Hi Everybody!

I am performing Immunoflorescence for rat's brain tissue containing GFP, fixed in 4% paraformaldehyde for 24 hours, two days in 30% sucrose solutions. And i am consistently getting background noise and No GFP signal. Used Anti-GFP antibody even. Kindly need your expert input. Here are the steps i performed and tried to trouble shoot:

1. Followed standard IF protocol, still background noise (BN).

2. Performed Antigen retrieval, still exist BN.

3. Increased washing duration, still BN.

4. Increased Antibodies concentrations, Still BN.

5. Increased BSA concentration from 1% to 2%, still BN.

Any suggestion how to reduce Background noise (BN)?

Your anticipated contributions are appreciated!

Thanks much!