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Masson's Trichrome Staining - Staining Protocol

Sample type required: Deparaffinized and rehydrated tissue section (3-5 microns) on positively (+) charged slides

Preferred fixative: 10% neutral buffered formalin (NBF)

Control: Internal; appendix, fallopian tube, uterus, or small intestine are recommended choices
ReagentTimeTechnical Notes
Bouin's solution15 minutes (in heated solution)
Used as both a fixative and mordant.
Microwave solution for 1 minute before placing slides inside.
Running water wash5 minutesEnsure that ALL picric acid (yellow) has been rinsed clear.
Weigert's working hematoxylin
10 minutes
Running water wash5 minutesRequired for nuclear bluing.
Distilled water3 changes
Biebrich scarlet acid fuchsin
5 minutes
Distilled water3 changes
Phosphotungstic/phosphomolybdic acid
10 minutesDiscard solution after use.
Aniline blue5 minutes
Distilled water3 changes
< 1% acetic acid
1 minuteDiscard solution after use.

Post staining procedure: Tissue section should be dehydrated with 95% and absolute alcohols. Follow with two changes of xylene and then coverslip.
    Expected results:
    • Nuclei - Black
    • Cytoplasm, muscle, erythrocytes - Red
    • Collagen - Blue