IF protocol for perfusion fixed tissue

Tissue preparation:

Subjects transcardially perfused with heparanized saline (0.9 w/v NaCl) followed by 4 w/v paraformaldehyde and 15 saturated picric acid in 0.1 M. phosphate buffer pH 7.4. Brains incubated in 20w/v sucrose in 0.1 M PB (24 hours at 4°C) followed by freezing in isopentane (-40°C). 30 micron coronal sections cut on freezing microtome/cryostat for free floating IF.

1. Wash free floating brains sections 4 times in 0.01M phosphate buffer saline (PBS). (The longer the sections are washed the better)
2. To quench non-specific binding, incubate sections in 10 v/v normal donkey serum (in PBS-triton 0.3 v/v (PBST)) for 1 hour at room temperature.
3. Incubate sections in primary antibody (at concentration stated on the datasheet) overnight at room temperature.
4. Wash tissue sections 3-4 times in 0.01M PBS.
5. Incubate sections in secondary antibody (as appropriate: donkey anti-mouse or donkey anti-rabbit (e.g. Alexa 488 at 1/2000 in PBST) for 2h at room temperature. 
6. Wash brain sections 3-4 times in 0.01M PBS.
7. Mount sections onto gelatinised or superfrost plus slide (BDH) with a fluorescence mounting medium (eg.Vectashield; Vector Laboratories).

Important controls to include in your experiment:

• control for primary antibody staining (eg. Tissue without primary antibody incubation)
• control for secondary antibody staining (eg. Tissue without secondary antibody incubation)
• positive controls (eg. Tissue/area where staining is expected as protein is reported to be present)
• negative controls (eg. Tissue/area where staining is not expected as protein is not present)