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 .
