Influence of cell fixatives and incubation schedule on PAP labeling of fibronectin in cultivated fibroblasts.
A simple combined method for differential PAP labeling of fibronectin (FN) in mouse embryo fibroblast cultures was developed. Methanol-5% glacial acetic acid in dry ice-fixed cell monolayers showed mainly intracellular FN staining. Fixation with neutral paraformaldehyde before labeling, developed membrane- and extracellular matrix-associated FN. A combination of both procedures, which required incubation with primary antibody, fixation with paraformaldehyde followed by chilled acid methanol, and re-incubation with primary antibody, yielded sharp intracellular and extracellular FN labeling. The outlined methods can be readily employed in association with other staining techniques.
D000818:Animals; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000648:ultrastructure; D005353:Fibronectins / Q000378:metabolism*; D005404:Fixatives; D007124:Immunoenzyme Techniques; D051379:Mice; D013997:Time Factors
D005353:Fibronectins; D005404:Fixatives