[Histonet] RE: Artifact in IHC

[Donna Maney]

Geoff and Terry,
Thanks so much for your input. All of the Histonet posts went into my spam so I just saw these today, but in the meantime by coincidence we ended up following your suggestions. To us, it looked like the tissue was drying out and cracking before being coverslipped. A new technician was being trained, and was perhaps trying too hard to drain all of the xylene off the slide. She noted that the longer she took to coverslip the slide, the worse the artifact was. So we soaked off the coverslips, rehydrated down through 70% and into 50% then water, then dehydrated and coverslipped again. I'd say the artifact is 95% gone. 

So, I don't know if it was just being speedier with the coverslip or the lower concentrations of alcohol and water soak that did the trick. But we will be implementing both now.

Thanks again,
Donna
--------------------------------------------
On Tue, 9/23/14, Geoff <mcauliff <@t> rwjms.rutgers.edu> wrote:

 Subject: Re: [Histonet] RE: Artifact in IHC
 To: histonet <@t> lists.utsouthwestern.edu
 Date: Tuesday, September 23, 2014, 3:40 AM
 
 Teri makes an excellent
 point about higher alcohols causing 
 precipitation of phosphate salts.
 
 Geoff
 
 On 9/22/2014 1:17 PM, Teri Johnson wrote:
 > Hi Donna,
 >
 > Interesting conundrum! A few questions:
 >   - Do you see it in tissues
 that have not undergone any staining, just floated in buffer
 and coverslipped?
 >   - Have
 you tried wet mounting them after staining and prior to
 dehydration and clearing to see if the artifact is there?
 >   - Have you recently changed
 glove manufacturers, could this be talc or some other powder
 from gloves?
 >   - Are you
 dehydrating out of PBS into an alcohol concentration higher
 than 70%? You could be getting phosphate precipitation. Try
 using a water rinse prior to alcohol or make sure your first
 alcohol concentration is 70% or lower.
 >
 > Those are the weird
 things that came to mind. I hope this helps,
 >
 > Teri Johnson
 > Manager Clinical Trial Testing
 > Genoptix, Inc.
 >
 Carlsbad, CA 92008
 >
 >
 >
 >
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 -- 
 --
 **********************************************
 Geoff McAuliffe, Ph.D.
 Neuroscience and Cell Biology
 Robert Wood Johnson Medical School
 675 Hoes Lane, Piscataway, NJ 08854
 voice: (732) 235-4583; fax: -4029
 mcauliff <@t> rwjms.rutgers.edu
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