We have found that the key to IHC staining consistency following HIER (esp. when using a pressure cooker) is consistent and SLOW cool down. Opening the cooker too quickly can cause flash boiling and is dangerous but removing the slides from the cooker too rapidly and plunging them into room temp buffer can also be problematic. We found that careful trickling of DI water into the cooker for a consistent amount of time until the buffer reaches ambient temp to be effective. We actually time the cooling process and check the temp to be sure the slides are gently and adequately cooled before transferring to buffer prior to staining.
Joe Galbraith -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Rene J Buesa Sent: Thursday, May 07, 2009 11:57 AM To: histonet@lists.utsouthwestern.edu; Margaryan, Naira Subject: Re: [Histonet] FW: prover' pojaluista 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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
