[Histonet] FW: prover' pojaluista

[Margaryan, Naira]

Dear Histonetters,

This is my question regarding IHC staining and water temperature: does IHC staining depend on water temperature in washing step?

As always, I am using the same protocol for IHC. But one day it works perfectly, another day, on the same tissue, it does not work. Everything is same, solutions are fresh, and the only thing that is different is water temperature. I am using running distilled water before and after Antigen Retrieval step, after DAB, after counterstaining and after bluing reagent. Dd-water goes into autostainer: all blocking steps and after primary, secondary and tertiary. I am not using usual running water to exclude different Chlorine concentrations in different days.

Have anybody of you ever paid attention to running distilled water temperature? What water temperature (cold or warm) you will recommend for washing steps? If this is critical, I can prepare my water up front.

Hope to get an answer soon.

Thanks,
Naira