[Histonet] Mallory-Azan stain

Tue Sep 10 10:54:48 CDT 2019

John, we love it when you "ramble!" It gives us an appreciation for the history and breadth of histo techniques.

Tim Morken
Supervisor, Electron Microscopy/Neuromuscular Special Studies
Department of Pathology
UC San Francisco Medical Center

-----Original Message-----
From: John Kiernan via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
Sent: Tuesday, September 10, 2019 8:43 AM
To: 'Histonet at lists.utsouthwestern.edu'; Betsy Molinari
Subject: Re: [Histonet] Mallory-Azan stain

Mallory's (easy) and AZAN staining (difficult) are different methods!

Frank B. Mallory's trichrome stain  (Journal of Medical Research 13: 113-136, 1905) is the earliest and one of the simplest of its kind: acid fuchsine followed by a solution containing orange G, aniline blue and phosphotungstic acid (PTA).  Martin Heidenhain's trichrome is usually called AZAN (from Azokarmin and Anilinblau, the German names of two of the dyes used. I've not read his original 1916 publiication, but a very thorough account was given by Manfred Gabe in his 1976 Histological Techniques book (ISBN 3540901620), pp. 219-223. I used this quite a bit in the 1990s mostly on paraffin sections of Bouin-fixed decalcified rats' heads. It is a 15-step procedure taking >2 hours and it includes two critical differentiations requiring careful microscopic control. Instructions based on my experiences can be found in Histological and Histochemical Methods (5th ed., 2015, pp.198-200).

AZAN gives a wider range of colours than Mallory's or Masson's trichrome or the various one-step trichromes (Cason, Gomori, Gabe). The related Romeis "cresazan" procedure was used to identify at least 6 anterior pituitary cell-types until the 1950s when more rational histochemically based stains were introduced by Adams, Herlant, Pearse and others. Nowadays, immunostainng accurately shows the hormones in pituitary cells, but much more expensively.

All trichromes give poor results after simple fixation in neutral formaldehyde. Bouin or (better) a mercuric chloride-containing fixative is needed. Zinc-formalin is probably also OK. (I haven't tried it myself for this purpose). If material fixed in NBF must be used, immerse hydrated paraffin sections in saturated aqueous picric acid either for 2h at 56-60C or overnight at room temperature, then wash well in water before staining. (Bouin's fluid is often used, but its ingredients other than picric acid are unnecessary.) Experiments are needed to learn the mechanism of this "rescue" of  staining properties of sections formaldehyde-fixed tissue, which is sometimes wrongly called "mordanting". My guess is that it's comparable to antigen retrieval. It has been claimed that citrate buffer is just as good, though the photos are unconvincing (J. Histotechnol. 26, 133).

It should be possible to identify Purkinje fibres with any staining method that shows nuclei and myofibrils, such as H&E or a trichrome method simpler than AZAN. A glycogen stain such as PAS might show this substance in the otherwise pale areas around the central nuclei of Purkinje fibres. I suggest persuading your researcher to let you try something simpler before attempting Heidenhain's AZAN. Wheater's Functional Histology has a nice photomicrograph of a section stained with H&E and for endocardial elastin (looks like orcein).

Enough rambling!
    John Kiernan
Anatomy & Cell Biology
University of Western Ontario
London, Canada
= = =

________________________________
From: Betsy Molinari via Histonet <histonet at lists.utsouthwestern.edu>
Sent: 09 September 2019 10:53
To: 'Histonet at lists.utsouthwestern.edu' <Histonet at lists.utsouthwestern.edu>
Subject: [Histonet] Mallory-Azan stain

Hi histonetters,
I have a researcher that wants to stain Purkinje fibers and has requested a Mallory-Azan stain.
I have no experience with this stain. I have looked online for information but am reaching out to you for personal advice.
Thanks.
Betsy Molinari HT,ASCP
Texas Heart Institute
6770 Bertner Ave.
Houston, TX  77030
832-355-6524 (lab)
832-355-6812 (fax)

Betsy Molinari
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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