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The page below is a sample from the LabCE course Histology Special Stains: Connective Tissue. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Masson's Trichrome Stain - 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: appendix, uterus
ReagentTimeTechnical Notes
Bouin's solution15 minutes (in heated solution)
Used as both a secondary fixative and mordant.
Microwave solution for 1 minute before placing slides inside. Microwaved Bouin's solution should not be reused.
Running water wash5 minutesEnsure that ALL picric acid (yellow) has been rinsed clear.
Weigert's working hematoxylin
10 minutes
Running water wash5 minutes
Distilled water3 changes
Biebrich scarlet acid fuchsin
5 minutes
Distilled water3 changes
Phosphotungstic/phosphomolybdic acid
10 minutes
Discard solution after use.
DO NOT rinse slides after this step as phosphotungstic/phosphomolybdic acid acts as a link for aniline blue stain to collagen.
Aniline blue5 minutes
Distilled water3 changes
< 1% acetic acid
1 minuteDiscard solution after use.

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