Re: [Histonet] Sirius red with low contrast between cells cytoplasm andstained fibers

From:John Kiernan

Instead of your steps 3 and 4, rinse in slightly
acidified water. (Your 0.01% HCl should be OK; I
use 0.2-0.5% acetic; about 30 s with agitation).
You can do 2 rinses if there's too much colour in
the water. This step does not extract any bound
dye. Shake off as much acidified water as you can
before the next step.

For your step 5 go directly into the first of 3
changes of 100% alcohol. Agitate slides for about
30 sec in each, then clear (xylene X2) and apply a
coverslip. You lose some of the picric acid in the
dehydration, but there's enough left in the
section to give a yellow colour to everything
except collagen. Nuclei are also yellow, unless
you stain them black before the picri-sirius red.
The long stay in the staining solution extracts
even Weigert's iron-haematoxylin, so a prior
nuclear stain needs to be decidedly overdone.
Junqueira at al (1979, Histochem. J. 11:447-455)
thoroughly studied this method; the paper is well
worth reading.

John Kiernan
Anatomy, UWO
London, Canada
Guillermo Palao wrote:
> Hello all:
>   I would really appreciate any suggestions to improve sirius red staining of kidney samples. Although collagen fibers in tissue nicely stain red, cells cytoplasm do not stain yellow, but sort of pinkish, making it very difficult to separate from red-stained extracellular fibers for quantification purposes.
>   My protocol is as follows:
>   1. Deparafinize and hydrate samples
>   2. Stain 1 h in 0.1% Sirius Red in saturated aqueous picric acid
>   3. Wash 2 min 0.01 N HCl
>   4. Rinse in water
>   5. Dehydrate and cover slides
>   I have tried shorter staining times and rinsing with water before HCl washing without improving results. Any help would be greatly appreciated.
>   Guillermo
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