Immunocytochemistry (cytospin preparations),
Immunohistochemistry (frozen sections, paraffin sections):
Adherent cells grown on coverslips or glass slides, cytospin preparations, frozen sections or paraffin-embedded materials may be used. Permeabilization of the cell membrane is a pre-requisite for exposing the epitope recognized by C494.
Frozen sections of cytospin should be air-dried and fixed in acetone for 10 minutes at -20°C.
Paraffin sections should be deparaffinized and rehydrated by conventional means. Secondary antibodies and fluorescent or histochemical detection systems available from other suppliers should be titered for optimal staining. Primary antibody may be diluted to >1:25 for biotin based detection systems. For optimal staining the primary antibody shold be incubated 20-60 minutes at room temperature.

Immunoprecipitation:
The C494 MAb can be used to immunoprecipitate P-glycoprotein from lysates of metabolically or surface radiolabelled cells.

Western Blot:
Protein concentration of specimens used should be in the range of  50-150µg per 50µl. There are many commercially available systems which may be used for direct Western blot. The MAb antibody should be used at a concentration of 1-10µl/ml in indirect Western blots. Exact concentration of antibody and incubation time will vary depending on the particular system used.

Optimal conditions must be determined individually for each application.