Laboratory Protocols
Histological Staining Techniques
Lendrum et al, 1962.
- Dewax sections, rinse in alcohol, rinse in water.
- Celestine blue - 5 minutes.
- Wash in tap water.
- Harris' haematoxylin - 5 minutes.
- Wash in tap water - 1 minute.
- Differentiate in acid alcohol (1% hydrochloric acid in 70% alcohol) - 10 seconds.
- Wash and blue in tap water - 5 minutes.
- Rinse in 95% alcohol.
- Shake excess alcohol from the slide.
- Stain in martius yellow solution - 2 minutes.
- Rinse the slide briefly in tap water on both sides, just enough to remove all the martius yellow.
- Rinse in deionised water.
- Stain in crystal ponceau solution - 10 minutes.
- Rinse in deionised water.
- Differentiate with 1% phosphotungstic acid - 5-10 minutes, or until most of the red dye comes out of the connective tissue.
- Wash in deionised water - 30 seconds.
- Stain in methyl blue solution - 2 minutes.
- Rinse in deionised water.
- Dehydrate, clear and mount.
Results:
Nuclei - brown/black.
Collagen - blue.
Red blood cells - yellow.
Muscle and fibrin - red.
Martius yellow solution
0.2g martius yellow
100mls. 95% alcohol
2g phosphotungstic acid
Dissolve the phosphotungstic acid in the alcohol before adding the
martius yellow.
Crystal ponceau solution
1% crystal ponceau 6R in 2% acetic acid made up in deionised water.
Methyl blue solution
0.5% methyl blue in 1% acetic acid
Note: Deionised water is used in place of distilled water for this method to alleviate problems of non-specific and/or difficult differentiation of the red stain.