Hello Beatriz María Fernández Santos,

Yes, TRIS-EDTA buffer pH 9 combined with autoclave can be too harsh on the tissue. The high pH of TE buffer is particularly effective at retrieving antigens after formaldehyde fixation, but it can be too strong, especially when combined with the heat of an autoclave.

DAPI staining is used to assess the effectiveness of the antigen retrieval process. The stain should be bright and uniform. But the blurrier/higher background in the TRIS-EDTA buffer indicates that the buffer is too aggressive.

On the other hand, citrate buffer (pH 6) is a gentler method of epitope retrieval. It will produce a less harsh unmasking compared to TE buffer and is less likely to cause excessive changes to the DNA structure, which could lead to a more consistent DAPI signal.

You should always test different buffers and conditions to find the best approach for your specific tissue and antibody combination. The optimal buffer for antigen retrieval is often tissue-dependent.

Best,

Thank you Malcolm Nobre for your reply. We have indeed tested Citrate Buffer pH 6 and our antibody (a very nuclear one) only worked with Tris-EDTA pH 9 and autoclave. Have you then experienced this before? Considering we already know this antibody does not work until harsh treatment with TE pH 9, could maybe lowering autoclave time be enough to improve DAPI?

You may reduce the autoclaving time and/or lower the autoclaving temperature (say to 90-95°C), and ensure your slides are electrostatically charged or strongly adhesive slides to prevent tissue loss.