Overview
Immunohistochemistry (IHC) localises specific proteins in tissue sections and is a cornerstone of neuroanatomical research. Reliable IHC depends on good sections, proper fixation, validated antibodies and careful controls.
Start with Good Sections
Uniform, undamaged sections with intact antigenicity give the best signal-to-noise. Fixed tissue cut on a vibratome or cryostat should be handled gently and kept flat to avoid folds that trap antibody unevenly.
Antigen Retrieval
For paraffin or heavily fixed tissue, antigen retrieval is often required to unmask epitopes — usually heat-induced epitope retrieval (HIER) with citrate or EDTA buffer. For lightly fixed vibratome sections, retrieval is often unnecessary.
Blocking and Incubation
Block non-specific binding with serum or BSA, then incubate with primary antibody at an optimised dilution and time. Overnight incubation at 4 °C typically gives better penetration and lower background than short room-temperature steps.
Controls
Always include a negative control (no primary antibody) and, where possible, a positive control. This distinguishes specific staining from background and is essential for trustworthy interpretation.
Mounting and Imaging
Mount sections flat with minimal coverslip pressure, use an anti-fade mounting medium for fluorescence, and capture images with consistent exposure settings so intensity can be compared across sections.