3.
Procedure for Immunofluorescence Double Staining
Note:
prior to perform double labeling, it is important to test each primary
antibody individually and select the best pretreatment(s) for each
antibody. It will be ideal if the two primary antibodies require same
pretreatment. Otherwise, one should do a further test by treating sections
with both pretreatments and then immunostain for each antibody
individually. If both antibodies survive the “double pretreatments”,
you are ready for immunohistochemistry double staining. Another
alternative is to do pretreatments separately for each antibody staining.
1.
Rinse Sections in washing
buffer for 2x2 min.
2.
Serum Blocking:
incubate sections in
normal serum blocking solution – species same as secondary
antibody (for example: primary antibodies are mouse and rabbit, and
secondary antibodies are horse anti-mouse, and goat anti-rabbit, so horse
and goat serum block should be used).
3.
Primary
Antibodies:
incubate sections in the mixture of two primary antibodies (mouse
and rabbit) at appropriate dilution in primary
antibody dilution buffer for 1 hour
at room temperature.
4.
Rinse in washing
buffer for 3x2 min.
5.
Secondary
Antibodies:
incubate sections in the mixture of two fluorescent conjugated
secondary antibodies (FITC conjugated Horse
anti-Mouse and Texas Red conjugated Goat anti-Rabbit)
in PBS for 30 minutes at room temperature).
6.
Rinse in washing
buffer for 3x2 min.
16.
Counterstain
with DAPI
if desired for 20 minutes at room temperature.
17.
Rinse in washing
buffer for 3x2 min.
9.
Coverslip with anti-fade fluorescent mounting medium
and seal with nail polish.
19. Store
slides in dark at 4 °C.
4.
Results
