For IHC antigen retrieval, should citric acid or EDTA be used? Which is more stable, autoclaving or microwaving?

Here is a set of stable parameters we commonly use: For NeuN, use EDTA pH 9.0, microwave on medium-high power at 95-98°C for 20 minutes, replenishing the solution every 5 minutes; for pressure cooking, use 110°C for 2.5-3 minutes, then allow to cool naturally for 30 minutes. For GFAP, use citric acid pH 6.0, microwave at 95-98°C for 15 minutes; pressure cooking is not necessary. After retrieval, wash 3 times with PBS, then block with 5% normal serum at room temperature for 30-60 minutes. When changing the retrieval solution or using a new antibody lot, be sure to rerun the same known positive brain section to confirm that the signal intensity does not drift.

High-pressure ratio microwave batch-to-batch stability is better because it heats more uniformly and is less affected by the liquid level in the container. For brain section NeuN, we commonly use EDTA pH 9.0 at 110°C for 3 minutes under high pressure, and the background does not increase significantly. High background is often not due to high pressure itself, but rather over-retrieval, excessively high primary antibody concentration, or insufficient serum blocking. The disadvantage of microwave is that when the power, liquid level, or container position changes, the temperature varies greatly. If you continue to use microwave, be sure to use a covered retrieval box and ensure the sections remain submerged throughout, checking and replenishing the liquid every 5 minutes.

EDTA pH 9.0 combined with high pressure poses a high risk of section detachment in brain and adipose tissue, and this cannot be solved by retrieval solution alone. For sectioning, it is recommended to bake at 58°C overnight; do not cut corners on the time for each step of dewaxing and graded alcohol; encircle the tissue edges with a PAP pen. The retrieval solution can be supplemented with 0.05% Tween-20 to improve infiltration, but the concentration should not be too high, otherwise background will increase. For NeuN, first try 1:500 at 4°C overnight; for GFAP, first try 1:500–1:1000, and if the signal is weak, increase the concentration. Prioritizing a low section detachment rate is more important than forcing a high signal.

NeuN and GFAP are best retrieved separately. NeuN is a nuclear antigen, and citrate-based microwave retrieval tends to yield weak staining, so it is recommended to switch to EDTA pH 9.0, with either autoclave or microwave retrieval, which will significantly enhance the signal. For GFAP, a cytoplasmic antigen, citrate pH 6.0 with microwave at 95-98°C for 15 minutes is sufficient. EDTA pH 9.0 does make brain tissue more prone to section detachment, so use positively charged or poly-L-lysine-coated slides, bake the slides at 58-60°C for more than 2 hours, and after retrieval allow them to cool naturally to room temperature before opening the lid; do not cool rapidly.