Re: ? about Bouins fixative

From:"J. A. Kiernan" <>

On Fri, 11 May 2001 wrote:

> I would like to know if there is any difference with staining results:
> On paraffin sections using any one of these steps.
> 1. If you first fix the tissue in Bouins.
> 2. If you use NBF fixed cut section on a slide in Bouins in an oven for 1hr .
> 3. If you use NBF  fixed cut section on a slide in Bouins on the countertop 
>     overnight.
> I know all three steps will work but will the end result stain better.
> I am using a elastic stain.

If you are staining only for elastic fibres and laminae it should
not make much difference what fixative you use. Treating sections
of formaldehyde-fixed objects with Bouin's or anything else should
have little or no effect on the staining of elastin.

If you are using a method in which elastin staining is only part
of the picture, with other dyes for nuclei, cytoplasm, mucus, 
collagen, elastin etc, you may well get improved differential 
staining of collagen and cytoplasm if you fix in Bouin. Immersing 
hydrated paraffin sections of formaldehyde-fixed objects in Bouin
is a widely practised alternative. You don't need to use complete
Bouin's fixative for this; a saturated aqueous picric acid solution
is just as good. The other ingredients of Bouin (formalin and acetic
acid) do not contribute to the improved brightness of staining with
anionic dyes. Research is needed to find the reason why picric acid 
works in this way. The various mooted explanations are based on
speculation, not on evidence.

You asked if Bouin overnight at room temp was equivalent to Bouin
at 60C for an hour or two. I've done both (with sat picric, not
Bouin) and both work (for Masson, AZAN etc). I do not know if
or why the times and temps mean anything. Possibly a 2-min
immersion in picric at room temperature would be OK. I can
thnk of no reason why not. 

I hope these remarks stimulate lots of suggestions and anecdotal 
tales of how to improve connective tissue staining!

John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1

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