Visualization of the intact endoplasmic reticulum by immunofluorescence with antibodies to the major ER glycoprotein, endoplasmin.
Antibodies to endoplasmin were used to examine the morphology of the endoplasmic reticulum (ER) by immunofluorescence on permeabilized plasmacytoma and fibroblastoid cells. In unfixed cells, permeabilization led to a pronounced vesiculation of the ER. Therefore cells were first fixed lightly prior to permeabilization with detergent. Fibroblastoid cells gave a characteristic reticular pattern surrounding the nucleus with clear staining of the nuclear membrane. Plasmacytoma cells, in the conventional fluorescence microscope, gave a cisternae-like pattern. Optical sectioning with a confocal scanning laser microscope gave a distinct pattern of concentric cisternae similar to those obtained with transmission electron microscopy on cell sections. The overall morphology of the ER in such cells could be revealed by serial optical sectioning. Evidence was obtained that the ER does not undergo extensive vesiculation during mitosis in plasmacytoma cells. Using anti-endoplasmin immunofluorescence monitoring, conditions were developed for the retention of ER morphology in unfixed, permeabilized cells. These studies illustrate the value of endoplasmin as a general marker for the analysis of ER morphology in different types of cells by immunofluorescence microscopy.
Koch
G L
GL
Medical Research Council Laboratory of Molecular Biology, Cambridge, England.
Macer
D R
DR
Smith
M J
MJ
D000911:Antibodies, Monoclonal; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000648:ultrastructure*; D005455:Fluorescent Antibody Technique; D007834:Lasers; D008562:Membrane Glycoproteins / Q000276:immunology; D008856:Microscopy, Fluorescence; D012503:Saponins / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins; D012503:Saponins; C053387:endoplasmin