RE: artifactual nuclear staining

From:"Monson, Frederick C."

"Wee", and I just finished a great read of Kidnapped!  Let's see.  Richard,
I remember a study once with FeHaematoxylin on human testis.  The report was
that there were two populations of cells based on nuclear staining.  Of
course, the section thickness was conveniently absent from the Mat's &
Meth's.  I have seen the same problem in IHC but had no time to follow-up as
follows.

If a typical neuro-type study, the sections may tend to be a bit thick and
many nuclei may be whole in the sections.  My first approach would be to
practice a bit more time in the rinse after my ployclonal or the
anti-lago-biotin.  Buffer rinses also tend to be somewhat pedantic in
formulation (I.e., PBS, etc.).   I would try slight modifications of
concentration.

 I would test my avidin fluorescein or rhodamine to see if the nuclei held
those probes more than they should.  If the sections are fresh-frozen or
frozen after fixation, the problem may be even more interesting, depending
on the 'Tween' of it.  

Finally, if the sections are thin and even so all the nuclei come up
positive, then the polyclonal might just have a nuclear bug in it, that,
depending on relative titers, might lend itself to some adsorption.
Otherwise, all else failing, I would ultimately resort to a mouse!    

My compliments to the post-grad for noticing.  Many just take the photos and
publish, depending on the editors and reviewers to live and let lie.

Regards and admiration for what you've writ,  and I hope my imaginings may
bear fruit.

Fred Monson

Frederick C. Monson, PhD 
Center for Advanced Scientific Imaging(CASI) 
West Chester University of Pennsylvania 
Schmucker Science Center II  
South Church Street                                                    
West Chester, PA, 19383
eMail:  fmonson@wcupa.edu
http://darwin.wcupa.edu/casi/



> ----------
> From: 	RichardWHorobin@aol.com
> Sent: 	Wednesday, November 7, 2001 10:23 AM
> To: 	Histonet@pathology.swmed.edu
> Subject: 	artifactual nuclear staining
> 
> G'day folks - Have an email from a non-HistoNet buddy, who asks: 
> 
> one of the post grads has a wee problem 
> he is using immunocytochemistry to localise some neural antigen (trysine 
> hydroxylase)(should present in the cytoplasm of his cells) he is using a 
> rabbit polyclonal followed by antirabbit biotin then avidin fluoroscein or
> 
> Rhodamine. 
> the problem is that all of the nuclei are staining - as if  you  used 
> propidium iodide - any suggestions - he has done a fair bit of immuno
> before 
> 
> Well I dont know - - Any suggestions? 
> Bye now - Richard 
> 
> Institute of Biomedical & Life Sciences, University of Glasgow 
> T direct 01796-474 480 --- E  RichardWHorobin@aol.com 
> "What should we expect? Everything."
> 




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