Rinse with TBS, replace with methanol, store at -20oC. Rehydrate by slowly adding TBS over a ten minute period. Antibody is added either as a cell culture supernatent, in which case it is used either neat or diluted with blocking solution, or as ascites or polyclonal in which case it is diluted in blocking solution. The dilution depends on the antibody. Block as above 15 to 30'.
Secondary antibody is added in blocking solution.
Incubate at 4 degC overnight on Nutator.