Most of our viruses have YFP as the fluorophore, we have two different protocols: a full multi-day protocol and a one day protocol. Both from the Gupta lab in Indianapolis provided by his then postdoc Mardones Diaz, Muriel Alejandra. We have had good luck with the single day protocol and haven't used the 2 day protocol.
SHORT (ONE DAY) IMMUNOHISTOCHEMISTRY PROTOCOL (GFP STAINING)
• Wash brain slices in PBS 3 x 10 min
• Permeabilize/block with PBS + 0.4% triton + 5% serum for 1 hour
• Wash 3 x 5 min in PBS
• Add GFP antibody (1:2000) in PBS + 0.4% +0.2% serum for 1 hour at room temperature
• Wash 2 x 10 min in PBS
• Add DAPi (1:10000) in PBS for 45 minutes
• Wash 2 x 5 min in PBS
• Mount
Long Staining Protocol (Secondary o/n)
-Permeabilize/block with PBS + 0.4% triton + 5% serum x1hr
-Add primary antibody (1:1000) in PBS + 0.4% triton + 2% serum overnight in fridge 4 degrees (16-24 hours)
-Wash 3x 5 minutes in PBS
-Add secondary antibody (1:1000) in PBS + 0.4% + 2% serum overnight in fridge 4 degrees (16-24 hours)
-Wash 2X for 10 minutes in PBS
-Add DAPI (1:10,000) for 45 minutes (optional)
-Wash 2x for 5 minutes in PBS
-Mount