Mechanically isolated hepatocytes are unsuitable to detect antibodies directed against plasma membrane determinants.
The ultrastructural morphology of mechanically and enzymatically isolated hepatocytes was compared. Plasma membrane integrity was evaluated by electron microscopy, both in the absence and in the presence of ruthenium red, which stains cell coat glycoproteins and makes it possible to identify even partially permeable cells, and by indirect immunofluorescence using experimental antibodies directed against epithelial submembranous components (prekeratin, actin). In order to evaluate the antigenic integrity of the hepatocellular plasma membrane after the isolation procedures, an immuno-electronmicroscopical technique (Staph. pA-colloidal gold) was applied using the IgG fraction from two liver-kidney microsomal antibody (LKM) positive sera, known to react with both plasma membranes and smooth endoplasmic reticulum (SER). Enzymatic procedure made it possible to isolate hepatocytes with a good preservation of both plasma membranes and subcellular organelles. Ruthenium red staining was confined to plasma membranes, thus indicating the preservation of both cell-coat glycoproteins and plasma membrane. Most cells were negative after exposure to experimental antibodies. After exposure to LKM positive sera colloidal gold was strictly confined to the plasma membrane. On the other hand, mechanically isolated hepatocytes showed wide interruptions of the plasma membrane and gross alterations of subcellular organelles. Most cells were stained by ruthenium red, thus confirming the plasma membrane permeability. In addition, a linear peripheral positivity was found in the vast majority of the cells tested with anti-prekeratin and anti-actin antibodies. LKM-colloidal gold complexes were found at the level of both residual plasma membrane and SER.(ABSTRACT TRUNCATED AT 250 WORDS)
M
M
Lenzi
P
P
Preda
F B
FB
Bianchi
G
G
Biagini
F
F
Cassani
U
U
Volta
E
E
Pisi
D000199:Actins / Q000276:immunology; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D002462:Cell Membrane / Q000276:immunology*; D002463:Cell Membrane Permeability; D002469:Cell Separation / Q000379:methods; D004721:Endoplasmic Reticulum / Q000276:immunology; D005455:Fluorescent Antibody Technique; D006651:Histocytochemistry / Q000379:methods; D007633:Keratins / Q000276:immunology; D008099:Liver / Q000166:cytology / Q000276:immunology* / Q000648:ultrastructure; D008297:Male; D051379:Mice; D011817:Rabbits; D051381:Rats; D012430:Ruthenium Red; D014343:Trypan Blue
D000199:Actins; D000906:Antibodies; D012430:Ruthenium Red; D007633:Keratins; D014343:Trypan Blue