Re: [Histonet] IHC background stain

From:=?iso-8859-1?q?Aidan=20Schurr?=

Hi Roger,
 
5um does seem a little on the thick side, although if this is what you always have done, then obviously something else has changed.  I tend to cut all my IHC slides at 3um...
 
Other reasons for excessive background could be:
* incorrect pH of buffer
* antibody / detection system 'off'/expired (has it been left out on the bench too often?!)
* sections dried out
* new batch of antibody - may need retitering
* Have you switched retrieval solutions?  EDTA can give notiveable background on its own...
 
These are just a few ideas - as you know, IHC is a bit of a black art, so your best bet to to look closely for any changes...
 
Best of Luck
Aidan
 


"Charles, Roger"  wrote:
Hello all, 
I do all the IHC for our diagnostic veterinary laboratory. Latley i have been getting a lot of background stain from certain protocols. I also over heard one of the pathologists state the sections are getting thicker. Could thick section cause background stain on IHC? I checked my histologist microtome and it is set at 5um. Could her microtome need calibrated?

Roger Charles
Microbiologist
Pennsylvania Veterinary Laboratory
2305 N Cameron St
Harrisburg, PA 17110
717-787-8808


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