[Histonet] post-fixation for Mallory Trichrome

[Rui TAHARA]

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