Years ago, I was taught by Jerry Fredenburg, a stain guru, never to microwave the Bouins step but do this for 1 hour at 60C or overnight at RT. The post-fixation/mordant is very important in order to acidify the connective tissue fibers properly for a trichrome stain, and the short time in MW will not do the job. Liz is correct here in letting the sections stand longer in Bouins after microwaving or simply just do the 1 hour at 60C.
I have removed Gills type hematoxylins from nuclei by over exposure to acetic acids so remember that ALL Masson's Trichrome reagents are acidified with acetic acid and will automatically do this, even on Weigert's Iron hematoxylin. We also examined our sections microscopically during staining to make sure the check the depth of red staining reagents on smooth muscle is correct - once again, Liz is correct about this fact. Gayle Callis -----Original Message----- From: Elizabeth Chlipala [mailto:l...@premierlab.com] Sent: Tuesday, June 30, 2015 2:30 PM To: Suzanne Martin; histonet@lists.utsouthwestern.edu Subject: Re: [Histonet] Trichrome troubleshooting Suzanne How many times have you used the kit and reagents, I did look up how the kit works but the trichrome stain can be tricky. First of all you need to make sure that the mordant (bouins solution) is at 60C prior to placing your slides in them. We normally heat up our bouins for at least an hour prior to placing the slides in the solution. We leave in bouins for an hour and a half rather than an hour. I see that this is a microwave protocol I cannot comment on that but I don't think that the hematoxylin is the issue, if you leave longer in 1% acetic acid that may pull some of the blue stain out or I would try dehydrating with lower alcohol percentage that can pull some of the blue stain out. I would also try leaving it a bit longer in the bouins after you microwave it - that might help. Trichrome staining works best with fresh reagents so if you have used these reagents too much that could cause problems. I'm also not a big fan of the one step trichromes, they are quicker but sometimes not as good as the two steps, just my opinion. FYI - to evaluate your staining look for a smaller vessel, the smooth muscle should be nice a red, if its greyish or blue you have not done the stain properly. Good Luck Liz Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC Premier Laboratory, LLC PO Box 18592 Boulder, CO 80308 (303) 682-3949 office (303) 682-9060 fax (303) 881-0763 cell l...@premierlab.com www.premierlab.com March 10, 2014 is Histotechnology Professionals Day Ship to Address: Premier Laboratory, LLC 1567 Skyway Drive, Unit E Longmont, CO 80504 -----Original Message----- From: Suzanne Martin [mailto:smar...@lcpath.com] Sent: Tuesday, June 30, 2015 12:37 PM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Trichrome troubleshooting Hi all, We are having trouble troubleshooting our trichrome. It is too blue. We are using Leica's kit with the Weigerts iron with Gills. Most of the small bowel controls have seen improvement but patient tissue is not... strange. We have tried lessening the time in Gills, adding time for the last acid step, even lessening time and adding time in the Weigerts. Thoughts? Thank you. Suzanne HT _______________________________________________ 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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet