AW: [Histonet] post-fixation for Mallory Trichrome

[Gudrun Lang]

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