I've had excellent results staining perfused mouse brain tissue fixed with 4% PFA using Iba1. However, I’m encountering difficulties when working with fresh frozen tissue—no staining is observed.

I understand that fresh frozen sections typically do not require antigen retrieval, as there are no covalent crosslinks like in PFA-fixed tissue. Still, I experimented with antigen retrieval methods, including heat-induced epitope retrieval (HIER) using citric acid (boiling for 30 minutes), both alone and in combination with 70% formic acid (5 minutes). After this treatment, I observed significant nuclear staining, which I suspect might be a false positive (image attached below).

I also came across a report from another researcher who experienced similar issues staining fresh frozen tissue with Iba1. They contacted Fujifilm Wako (the antibody supplier) but did not receive a conclusive solution.

Any insights or suggestions would be greatly appreciated.