Selective staining of large projection neurons by monoclonal antibody to a glycoprotein of PC12 cells.
A monoclonal antibody has been produced against glycoproteins prepared from PC12 cells. This antibody stains PC12 cells in a rimlike fashion, whether they are fixed or living. When neuronally differentiated PC12 cells are used, neurites as well as somata stain. In the presence of complement, the antibody is cytotoxic to PC12 cells. These lines of evidence suggest that at least some of the binding is to an exposed component of the plasma membrane in these cells. Biochemical experiments directed at identifying the antigen(s) indicate specific recognition of glycoproteins of apparent molecular weights of approximately 160 and 95 kDa by SDS-PAGE. The antibody appears to be directed against a determinant on the protein moiety. The larger species is likely to be the same as a glycoprotein previously described (Lee et al., 1981), which has been found in PC12 cells, but not in brain. The monoclonal antibody we have produced, however, selectively stains a subset of neurons in brain. Specifically, it binds to Purkinje cells in cerebellar cortex, pyramidal cells in cerebral cortex and hippocampus, ganglion cells in retina, and some neurons, generally large, in most brain structures examined. No glia or nonneural tissues stain. We suggest that the neurons recognized by this antibody may be the projection cells of each structure. However, in contrast to PC12 cells, the antigen(s) bound in this case appears to be predominantly internal. This interpretation is supported by the appearance of stained cells in tissue; by the failure of the antibody to stain live dissociated neurons from brain; and by its failure to lyse sympathetic neurons in the presence of complement. Western immunoblots show that there is glycoprotein present in brain that is recognized by the antibody, and that it is enriched in a crude microsomal fraction prepared from that tissue. This material migrates on SDS gels as two bands of 130 and 95 kDa.
P
P
Sajovic
E
E
Moraru
L A
LA
Greene
M L
ML
Shelanski
D000310:Adrenal Gland Neoplasms / Q000032:analysis*; D000818:Animals; D000911:Antibodies, Monoclonal; D002460:Cell Line; D003602:Cytotoxicity, Immunologic; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000276:immunology*; D007124:Immunoenzyme Techniques; D051379:Mice; D008854:Microscopy, Electron; D008970:Molecular Weight; D009474:Neurons / Q000166:cytology*; D010673:Pheochromocytoma / Q000032:analysis*; D011689:Purkinje Cells / Q000166:cytology; D011712:Pyramidal Tracts / Q000166:cytology; D051381:Rats; D013194:Staining and Labeling
D000911:Antibodies, Monoclonal; D006023:Glycoproteins