AW: [Histonet] post-fixation for Mallory Trichrome

[Rui TAHARA]

Thank you!

I have added extra step of 5 min of 1% phosphomolybdic before aniline blue-orangeG, which increased the blue contrast in late stage embryos but not early stage ones.  

I did not realize that Mallory trichrome react differently between embryonic and adult samples. I just assumed the formalin fixation is causing the staining problem for early embryos. 
Does formalin fixation cause the different colors or weak staining? 
If the Mallory Trichrome works differently with developing stages, is mallory trichrome preferably used for adult samples and late stage samples? 
I found the following ref about modified Mallory Trichrome for embryonic samples. This method is basically use three dyes separately that can help differentiate staining colors like optimized colors achieved in adult samples. I have tried the protocol yet the cartilage does not show sharp enough and most of other tissues are stained purple-ish. 

Would you suggest some articles that i can refer to for Mallory trichrome in embryos? 
Thank you, 

Rui 

> From: gu.lang <@t> gmx.at
> To: ruio7 <@t> hotmail.com
> CC: histonet <@t> lists.utsouthwestern.edu
> Subject: AW: [Histonet] post-fixation for Mallory Trichrome
> Date: Sat, 17 Aug 2013 09:26:38 +0200
> 
> Hi,
> I think staining would be improved, when you use Bouin as postfixative ( 1h
> hour, 60°C).
> Try to invite a further step between Fuchsin and Anilinblue. When you
> incubate the slides in 1% phosphomolybdic acid for 5-10 min, you can see
> decolorization of cardilage. This improves later on anilin-binding.
> You may control the differentiation-step under microscope. Anilinblue can be
> reduced to 5 min afterwards.
> 
> You may also consider, that there's a real difference in
> cardilage-architecture of the early and late samples, that takes influence
> on staining-abilities.
> 
> Gudrun Lang
> 
> -----Ursprüngliche Nachricht-----
> Von: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] Im Auftrag von Rui TAHARA
> Gesendet: Freitag, 16. August 2013 23:33
> An: histonet <@t> lists.utsouthwestern.edu
> Betreff: [Histonet] post-fixation for Mallory Trichrome
> 
> 
> Hello, 
>  
> There seems to be a lot of suggestions for Mallory Trichrome staining
> involved in Acid fusin, Aniline blue, and orange G.
> 
> My sample of avian embryos were
> fixed with Formalin based fixative (4% paraformaldehyde in
> phosphate-buffered saline and 1% glutaraldehyde) overnight, in case of late
> embryos bones were decalcified, and followed standard  alcohol series,
> paraffin embedded, and 10 micron sectioned. 
> The slides were dehydrated,
> stained with 0.5 % Acid fushin for 10min, then without wash, 0.5% Aniline
> blue and 2 % orange G in 1% phosphomolybdic acid solution for 20  min, then
> undergone ethanol series, cleared and mounted.
> 
> Now I know that formalin fixed
> sample do not present optimized trichrome colors based on your websites and
> references. Yet the stained sections of late stage embryo do still show near
> optimized colors whereas those of  early stage embryo show almost no blue or
> very dark blue, almost gray color for cartilages and most of morphologies as
> purple-redish colors.
> 
> Since I tested staining on late
> embryos first I thought staining would work on early embryos. Does anyone
> provide me explanation why the staining mostly shows red-ish color and not
> optimized color for cartilage in early  embryos? Is that because of the
> formalin-fixed embryos although late stage embryos fixed with formalin still
> show the blue for cartilage?
> 
>  
> Another question is related to
> fixative. I prefer not to use bousin fixative due to picric acid and it
> seems that Citrate buffer or Gram’s iodine solution can be substituted to
> bousin post-fixative. Have anyone tried  these solution for Mallory
> Trichrome? Do those methods show near optimized color for Mallory trichrome?
> 
>  
> Any suggestion is appreciated. 
> Thank you, 
>  
> Rui TAHARA
> PhD candidate
> McGill University, Biology Department
>  		 	   		  
>