Azan Stain (Hasumi)

Azan Staining Method

Azan stain is a complex method for general histology. This stain is suitable for color photographs (Vasa Deferentia Fluctuation 1; Vasa Deferentia Fluctuation 2; Testicular Lobules; Testis-Ova; Female Genital System; Prolactin & Cutaneous Changes). Nuclei, erythrocytes, fibrin, fibrinoid, acidophilic cytoplasm, epithelial hyalin, and so on are stained "red" with azocarmine G. Collagen fibers, basophilic cytoplasm, mucus, and so on are counterstained "blue or orange" with aniline blue or orange G. The following manipulations should be conducted under room temperatures (around 24 C: heating the azocarmine G-drifting solution by 60 C is not necessary, unlike some other references).

[Deparaffinization]
(1) Xylene down I: 10 min
(2) Xylene down II: 10 min
(3) 100% ethyl alcohol (= ethanol) down I: 10 min
(4) 100% ethanol down II: 10 min
(5) 90% ethanol down: 10 min
(6) 70% ethanol down: 10 min
(7) Distilled water (= D.W.): 5 min

[Staining]
(1) Mordant solution: 10 min
(2) D.W.: 5 min
(3) Orange G solution: 15 min
(4) D.W.: 5 min
(5) Azocarmine G-drifting solution: 10 min
(6) Washing with running tap water: 5 min
(7) Aniline-ethanol: moderately (move a micro slide glass up and down for differentiation, by which excess and easily soluble dye in the tissue is removed after staining)
(8) Acetic acid-ethanol: 5 times (to remove aniline)
(9) Washing with running tap water: 2 min
(10) 5% H₃[P(W₁₂O₄₀)].30H₂O solution: 1-3 h
(11) Washing with running tap water: 2 min
(12) Aniline blue solution: 15 min
(13) 100% ethanol: moderately (move a micro slide glass up and down for differentiation)

[Dehydration and Immersion]
(1) 100% ethanol up I: 3 min
(2) 100% ethanol up II: 5 min
(3) Creosote-xylene up: 5 min
(4) Xylene up I: 5 min
(5) Xylene up II: 10 min
(6) Mount of sections in 6-7 drops of a mixed solution of Canada balsam and xylene with a 24 x 45-55 mm micro cover glass

[Components of each solution]
(1) Mordant solution (200 ml): Mix 10% K₂Cr₂O₇ solution (100 ml) with 10% Cl₃C.COOH solution (100 ml)
(2) Orange G solution (200 ml): Dissolve orange G (1.5 g) in D.W. (200 ml), and then add 4 drops of glacial acetic acid to this solution
(3) Azocarmine G-drifting solution (200 ml): Grind azocarmine G (0.4 g) with some amount of D.W. in a mortar minutely, and then add D.W. (up to 200 ml) to it. Heat this solution first with gentle fire, and gradually heat it up for approximately 1 h. After cool this solution down in a room temperature, add glacial acetic acid (2 ml) to it. Filtrating this solution is not very necessary
(4) Aniline-ethanol (200 ml): Mix aniline (0.2 ml) with 90% ethanol (200 ml)
(5) Acetic acid-ethanol (200 ml): Mix glacial acetic acid (2 ml) with 90% ethanol (200 ml)
(6) Aniline blue solution (200 ml): Dissolve aniline blue (0.5 g) in D.W. (200 ml), and then add glacial acetic acid (8 ml) to this solution
(7) Creosote-xylene (200 ml): Mix creosote (50 ml) with xylene (150 ml)