Re: [Histonet] IHC background stain

From:Caroline Stott

Hi Roger,
I don't think the 5 micron sections should be too much of a problem.  We 
cut a lot of immuno sections at 7 microns and they come up with great 
results.  Are you using a polyclonal antibody?  We had trouble with 
background staining when using them.
It might be a good idea to calibrate the microtome though.

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

"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
Pennsylvania Veterinary Laboratory
2305 N Cameron St
Harrisburg, PA 17110

Caroline Stott

Histology Service Unit
Medical School
University of Otago
(03) 479 7152 

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