Immunocytochemical visualization of the Golgi apparatus in several species, including human, and tissues with an antiserum against MG-160, a sialoglycoprotein of rat Golgi apparatus.

Abstract

We used a monoclonal antibody (10A8), derived from mice immunized with fractions enriched in Golgi apparatus of rat brain neurons, to isolate an intrinsic membrane sialoglycoprotein of 160 KD from rat brain. By immunoelectron microscopy the sialoglycoprotein, named MG-160, was localized in medical cisternae of the Golgi apparatus of neurons, glia, adenohypophysis, and cultured rat pheochromocytoma (PC 12). The monoclonal antibody (MAb) reacted only with rat tissues. Because the epitope(s) recognized by a monoclonal antibody may be restricted, localization of an antigen by a single MAb may not reflect the extent of the distribution of antigen in various species and tissues. Therefore, to further investigate the presence and localization of MG-160 or of an antigenically related protein in several species and tissues, we used a polyclonal antiserum raised against MG-160 purified by antibody (10A8) affinity chromatography. Immunoblots of crude microsomal fractions from rat brain probed with the antiserum against MG-160 showed two to three prominent bands of approximately 160, 150, and 68 KD. Immunoblots of crude microsomal fractions from human, chicken, and frog brains showed prominent bands of 130-140 and 68 KD. Immunoblots of crude membrane fractions from Saccharomyces cerevisiae showed prominent bands of approximately 110-120 and 80 KD. Light microscopic immunocytochemical studies with frog, chicken, mouse, rat, rabbit, bovine, and human brains and with several other rat and human tissues showed a staining pattern consistent with the Golgi apparatus. Immunoelectron microscopy with rat and human brain and with rat myocardium and pituitary showed prominent and exclusive staining of cis, medial, and occasionally trans cisternae of the Golgi apparatus. The cisternae of the trans Golgi network were not stained. These findings are consistent with the hypothesis that a polypeptide related to MG-160 is present in the Golgi apparatus of several tissues in human, rodents, chicken, and frog and possibly in Saccharomyces cerevisiae. The antiserum to MG-160 represents a reliable reagent for immunohistochemical visualization of the Golgi apparatus in brain and several other human tissues obtained at autopsy, fixed with Bouin's, and embedded in paraffin.

The Brefeldin A-induced Retrograde Transport from the Golgi Apparatus to the Endoplasmic Reticulum Depends on Calcium Sequestered to Intracellular Stores

“…This is demonstrated by immunofluorescence (Fig. 3A) using an antibody to MG-160, a sialoglycoprotein of the medial cisternae of the Golgi apparatus (Croul et al, 1990;Gonatas et al, 1989). As in the case with other cell types , treatment of HeLa cells with BFA for 30 (D) or 60 (G) min leads to the redistribution of the Golgi marker to the ER, as indicated by the reticular fluorescence pattern of the cytoplasm obtained with the antibody to MG-160.…”

“…Antibodies-A polyclonal rabbit anti-rat ribophorin I antibody has been described previously (Marcantonio et al, 1984;Yu et al, 1990;Tsao et al, 1992). A polyclonal rabbit anti-MG-160 antibody that reacts with a 160-kDa membrane sialoglycoprotein located mainly in the medial cisternae of the Golgi apparatus (Gonatas et al, 1989;Croul et al, 1990;De Lemos-Chiarandini et al, 1992), was generously provided by Dr. N. K. Gonatas (University of Pennsylvania School of Medicine, Philadelphia, PA). The polyclonal rabbit anti-bovine galactosyltransferase antibody, a marker for the trans cisternae of the Golgi apparatus (Roth and Berger, 1982;Eckstein and Shur, 1989;Tooze and Hollinshead, 1992) was a gift from Dr. B. D. Shur (University of Texas, M. D. Anderson Cancer Center, Houston, TX).…”

The Golgi apparatus of spinal cord motor neurons in transgenic mice expressing mutant Cu,Zn superoxide dismutase becomes fragmented in early, preclinical stages of the disease.

“…Perfusion was continued with -50 ml of freshly prepared 1% paraformaldehyde in PBS. The spinal cord was removed, cut into 2-to 3-mm cross sections, kept in the same fixative at room temperature for 6-8 hr, and processed as previously described (32).…”

Developmental regulation of membrane traffic organization during synaptogenesis in mouse diaphragm muscle.

“…The medial-Golgi cisternae were labeled with either an affinity-purified rabbit antibody raised against a 160-kD medial-Golgi sialoglycoprotein (MG-160) (Gonatas et al, 1989;Croul et al, 1990), or with an affinity-purified rabbit antibody to ¢t-mannosidase II (Moremen and Touster, 1985). The TGN was labeled using an affinitypurified rabbit antibody against TGN38, an integral membrane protein of the trans-Golgi network (Luzio et al, 1990).…”