Immunofluorescent Staining of Foxp3 in Frozen Sections
Thaw frozen sections in a sealed environment to avoid damage by frozen watercrystals. (Approximately 1 hour.)
Fix for 5 minutes in acetone (-20°C) (note: do not use more often than 5 times).
Dry for 10 minutes at room temperature.
Incubate sections in TBS-T (50 mM Tris, 150 mM NaCl, adjust pH with HCl to 7.6 with 0.05% Tween 20) for 15 minutes.
Pretreatment: Perform antigen retrieval or enzyme digestion if needed.
Wash twice for 2 minutes each with TBS-T (Optional: staining of surface marker can be performed before “Cleaning” step)
Cleaning: Incubate sections with 1% Triton X-100 diluted in TBS for 30 minutes at room temperature.
This step will help to reduce background staining (longer incubationmay be more effective, especially for sections thicker than 10 μm).
Normal Serum Blocking: Without washing, incubate sections directly with 5% normal mouse/rat/rabbit serum blocking solution for 30 minutes at room temperature.
Note: Normal serum should be the same species of which the secondary antibody is raised.
Wash three times for 2 minutes each with TBS-T.
Endogenous Peroxidase Blocking: Incubate sections with 3% H2O2 in TBS for 10 minutes to block endogenous peroxidise. (Recommended for liver sections, but optional for other tissues.)
Wash 3 times for 2 minutes each with TBS-T.
Primary Antibody: Incubate sections with primary antibody at its optimaldilution (Mouse Foxp3-Alexa Fluor® 647, clone MF-14, cat # 126408) in “antibodydilution buffer“ for 30-60 minutes at room temperature.
Note: Using a purifiedunconjugated antibody along with a secondary reagent may improve staining due tosignal amplifcation.
Wash twice for 2 minutes with TBS-T.
Counterstain twice for 3 minutes each with DAPI Wash twice for 2 minutes with TBS-T.
Coverslip with Mowiol or mounting medium.
