[Histonet] Immunogold question

[Robert Chiovetti]

Hi Danielle,

Just a guess here, but the higher pH may help reduce nonspecific background staining.  

I worked in an EM lab that occasionally played with pH and salt concentrations for immunogold labeling when it was necessary.  I know for sure that high salt washes after the primary and secondary Ab's, followed by a return to normal TBS, does a super job of getting rid of background.  I'm not so certain about the pH.  I only used it a couple of times and I really couldn't tell much difference...

Bob
 
Robert (Bob) Chiovetti, Ph.D.
Southwest Precision Instruments
Arizona's Microscopy Resource
132 North Elster Drive
Tucson, AZ 85710-3212
Tel./Fax 520-546-4986
Member, Arizona Small Business Association
(www.asba.com)

----- Original Message ----
From: Danielle Crippen <dcrippen <@t> buckinstitute.org>
To: histonet <@t> lists.utsouthwestern.edu
Sent: Monday, January 29, 2007 10:27:46 AM
Subject: [Histonet] Immunogold question

Dear EM experts,

I've been using the following protocol for immunogold labeling for the past couple years with good success.  This morning one of our users has inquired as to why the secondary is incubated at a higher pH than the primary.  I do not know the answer...do any of you???




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