℃下孵育5-30分钟，不添加标记抗体或Fab片段。使用二氨基联苯胺（DAB）方法，在光学显微镜和电子显微镜下研究过氧化物酶的分布。抗大鼠Ig抗体的Fab片段用（125）I进行碘化，随后与辣根PO偶联。通过电子显微镜放射自显影和过氧化物酶细胞化学检测质膜和内化的免疫球蛋白。单标记（Fab-PO）和双标记（[(125)I]Fab-PO）的淋巴细胞显示出免疫球蛋白在表面或内部分布的相同模式。在电子显微镜下，0℃-4℃下的Fab-PO偶联物导致淋巴细胞和浆细胞质膜的弥漫性特异性染色。大多数小的深色淋巴细胞（T细胞？）未显示质膜Ig。巨噬细胞未显示质膜染色，但在20℃或37℃下与抗体或Fab-PO偶联物孵育后显示非特异性细胞质染色。淋巴细胞和浆细胞在0℃-4℃下与抗体-PO偶联物孵育后，其质膜上有氧化DAB的斑块状沉积。同样处理的巨噬细胞没有质膜染色。在37℃下用抗体-PO孵育后，淋巴细胞和浆细胞质膜上经常可见斑块和帽状形成。淋巴细胞和浆细胞的内化模式不同。在淋巴细胞中，在细胞一极聚集的小圆形或椭圆形小泡中观察到过氧化物酶染色（37℃下30分钟）。在浆细胞中，在类似于高尔基体的小管簇中可见过氧化物酶染色。与Fab-PO标记相比，抗体-PO标记后质膜IgG的内化不太明显。
