May I suggest first staining the heparin in the mast cells with alcian blue, pH 1.0, which gives an indestructible coloration. (Cartilage matrix and some kinds of mucus are also stained; shouldn't cause confusion unless you're working with gut mucosa. Nuclei are not stained.) Then do the immunohistochemistry. If there is a problem with nonspecific binding of DAB to mast cells, followed by its oxidation to a brown product, use a different labelling enzyme/chromogen. Remember that in many species mast cell granules are largely removed by commonly used aqueous fixatives, including neutral formaldehyde, though this is OK for rats and mice. See "The Mast Cells" by Hans Selye (Washington: Butterworths, 1965, pp 25-26) for summaries of 13 papers and citations of 5 review articles on fixation of mast cells. 
-- 
-------------------------------
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
   kiernan[AT]uwo.ca
   http://publish.uwo.ca/~jkiernan/
   http://instruct.uwo.ca/anatomy/530/index.htm
_______________________________



----- Original Message -----
From: "Johnson, Teri" 
Date: Monday, September 24, 2007 10:43
Subject: [Histonet] Re: IHC together with toluidine blue staining for mast cells
To: histonet@lists.utsouthwestern.edu

> Moran, I find that in IHC procedures mast cell granules take up the
> secondary label. If you are doing this to try to see if your IHC is
> labeling mast cells, I agree with Rene's assessment - I doubt 
> you will
> get histochemical staining over the chromogen, but it might be 
> worth a
> try.
> 
> Do your IHC procedure first with DAB, then you will stain with Tol.
> Blue, air dry, and mount with permanent mounting medium. The Tol blue
> will also stain your nuclei, just as if you were doing a regular 
> nuclearcounterstain.
> 
> If this stain will obscure nuclear IHC signal, consider doing the
> Tolduidine blue stain first, coverslip, photograph, decoverslip and
> hydrate back to water, and then perform your IHC procedure, coverslip,
> and photograph.  No need to remove the Tolduidine blue 
> before doing your
> IHC procedures, as it should wash out in exposure to alcohols 
> and water
> prior to and during the IHC procedures.
> 
> Hope this helps!
> 
> Teri Johnson, HT(ASCP)QIHC
> Managing Director Histology Facility
> Stowers Institute for Medical Research
> 1000 E. 50th St.
> Kansas City, MO 64110
> 
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