Re: [Histonet] DAB and fading

From:Geoff McAuliffe

    Sounds like the DAB is being oxidized by something in the mounting 
medium. John Kiernan has recipes for non-fading mounting media on his 
website that you can make yourself. Ringing the coverslips with nail 
polish may not be "necessary" but if air is getting to the sections that 
may be making the problem worse. Do the experiment yourself, ring some 
with nail polish and see if it helps. DAB (even with intensification) 
does fade (after a few years) in Permount too, that is why I use only 
DPX these days.


David Laidley wrote:

> I am currently staining brain tissue (perfused 4% paraformaldehyde) 
> that has been cut in a cryostat at 50 microns and I am staining for 
> NeuN using DAB tablets with Cobalt from Sigma.
> The stain works perfectly. I am using a mounting medium that is 
> glycerol based (PVA-glycerol). This mounting medium also works 
> perfectly as it can be quickly applied to the sections after they have 
> been mounted on a slide which perserves the thickness of the sections 
> (I am interested in doing stereology) and it polymerizes overnight so 
> that nail polish is not necessary to keep the coverlip on. (I have 
> used the same medium with fluorescent tissue except DABCO was added)
> But I am noticing that over a number of days that may sections which 
> have neurons stained black, turn brown and pale. Is this fading a 
> product of the mounting medium? Does the fading progress to where the 
> precipitate can no longer be seen? Has anyone used aqueous mounting 
> mediums with DAB and had fading? It was my understanding that DAB 
> didn't fade (or at least not too much).
> Comments? Suggestions? Please help me put my concerns to rest.
> David Laidley
> MSc student
> Memorial University of Newfoundland
> Division of Basic Medical Sciences - Neurosciences Group
> ------------------------------------------------------------------------
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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

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