I made up everything fresh just before I did the stain... :oP    I'll 
try the 10% chromic acid.

Thanks,
Kathleen

Wayne Holland wrote:

> Make sure your solutions are fresh, especially the silver. ALso try 
> 10% chromic for 10 minutes.
>
>
>> From: Kathleen Roberts 
>> To: "'histonet@lists.utsouthwestern.edu'" 
>> 
>> Subject: [Histonet] Grocott's Methenamine Silver Stain
>> Date: Wed, 05 May 2004 13:54:18 -0400
>>
>> 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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