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-boun...@lists.utsouthwestern.edu [histonet-boun...@lists.utsouthwestern.edu] namens Rui TAHARA [ru...@hotmail.com] Verzonden: vrijdag 16 augustus 2013 23:32 To: histonet@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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet