Ethanol concentration may be  very high (50%). You can minimize ethanol concentration

30 min is long for the vacuum infiltration, this could damage the plant tissue. A few minutes should be sufficient.

You need to try high concentration low period of 5 minutes.

Thanks for the answers, I tried 4% PFA and 2% Triton and it worked better. Anyway I'll try to reduce the ethanol concentration. What would you recommend Omaima? 30%? Have you had that experience before and reducing ethanol helped you?

I have tried less vacuum, but that does not seem to be the problem. The brownish starts almost 3-5 minutes after dissections. Lately, I've being dissecting the tissue submerged into the fixative under the hood and that's been helping a bit.

Hello Suresh, for which reagent you mean high concentration?

Thanks to all!