Immunolocalization, quantitation and cellular heterogeneity of apolipoprotein B in rat hepatocytes.
Hepatocyte autofluorescence represents a major problem in immunofluorescence studies with fluorescein conjugates because of significant spectral overlap. We describe a method for immunostaining hepatocytes with R-phycoerythrin (a fluorochrome with minimal overlap with autofluorescence) with paraformaldehyde fixation and Triton X-100 permeabilization for better antibody penetration. This method produced both perinuclear (presumed Golgi apparatus) and dispersed, reticular staining (presumed endoplasmic reticulum) in rat hepatocytes in culture stained with a monoclonal antibody to rat apolipoprotein B. Treatment with brefeldin A resulted in loss of apolipoprotein B perinuclear staining and increased reticular immunofluorescence consistent with known properties of brefeldin A (inhibition of protein transport within the secretory pathway by dissolution of Golgi bodies). This suggests that apolipoprotein B epitopes are present in both Golgi bodies and endoplasmic reticulum. To demonstrate the utility of the technique for quantitative studies, static cell cytofluorometry of brefeldin A-treated cells was performed, demonstrating increases in specific immunofluorescence of apolipoprotein B corresponding closely to results estimated by monoclonal antibody radioimmunoassays of cellular homogenates. The technique was then used with flow cytometry of single-cell suspensions of control rat hepatocytes derived from immunostained primary cultures to reveal cell-to-cell heterogeneity of apolipoprotein B epitope expression manifested as apolipoprotein B-negative and positive populations. Results for brefeldin A-treated cells revealed even clearer delineation of heterogeneity as indicated by frank bimodality of the populations, along with not only higher mean apolipoprotein B levels but also a significantly higher proportion of apolipoprotein B-positive cells than in the control.
Corsetti
J P
JP
Department of Pathology and Laboratory Medicine University of Rochester School of Medicine and Dentistry, New York 14642.
Way
B A
BA
Sparks
C E
CE
Sparks
J D
JD
D000818:Animals; D001055:Apolipoproteins B / Q000032:analysis / Q000378:metabolism*; D020126:Brefeldin A; D002478:Cells, Cultured; D003517:Cyclopentanes; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D010799:Phycoerythrin; D051381:Rats; D011919:Rats, Inbred Strains; D013194:Staining and Labeling / Q000379:methods
D001055:Apolipoproteins B; D003517:Cyclopentanes; D010799:Phycoerythrin; D020126:Brefeldin A