Mounting solution
Dissolve 100 mg of p-phenylenediamine in 10 ml PBS, adjust the pH to above 8.0 with 0.5 M sodium carbonate buffer (pH 9.0), and bring the volume to 100 ml with glycerol. Add DAPI to 50 ng/ml. Mix thoroughly and store at -80°°C. When the mount gets old, it turns dark brown and becomes autofluorescent; at this time, it should be disposed of and fresh mount should be made.
Note:
Most antibodies require the methanol+acetone fix/denaturation step for their epitopes to be effieciently recognized. However, some antibodies do not require this step, thereby allowing the avoidance of artifacts that are sometimes associated with the methanol/acetone treatment. If the methanol/acetone fixation step can be avoided, include 0.1% Triton X-100 in the block, as well as antibody solutions to help permeailize the cells.
