Re: [Histonet] Grocott's Methenamine Silver Stain

From:Amy Porter

I would be suspicious about the borax.  We do decalcified mouse turbinates with this method for fungal sinusitis.  Any time I have no or weak reaction i make up fresh borax.  Since you are making everything up fresh, ?????.  If your control hasn't been decaled then it seems like that should be working if all your solutions are fresh. I use a water-bath and prewarm my working silver before i add my slides.  Hope you figure out what is wrong, very frustrating.

Kathleen Roberts  wrote: Hey, all-

I've been trying to stain some FFPE slides of decalcified rat sinuses 
for fungi using the AFIP protocol for Grocott's stain, and despite using 
fresh reagents, the positive control and unknowns do not stain at all, 
except for some random deposition of silver that does not even begin to 
look like fungi. The positive control is loaded with Candida, and can 
be seen with H&E easily.

So far, we don't see any fungi in the rat sinuses and lungs (they were 
sneezing, and the bedding came back positive for Aspergillus) with H&E 
staining, which is why we decided to try the Grocott's-one way or 
another we should be able to visualize it in the tissues.

Any ideas why the Candida-loaded positive control would not stain with 
Grocott's? Right now I am thinking that perhaps they didn't sit long 
enough in the 4% chromic acid, even though the protocol says to soak the 
slides for an hour to oxidize them. Either that or a longer soak in the 
methenamine-silver solution at 60 degrees C (also for one hour).

Thanks in advance for your help-
Kathleen Roberts
Principal Lab Technician
Neurotoxicology Labs
Dept of Pharmacology & Toxicology
Ernest Mario School of Pharmacy
Rutgers University
41 B Gordon Rd
Piscataway, NJ 08854

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Amy S.Porter, HT(ASCP) 
Michigan State University 
Department of Physiology 
Division of Human Pathology 
College of Human Medicine

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