Thank you all very much for you help. I really appreciate it. Gudrun: I tried the PTA before the WSAF and it worked beautifully (1 min PTA, dH2O, 5 min WSAF, dH20, AA, 10 min PTA). Red muscle and clean collagen.Thank-you
Liz: I didn't get to trying the 60*C Bouin's but I will make a note for next time I am optimizing. Thanks again Krista On Sat, May 11, 2013 at 4:04 AM, Gudrun Lang <gu.l...@gmx.at> wrote: > Try to stain first in PTA/PMA solution to impregnate the collagen fibers - > perhaps testing with different times. Then follow up with red stain and the > usual procedure. > > We use a stain called SFOG, that first impregnates 2 min with PMA and > afterwards with the mixture of Chromotrop, Acidfuchsin and Anilinblue for > 10 > min. The longer we do the Polyacid-step the more intensive are the fibres > and less intensive is the cytoplasma. > I think, if after this trial the collagen is still red, that there are > binding-sites in the collagen, that can't be influenced. Maybe acidfuchsin > is here the main partner and a pure solution of Scarlet Red may help. > > Gudrun Lang > > -----Ursprüngliche Nachricht----- > Von: histonet-boun...@lists.utsouthwestern.edu > [mailto:histonet-boun...@lists.utsouthwestern.edu] Im Auftrag von Krista > Sider > Gesendet: Freitag, 10. Mai 2013 23:13 > An: histonet@lists.utsouthwestern.edu > Betreff: [Histonet] Movat Pentachrome - Can’t Remove Woodstain Scarlet-Acid > Fuchsin from Collagen > > Hello All, > > I have successfully stained porcine and mouse paraffin embedded heart > tissues with Movat’s Pentachrome (MP), using EMS’ MP solutions and protocol > (http://www.emsdiasum.com/microscopy/technical/datasheet/26385.aspx), with > some optimization on times. > > However, now I am working with archival human tissue that has been fixed > for > much longer and is much older than my other samples (Human Aorta & Aortic > Valve, formalin fixed 2-6 months or 2-4 years, embedded in paraffin and > stored at RT for ~6 years). I cannot seem to get the Woodstain Scarlet-Acid > Fuchsin (WSAF) to pull out of the collagen with the 5% Phosphotungstic Acid > (PA) or 1% Phosphomolybdic acid (PM) Acid. I have tried increasing the > Bouin’s initial fixation (max 2 hrs 50*C), diluting the standard WSAF > solution by 50% (1 dip) and taking the PA or PM up to 1 hr, yet there is > always a lot of red still left in the collagen, in some regions as strong > as > the muscle (which I am sure are not muscle or dense cells). I see virtually > no change with increased PA or PM time. As I have aorta in my samples I > can’t just leave out the muscle stain. > > I would be very grateful for your insights into anything I could try to get > clean collagen and stained muscle in the MP stain. Why might my method not > be working? Is there something I can substitute for the WSAF that might be > appropriate? > > Thank you very much for you help, > > Krista > _______________________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > > _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet