[Histonet] Grocott's Methenamine Silver Stain

[Kathleen Roberts]

One thing-this wasn't a kit that I used, and I *am* following the 
protocol in the AFIP manual.  The chromic acid is a 10% solution from 
LabChem, and I diluted it down to 4%.  I suppose that they could have 
made a mistake in making the solution.

How do you make up your chromic acid?

Thanks for your help-
Kathleen

Gayle Callis wrote:

>I don't thing 10% chromic is the answer, the method calls for 4%.  Maybe
>they made a mistake making it? Is it actually stronger than they say?  
>
>You don't want to have Chromic too concentrated as if you overoxidize you
>will take the vicinal OH groups to aldehyde and then beyond and get zip for
>results.  I have never used ready to use kits, and make up my 4% chromic
>acid fresh each time. It is possible to OVEROXIDIZE with this method.  
>
>A good read on this method is AFIP manual, Lee Luna tells HOW it should be
>done very clearly. 
>
>
>
>At 03:18 PM 5/5/2004 -0400, you wrote:
>  
>
>>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 <kgrobert <@t> rci.rutgers.edu>
>>>>To: "'histonet <@t> lists.utsouthwestern.edu'" 
>>>><histonet <@t> 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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>>    
>>
>Gayle Callis
>MT,HT,HTL(ASCP)
>Research Histopathology Supervisor
>Veterinary Molecular Biology 
>Montana State University - Bozeman
>PO Box 173610
>Bozeman MT 59717-3610
>406 994-6367 (lab with voice mail)
>406 994-4303 (FAX)
>
>
>  
>