Re: [Histonet] IHC background stain
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.
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:
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?
Pennsylvania Veterinary Laboratory
2305 N Cameron St
Harrisburg, PA 17110
Histology Service Unit
University of Otago
(03) 479 7152
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