Dear Betsy, Don't say you are sorry for putting a long post on Histonet! To get troubleshooting help you need to say exactly what you did. If you wrote only, "why are my sections brown after Movat staining", nobody would understand your problem.
Your procedure starts with an hour in hot Bouin. For many years this has been a routine prior to trichrome stains done on sections of specimens fixed in neutral formaldehyde. It isn't part of Movat's original method (Arch. Path. 60:209-295, 1955), which probably was devised for sections optimally fixed for trichrome staining (in mixtures containing mercuric chloride). Movat's pentachrome is a trichrome method preceded by alcian blue (for no obvious reason) and an iron-haematoxylin for nuclei and elastin. It differs from older trichromes in using a mixture of yellow polyene dyes (saffron) to stain the collagen, instead of the blues or greens as in the Mallory and Masson methods. Your method includes "5% sodium thiosulfate -1 min" after the iron-haematoxylin stain for black nuclei and elastic fibres. This also isn't part of Movat's pentachrome method, and I wonder why. Did you inherit an informal list of instructions passed on within the lab? After a mercuric fixative, hydrated sections are dipped in iodine, followed by thiosulphate, before staining, to remove a black deposit (probably mercurous chloride) introduced by the fixative.I've been seeing similar informal passing of bad staining instructions in research labs for many years. Are you a victim of this trend? The thiosulphate step in your procedure obviously does no harm, because you got the right results with the dog tissues. There may be something different about your human specimens: perhaps inadequate fixation, or excessive acid treatment (if that's what Cal rite is) for decalcification. If the sections of human arteries look OK with a microscope, it might not matter that grossly they are a different colour from the dog small intestine sections. They are, after all, different tissues. A rather long, and not very helpful reply! John Kiernan London, Canada = = = ________________________________ From: Betsy Molinari via Histonet <histonet@lists.utsouthwestern.edu> Sent: May 5, 2021 9:46 AM To: histonet@lists.utsouthwestern.edu <histonet@lists.utsouthwestern.edu> Subject: [Histonet] Movats Hi Histonetters, I have received several human vessels for paraffin processing and to stain the sections for H&E and Movats. The H&E were fine. The human sections turned brownish yellow with the Movats.The control which is canine small intestine was perfect. The protocol is standard Bouins 1hr in 58C waterbath Rinse till yellow disappears Rinse in DH2O 1% Alcian Blue -20 min Rinse in running tap H2O -5min Alkaline alcohol-1hr Rinse 10 min tap H2O Rinse in DH2O Verhoff's Hematoxylin -15 min 3 changes DH2O Differentiate in 2% FeCl Rinse in DH2O 5% sodium Thiosulfate -1min Rinse in running tap-10 min Rinse in DH2O Woodstain scarlet/acid fuchsin-1.5 min Rinse in DH2O Rinse in 0.5% acetic acid water 5% aqueous phosphotungstic acid -2 changes 5 min each Rinse in 5% acetic acid water Rinse in 3 changes absolute ETOH 6% alcoholic Safran solution Absolute alcohol-xylene-coverslip The human slides were fine until the Safran step. When I removed them from the stain into the 100% they were a yellowish brown .Under the scope the colors were there, blue, red, yellow and black. But on the slide the tissue was that brownish yellow. The researcher does not like to strong yellow color. Since my control was fine I question if something was going on with their tissue. I do not know how the tissue was handled before it came into the lab. They were very calcified and were decaled for 1-3 days in Cal Rite. I do know they were not rinsed after decal and were put straight back into 10% NBF before I got them for processing. Should I have used a human control instead of canine? These were very large pieces that were crammed into the cassette. Thanks for the help. Sorry for the long post. Betsy Molinari HT(ASCP) Texas Heart Institute Cardiovascular Pathology 1101 Bates St. Houston,TX 832-355=6524 (lab) 832-355-6812 (fax) Betsy Molinari, HT (ASCP) Sr. Histology Research Technician CV Pathology Research Texas Heart Institute 6770 Bertner Avenue, MC 1-283 Houston, TX 77030 Office: 832-355-6524 | Fax: 832-355-6812 Email: bmolin...@texasheart.org texasheart.org<https://www.texasheart.org/> | texasheartmedical.org<https://www.texasheartmedical.org/> | facebook<https://www.facebook.com/Texas.Heart.Institute> | twitter<https://twitter.com/Texas_Heart> CONFIDENTIALITY NOTICE: This email and attachments contain information that may be confidential or privileged. If you are not the intended recipient, notify the sender at once and delete this message completely from your information system. Further use, disclosure, or copying of information contained in this email is not authorized, and any such action should not be construed as a waiver of privilege or other confidentiality protections. _______________________________________________ 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
