Re: [Histonet] Excessive background staining with Millers elastic stain
I believe the problem you are having is due to the presence of very
similar reactive groups in both cartilage and elastic fibres, Elastin
(the protein ) is heavily sulphated with an abundance of disulphide
groups (hence the yellowish appearance of elastic fibres when viewed
unstained or even macroscopically). When you oxidize the sections in
potassium permanganate, you are converting the disulphides to sulphates,
the sulphates react with Miller's elastin stain. Miller's stain is
essentially as modification of aldeyde fuchsin which iis a well known
method for demonstrating sulphated groups in mucopolysaccharides, etc.
The extracellular matrix of cartilage consists of chondroitin sulphate
(plus a few other assorted bits of connective tissue). These groups will
react quite strongly with Miller's stain solution.
So unfortunately, I think you may well be out of luck as far as
improving the specificity of the staining reactions. The same low
specificity reactions occur with most other elastin staining methods
when they are used on cartilage, the reactive groups present in both
locations stain with the dye in use. I cannot think of a method that
stains only elastin and not cartilage matrix.
Thinner sections may give you a better chance of distinguishing one from
the other. Try sections at several thinner settings and see what the
outcome is. Maybe there is a thickness at which you can see sufficient
enough detail in the elastic fibres without the cartilage obscuring it.
Lachlan Smith wrote:
>I am staining cartilage using Millers elastic fibre stain, and while elastic
>fibres are staining well, there is a lot of blue background staining which
>makes thresholding for histoquantitation almost impossible. My sections are
>micron paraffin embedded. I am also oxidising with 0.5% aqueous potassium
>permanaganate and bleaching with 2% oxalic acid.
>Any suggestings for reducing this background staining would be greatly
>Institute of Medical and Veterinary Science
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