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Protocols: immunofluorescence, cryostat sections, sample preparation ...
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This immunofluorescence protocol is useful for labelling cryostat sections of sample tissue.
1. Submerge the slides in paraformaldehyde solution
(make fresh solution) for 20 min at room temperature (RT)
2. Wash the slides in PBS
(5 min.). Repeat 3 times.
3. Using a PAP pen, encircle the tissue section on each slide. This
will help prevent excessive spreading of the reagents allowing one to
use less. Do not allow the sections to dry.
4. Permeabilize and block tissue sections (permeabilization
solution) for 60 min RT: using a micropipettor, cover each tissue
section (volume will depend on tissue size) with the solution.
5. Primary antibody application. Dilute the primary antibodies in 1%
BSA (Bovine Serum Albumin) and 0.05% Triton X-100 in PBS. Incubate the
antibody and samples overnight at 4 C.
6. Wash the sections (3 x 5 min.) in PBS.
7. Secondary antibody application (apply in a darkened room). Dilute
the secondary antibodies in 1% BSA and 0.05% Triton X-100 in PBS. Apply
the antibodies to the samples and allow to incubate for 60 min. RT in
the dark.
8. Mount the slides using a mounting medium.
9. Store in a darkened place to minimize photo bleaching.