Immunoblotting of keratin polypeptides extracted from tissues preserved in standard histologic fixatives.
Cytoskeletal polypeptides from fresh placental tissue, tissue stored at -30 degrees C, and tissue fixed in 10% buffered formalin, Bouin's solution, and Carnoy's solution were extracted, separated by electrophoresis, and immunoblotted using monoclonal antibodies immunoreactive with keratin polypeptides. Storage of the placental tissue at -30 degrees C, or fixation in Carnoy's solution did not alter the extractability, migration pattern, or immunoreactivity of the keratin polypeptides. Keratin polypeptides could not be adequately demonstrated in extracts prepared from formalin- or Bouin's solution-fixed tissues. Several unmasking procedures used on tissues before extraction and on nitrocellulose blots before application of primary antibodies failed to unmask keratin polypeptides, either in Coomassie blue-stained gels or in immunoblots reacted with anti-keratin antibodies. These data indicate that Carnoy's solution is the fixative of choice for tissues in which electrophoretic and immunoblotting analyses of keratin polypeptides might be required.
R K
RK
Clark
I
I
Damjanov
D000911:Antibodies, Monoclonal; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007163:Immunosorbent Techniques; D007633:Keratins / Q000032:analysis*; D008722:Methods; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis*; D010920:Placenta / Q000032:analysis; D011247:Pregnancy; D014021:Tissue Preservation
D000911:Antibodies, Monoclonal; D010455:Peptides; D007633:Keratins