Immunohistochemistry-Frozen Protocol for FoxP3 Antibody (NB600-246)

IHC Protocol for NB600-246
Control Tissues: Thymus from healthy (C57BL6/6) and Influenza infected mice.
-Cut 5 um sections in a cryostat and fix them in cold acetone for 10 mins.
-Dry the slides completely at room temp for 30 mins.
-Permeabilize tissues by immersing them in 0.1% saponin, 3 times (5 mins each time).
-Block unspecific binding to the slides using 5% normal donkey serum, 5% normal rat serum and Fc block (1:500). Everything has to be dissolved in 0.1% saponin.
-Incubate slides in a wet chamber at RT for 30 mins.
-Dip off the blocking solution from the slides and add streptavidin (0.1mg/ml) for 15 mins at RT.
-Wash one time with 0.1% saponin.
-Add similar volume of biotin (0.1) and incubate 15 min at RT.
-Wash one time with 0.1% saponin.
-Add NB600-246 (1:150) ON at RT.
-Next day: Wash slides with 0.1% saponin
-Add biotin-donkey anti-rabbit secondary (NB120-6801) for 3 hrs at RT.
- Wash slides with 0.1% saponin
-Add fluorescently labeled streptavidin (Molecular probes) in PBS for 1 hr at RT.
-Wash 3x with PBS.
-Mount with prolong gold antifade (Molecular probes)