[Histonet] post-fixation for Mallory Trichrome

Jonathan Cremer Jonathan.Cremer <@t> med.kuleuven.be
Mon Aug 19 01:52:09 CDT 2013


Post-fixation is important in the trichrome stains, if you did not fix your tissue with picric acid or mercury (please never use mercury).
If you wish to avoid picric acid, you can also use 1% zinc sulfate in 4% formaldehyde (in water) 2 hours at 55-60 °C. Or you could fix your tissues in this fixative.

I have also noticed when doing the MSB stain, that I get much more vibrant and deeper colours when I leave the slides for a few weeks after cutting before doing the stain. I have not found an explanation for this in the literature, best thing I can think of is "oxidation".

---
Jonathan Cremer


________________________________________
Van: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] namens Rui TAHARA [ruio7 <@t> hotmail.com]
Verzonden: vrijdag 16 augustus 2013 23:32
To: histonet <@t> lists.utsouthwestern.edu
Onderwerp: [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



More information about the Histonet mailing list