⑫ Staining

Azan staining

The Azan staining method stains the collagen fibers within fibrous connective tissue blue.

Staining procedure

1DeparaffinizationXylene3 changes, 10 minuntes each
2Removal of xylene100% ethanol3 changes, 5 minutes each
3Hydration95%, 70% ethanol5 minutes each
4WashingRunning tap water2 minutes
5RinsingDistilled water 
6StainingAzocarmine G solution (preheated at 50℃)1 hour at 50℃ and 1 hour at RT (Filter before use)
7 WashingRunning tap water 
8DifferentiatingAniline-alcohol solutionDifferentiate until nuclei stand out sharply (This step may be omitted)
9RinsingAcetic alcohol 1 to 2 minutes (This step is omitted when sept8 is omitted.)
10WashingRunning tap water 
11Mordanting5% phosphomolybdic acid solution3 hours or overnight
12WashingRunning tap water 
13RinsingDistilled water  
14StainingAniline blue-orange G mixtureApproximately 15 to 30 minutes. After staining, wipe slides with filter paper to drain off the solution.
15Differentiating100% ethanol3 changes. Agitate the slides during this step.
16Microscopic check Differentiate until blue and red color can be distinguished.
17ClearingXylene3 changes, 10 minuntes each

Staining solution

Azocarmine G solution

Azocarmine G0.1g
Distilled water100ml
Acetic acid1ml

Aniline blue-Orange G mixture

Aniline blue (water-soluble)0.5g
Orange G 2g
acetic acid8ml
distilled water100ml

Boil, cool, and filter the mixture and then dilute the stock solution 3–5 times for use.

Differentiating solution

Aniline-alcohol solution

95% ethanol100ml

Acetic alcohol

Acetic acid1ml
100% ethanol100ml

Phosphotungstic acid solution

Phosphotungstic acid (substituted by phosphomolybdic acid)5g
distilled water100ml

Staining results