Hi,

Why do you say that the brains were over-fixed due to old PFA? Were they left for too long in the PFA during post-fixation? If PFA is too old, its oxidation might reduce the % of formaldehyde, resulting in under-fixation, rather than over-fixation. In this situation, you can incubate your brain slices in fresh PFA before starting the IHC protocol.

In case of a real over-fixation, you can try several pH for the antigen retrieval, such as citrate buffer pH=6 or Tris buffer pH=8. If it doesn't work, you can also try amplifying the signal (if you get any) with a biotinylated secondary antibody followed by a fluorescent streptavidin, or a HRP-labelled secondary antibody followed by TSA amplification.

I would try VGLUT and VGAT antibody from Synaptic Systems, wich are very good in labelling synapses. Both are presynaptic markers. You can also use Homer1 (post synaptic excitatory) or Gephyrin (post-synaptic inhibitory). Also in this case, the Synapt Systems ones, works really good.

Over-fixation can indeed mask epitopes and hinder antibody binding, making immunolabeling challenging. Since you have already tried antigen retrieval without success, here are several alternative approaches and troubleshooting ideas that may help you achieve successful synaptic labeling:

Alternative Synaptic Markers

Trying other synaptic markers like PSD95 and Synapsin is a good strategy. Here are a few additional markers to consider:

Enhanced Antigen Retrieval Techniques

Since standard antigen retrieval has not been successful, consider trying enhanced or alternative antigen retrieval methods:

Secondary Antibody Optimization

Sometimes, optimizing the secondary antibody can improve signal detection:

Amplification Techniques

Enhancing the signal can sometimes overcome the reduced labeling due to over-fixation:

Alternative Fixatives

If possible, try re-fixing the tissue sections with a milder fixative:

Post-Fixation with Sodium Borohydride

Sodium borohydride treatment can reduce aldehyde groups and potentially unmask epitopes:

Peptide Blocking

Pre-incubate the primary antibody with a specific blocking peptide to ensure specificity:

Professional Consultation and Services

Consider consulting with companies or laboratories that specialize in difficult immunohistochemistry (IHC) cases:

Trial and Error

Since over-fixation is a common issue, it may take several trials to find the optimal solution. Documenting each step meticulously will help in troubleshooting and identifying the most effective protocol.

All the best