Systematic Veterinary Virology

PROCEDURE

• To the cell growth on cover slip add 1 ml of 3.7 % formalin in PBS solution to the dish. Allow to react at room temp for 1 minute.
• Take off the formalin/PBS and add 1ml of cold methanol (-20°C, kept in well sealed bottle in fridge). Allow to react for not more than 1 minute.
• Take off methanol and add 1ml of PBS, not letting the specimen dry out. To block nonspecific antibody binding can add ~10ml (=1%) of goat serum and can incubate for 30 minutes.
• Add primary antibody reagent (Serum). Incubate for 1 hour at room temp. (or can go at 37°C for 30 minutes to 1 hour or at 4°C overnight.
• Remove primary antibody and replace with 1 ml of PBS. Allow to react for 5-10 minutes, replace PBS and repeat twice, to give three washes in PBS.
• Add secondary antibody. These are fluorescently labeled Goat anti mouse or rabbit antibodies and are conjugated to FITC. Typically make 1:2,000 dilutions of these secondaries in PBS plus 1% goat serum, BSA or non fat milk carrier. Incubate for 1 hour at room temp or at 37°C for 30 minutes to 1 hour, or at 4°C overnight).
• Remove secondary antibody and replace with 1 ml of PBS. Allow to react for 5-10 minutes, replace PBS and repeat twice, to give three washes in PBS.
• Drop on one drop of Fisher mounting medium onto dish and apply 22mm square coverslip. View in the Fluorescent microscope.