RE: trichrome

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From:Ms Louise Taylor <>
Date:Tue, 16 Mar 1999 17:16:33 +0000 (GMT)

Hello everybody (Sayeed and Katrina especially)

Here is the recipe and method for the iron haem that we use routinely 
for all trichromes.
Ref: J clin Path 1972.25; 373-396

Dissolve a) 1g Heamatoxylin in 100ml 95% alcohol
         b) 10g Aluminium chloride hexahydrate and 10g 
            ferrous sulphate in 100g dH20.
Combine these 2 solns. and add 2ml HCl (conc) and 2ml stock aqueous 
9% sodium iodate.
Mix and allow 48hrs to ripen. Solution should remain active  for 2 
months if kept at 4deg C.
1. Bring sections to water                                    
2. Stain in stable haematoxylin 5 mins            
3.*** Differentiate in 1% acid alcohol - check microscopically to note 
whether only nuclei are staining, and that background is clear***     
4. Wash well in tap water before counterstaining.
Note: This stain can be reused for about a week ( until soln turns 
brown), but do not pour used soln back into stock bottle.
****Degree of differentiation will depend on stain to follow; less 
differentiation is required if to be used for a Van Gieson rather 
than a Masson's trichrome.
best regards
louise taylor
South Africa

On 16 Mar 99 at 8:17, Mohammed, Sayeed wrote:

Hi Louise,
I am working on developing a modified Trichrome for a prstate research
project. It will be very intresting try your stable Iron-Heamotoxylin. I
will appreciate very much if you kindly e'mail me your procedure. I will
keep you informed of the out come of it.     Thanks in advance.
      Baylor College of Medicine
      Phone# (713) 798-4694

> -----Original Message-----
> From: Ms Louise Taylor []
> Sent: Tuesday, March 16, 1999 6:40 AM
> To:
> Subject:  Re: trichrome
> Hi Katrina,
> we routinely use a "stable" iron heamatoxylin - if you like I can 
> send you the method - you may have to make it up yourself though, 
> I don't know if it is commercially available.
> Best regards
> Louise taylor
> On 15 Mar 99 at 13:36, Barry Rittman wrote:
> Katrina,
>            working in a dental school we don't stain many sections of
> uterus
> The differentiation you refer to could be a result of too acidic a
> trichrome
> stain or oxidation by components which  will convert  Hx to tri- tetra- or
> pentoxy-hemateins. The tetroxy hematein is usually brownish (depending on
> mordant) and the pentoxy is colorless. This is time dependent so that if
> for
> example you stain 7 micron sections with van Gieson for 3 minutes the Hx
> looks
> great, if for 5 minutes (often necessary for teeth) then the Hx will often
> appear brownish or wishy washy. t
> This may depend on which variant of Mason trichrome that you are using.
> I cannot believe that the picric acid in Bouin's is causing the problem
> unless
> there is so much of it left in the section that the Hx is becoming over
> oxidized. You can remove the picric acid by soaking in 70% ethanol (with a
> trace
> of lithium carbonate) .
>             Have you considered using celestine blue instead of iron
> hematoxylin? Celestine blue is much more resistant to decolorization than
> iron
> Hx which is essentially over oxidized in many of the trichrome stains. We
> have
> used celestine blue routinely as a nuclear stain in  van Gieson staining.
> If you wish I can send the method for staining
> Barry
> Katrina Knott wrote:
> > We have been working on a the Masson's trichrome stain on human uterus.
> The
> > stain looks nice but we are not getting nuclear staining.  We are using
> > Weigert's Iron Hematoxylin.  It seems to look well when we apply it, and
> > when we take the tissue out of the stain.  Is it possible that we are
> losing
> > staining is subsequent steps due to the acidity, etc?  What can be done
> > about this?  Increase the mordant concentration?  Change one of the
> other
> > steps in the procedure?  It seemed to stain when we did not use Bouin's
> to
> > post fix... but then the other dyes did not look well.
> >
> > thanks,
> > Katrina

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