Strictly speaking, IHC reactions do not depend on water temperature temperature. What you describe does not include dist. water between the IHC steps therefore the water temp. should not be affecting your reactions. If you use dist water after HIER you have to remember placing the slides into room temp. buffer before starting the IHC procedure, so if there was any effect due to the dist. water, that should have take care of it. The inconsistency in your staining I think is not due to the dist. water temp. René J.
--- On Thu, 5/7/09, Margaryan, Naira <nmargar...@childrensmemorial.org> wrote: From: Margaryan, Naira <nmargar...@childrensmemorial.org> Subject: [Histonet] FW: prover' pojaluista To: "histonet@lists.utsouthwestern.edu" <histonet@lists.utsouthwestern.edu> Date: Thursday, May 7, 2009, 11:23 AM 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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
