[Histonet] Movats

Thu May 13 09:43:13 CDT 2021

Hi John,
Thank you so much for your reply. This staining protocol is a "hand me down" . I have been using it for years with positive feed back so I had the "if it ain't broke don't fix it" mentality. This changed with this batch of samples. I just finished the study with 40 Movats total. The controls with each batch were fine and all the tissue from the client stained consistently with the strong yellow staining. They are satisfied with the results and we have discussed how to treat future samples.
  I did run controls skipping the Bouins step and the sodium thiosulfate . There was no discernable difference , so I saved at least an hour. I will miss the aroma of hot Bouins (NOT!) .
I have always learned something new or thought provoking with your posts.
Thank you!

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: BMolinari at texasheart.org
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From: John Kiernan <jkiernan at uwo.ca>
Sent: Friday, May 7, 2021 5:12 PM
To: histonet at lists.utsouthwestern.edu; Betsy Molinari <BMolinari at texasheart.org>
Subject: Re: Movats

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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 at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu>>
Sent: May 5, 2021 9:46 AM
To: histonet at lists.utsouthwestern.edu<mailto:histonet at lists.utsouthwestern.edu> <histonet at lists.utsouthwestern.edu<mailto:histonet at 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: BMolinari at texasheart.org<mailto:BMolinari at texasheart.org>
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