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Renal Masson Trichrome Staining Protocol
NovaUltra Special Stain Kits
Principle
A differential visualisation of tissue elements is achieved with two similar acid dyes. A dye with a small molecule size (Biebrich scarlet/acid fuchsin) is used first which stains all tissue elements in the section. This red stain is selectively removed from unwanted areas by differentiating with phosphomolybdic acid, which also acts as a mordant for the next step of the procedure. A dye with a large molecule (light green) is then progressively applied to the section. A celestin blue/haematoxylin is used to stain the nuclei as it is more resistant than alum haematoxylins to removal by the acid solutions used in the method. This technique differentiates oedema from fibrosis and visualises immunological deposits. Post mordanting in bouins before staining with this method, gives enhanced acidophilic staining.
Technical Points
1. (step 8) - The mordanting by dodecamolybdophosphoric acid is easily removed by washing
Method
1. Sections to distilled water
2. Stain in Celestin blue 5 mins
3. Rinse in distilled water
4. Stain nuclei with alum haematoxylin 5 mins
5. Wash in running tap water 5 mins
6. Rinse in distilled water
7. Stain with Biebrich scarlet-acid fuchsin stain 10 mins
8. Rinse in distilled water
9. Treat with freshly prepared dodecamolybdophosphoric acid 10 mins
10. Drain, do not rinse
11. Stain with light green 10 mins
12. Rinse excess stain from slide with alcohol
13. Dehydrate, clear and mount.
Results
fibrinoid.................................red
immune deposits.......................red
basement membrane..................green
collagen.................................green
nuclei....................................blue
Reagent Formulae
1. Celestin blue
2. Biebrich scarlet
Biebrich scarlet (C.I. 26905) 0.9 g
Acid fuchsin (C.I. 42685) 0.1 g
Glacial acetic acid (Analar) 1.0 ml
Distilled water 100 ml
Mix the powders together then dissolve in distilled water. Add acetic acid.
2. 5% aq dodecamolybdophosphoric acid (Phosphomolybdic acid)
Prepare weekly
Phosphomolybdic acid 5 g
Distilled water 100 ml
4. 1% Light Green in
4% Acetic Acid stain
light green SF (CI 42095)
1.0 g
distilled water
96.0 ml
Glacial Acetic Acid
4.0 ml
Dissolve
the dye in the water, add Acetic Acid. Mix well. The stain
keeps well.
References
Masson PJ, (1929), J.Technol.Methods