[Histonet] Bouin's, formic acid & and sirius red staining

[John Kiernan]

Fixation must be adequate before decalcifying. 12-24h in Bouin is OK. My favourite formic decalcifier is that of Clark (1954) Am. J. Clin. Path. 24: 1113-1116. It's a buffer (pH2.0) made by mixing 90% formic acid 250ml, water 750 ml and sodium formate (anhydrous) 34g. Keeps for ever. Check the reference. Never trust a recipe you read on the internet!

The picric acid in picro-sirius red is a necessary component of the staining solution. See Junqueira et al 1979 Histochem. J. 11: 447-455. It's important to have the correct red dye; there is more than one "sirius red". Use C.I. 35780, Direct red 80. 

John Kiernan
London, Canada
= = =
On 15/04/15, Tyrone Genade  <tgenade <@t> gmail.com> wrote:
> Hello,
> 
> Some questions regarding Bouin's solution.
> 
> I was told, back when I was doing my PhD and new very little, that I should
> fix my fish in Bouin's as it will decalsify the bones. Well, Bouin's fixed
> fish were easier to cut than PFA fixed fish... but I read today that by
> adding formic acid the decalsification is better. (I must confess, that
> after Buoin's fixation I still had to soak the tissue face in some dilute
> nitric acid now and then...) Another reference said that the formaldehyde
> should be replaced with formic acid. So which is it: add formic acid or
> replace formaldehyde? And if the former, how much do you add?
> 
> Second question: a colleague and I want to stain for collagen in diseased
> kidneys. The fixative of choice for soft tissue is Bouin's... But the
> staining protocol called for a solution of picric acid and sirius red. Is
> the picric acid needed if I haven't washed the picric acid from the Bouin's
> fixation out of the tissue? I was told once that the picric acid was for
> contrast... Is this BS? Does the picric acid play an important chemical
> role in the staining? I would like to avoid the need for a bottle of
> saturated picric acid on the lab shelf here in Iowa where the winter low
> humidity desiccates everything... I'm hoping this protocol,
> http://www.ihcworld.com/_protocols/special_stains/sirius_red.htm , can be
> modified to omit the picric acid.
> 
> Thanks
> -- 
> Tyrone Genade
> Orange City, Iowa
> tel: (+1) 712 230 4101
> http://tgenade.freeshell.org
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