Simplified Protocol for immunostaining:
1. Cells (or tissue sections) can be fixed in the PBS with 4%
paraformaldehyde (or other fixatives) for 10-15 min.
2. Rinse samples with PBS 2-3 times ( 3-5 min. each time)
3. Normal goat serum blocking (with Triton-X if necessary) for 20-30
min. (Optional)
4. Prepare primary antibodies. For example: Mouse monoclonal antibody
and/or rabbit polyclonal antibody. ( Dilutions depend on the titers of
antibodies).
5. Immunostaining: add primary antibodies to cover the samples, 4 degree
for overnight (or 37 degree for 2 hr)
6. Rinse samples with PBS 3 times (5 min. each)
7. Apply the secondary antibodies. For example: FITC-conjugated goat
anti-mouse IgG (1:100 dilution) and/or Rhodamine-conjugated goat
anti-rabbit IgG (1:200 dilution) at room temperature for 1 hour.
8. Wash the samples with PBS for 5 times (5 min. each).
9. Mount the samples with Crystal Mount.
10. Observe the samples under the Confocal.