[Histonet] Heidenhain's AZAN modification of mallory's triple stain: protocol, hand staining

From:yvan lindekens

Acronym of AZocarmine-ANilin blue. 
Martin Heidenhain really loved acronyms: AZAN, SUSA...

(By hand)

Fixation: any, but best results after fixation in
fixatives containing corrosive sublimate.

- Bring sections to water
- Stain in azocarmine G solution, warmed up to about
56C for 10 - 15 min or in azocarmine B @ RT for an
- brief rince in distilled water
- differentiation in anilin-ETOH untill only nuclei,
erytrocytes and musle (the latter depending on
fixation) are stained a vivid red (2)
- thorough rinse in ETOH-acetic acid to stop
differentiation and to remove anilin (3)
- Mordant in phosphotungstic acid solution for 1-3hrs
- brief rince in distilled water
- Stain in anilin blue-orange G solution for 1-3hrs(5)
- rinse in distilled water
- dehydrate/differentiate the counterstan in ETOH/IPA.
- Clear and mount

Results: collagen and reticular connective tissue
sharp blue, nuclei: vivid red, muscle orange to red
(depending on fixation), erytrocytes: red, glia
fibers: red.

(1) Azocarmine staining solution
Dissolve 0.1gm azocarmine G in 100 ml boiling
distilled water. Cool down, add 1ml glacial acetic
acid OR:

Dissolve 0.1gm azocarmine B in 100 ml distilled water
and add 1ml of glacial acetic acid

(2) Anilin-ETOH differentiation solution
ETOH 96%: 1000ml
Anilin: 1ml
If anilin isn't available: use crude wood spirit as
the differentiator OR:
ETOH 96%: 1000ml
pyridin: 1ml

(3) ETOH-acetic acid rinsing fluid
ETOH 96%: 1000ml
Glacial acetic acid: 10ml

(4) Mordanting solution
Distilled water: 1000ml
Phosphotungstic acid: 50gm

(5) Anilin blue-orange G staining solution

Distilled water: 1000ml
Anilin blue: 5gm
Orange G: 20gm
Glacial acetic acid: 80ml
Bring to the boil and cool down after dissolution of
the dyes and filter.

There are still a few other languages besides English.
Less important, I know, but nevertheless: they exist.
Perhaps it would be a good idea to import some German,
French, Italian, Spanish slide prep manuals into the States...

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