Gladstone Institutes Histology and Light Microscopy Core Massons Trichrome Protocol

Massons trichrome is a three colour staining protocol used to identify cells and surrounding connective tissues. Often a Weigert nuclear counterstain is added. Most MT protocols produce red keratin and muscle, green collagen and bone, light red to pink cytoplasm and black cell nuclei.

An iron haematoxylin (weigerts) is used to stain the nuclei, then the Briebrich scarlet (an acid dye) attaches to the acidophilic parts of the tissue, the phopho acid then removes the scarlet from the collagen leaving it open to staining with light green, or alternatively, aniline blue.

Pretreatment of the dewaxed sections in Bouins or aqueous saturated picric acid increases the vibrancy and crispness of the colors in the resulting slide.

Tissue: Formalin fixed paraffin embedded tissue or cryosections (with less resolution and quality), sectioned at 2-10m

Controls: Skin, lung, intestine, stomach

Solutions:Bouins Solution American Mastertech FXBOUGAL

Or saturated (1.2%) aqueous picric acid

Biebrich scarlet-acid fuchsin Stable for 6 monthsBiebrich scarlet 2.7gAcid fuchsin 0.3gdiH2O 300mlGlacial acetic acid 3ml

Phosphomolybdic - Phosphotungstic acid Stable for 6 monthsPhosphomolybdic acid 2.5mlPhophotungstic acid 2.5mldiH2O 100ml

1% Light Green with acetic acid Stable for 6 monthsLight green 5gGlacial acetic acid 2mldiH2O 250ml

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Alternate final stain:2.5% Aniline blue with acetic acid Stable for 6 months

Aniline blue 2.5gGlacial acetic acid 2mldiH2O 100ml

Procedure:1. Dewax and rehydrate to water2. Mordant in picric acid solution overnight at RT or 1hr in a 60oC waterbath3. Allow to cool for 10 min (if required) and wash in running tap water until yellow

disappears from tissue 10min4. Stain in Weigert’s working solution 10 min5. Wash in running tap water 10 min6. Stain in Biebrich solution 15 min7. Place in the phosphomol/ phosphotung acid 10-15min

Check collagen is unstained microscopically8. Stain in light green 20min9. 95% alcohol 10 dips10.100% alcohol 2 x 1 min11.Xylene 3 x 1 min12.Mount in DPX (Gurr)

Results: Nuclei - BlackMuscle, cytoplasm, keratin - RedCollagen - Green

Images:Adult mouse heart 4-chamber view stained with Masson’s Trichrome

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Mouse tongue with Masson’s Trichrome (2x)

Mouse tongue with Massons Trichrome (10x)

Mouse liver with Massons Trichrome (10x)

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