Laboratory Protocols

• Cryotomy
• Histological Staining Techniques
  • Index
• Histological fixatives
• Peroxidase-anti-peroxidase Technique
• Preparation of tissue section adhesives
• Processing tissues for light microscopy (basics) (PDF 1.1mb)
• Processing tissues for light microscopy (detailed)
• Sample collection guidelines
• Setting up the light microscope
• Specimen preparation for electron microscopy

Histological Staining Techniques

Masson's Trichrome Stain

Lillie, R.D., 1948

• Dewax sections, rinse in alcohol, rinse in water.
• Stain in Weigert’s iron haematoxylin - 20 mins.
• Wash in running tap water - 1 minute.
• Differentiate in acid alcohol (1% hydrochloric acid in 70% alcohol) - 10 seconds.
• Wash in running tap water - 5 minutes.
• Stain in red solution - 15 mins (For C.N.S. stain for 1-2 mins).
• Rinse in distilled water.
• If using blue dye - treat in 2.5% phosphotungstic acid in 2.5% phosphomolybdic acid for 5-10 mins.
  If using green dye - treat in 5% phosphotungstic acid for 10-15 mins.
• Rinse briefly in distilled water.
• Shake off excess water and check the differentiation to make sure the red is removed sufficiently from any collagen and re-treat as necessary.
• Blue dye - stain for 5-10 mins,
  Green dye - stain for 1 min.
• Rinse in distilled water.
• Treat with 1% acetic acid for 1-3 mins.
• Rinse in distilled water.
• Shake off excess water and check the blue or green staining, re-treat with dye or acid as necessary.
• Dehydrate, clear and mount.

Results:

Nuclei - Black
Cytoplasm, keratin, muscle fibres, intercellular fibres - red.
Collagen, mucins - blue (or green).

Notes:

1. Red solution:
   1% Biebrich scarlet - 90 ml
   1% Acid fuchsin - 10 ml
   Glacial acetic acid - 1 ml

2. Blue dye: 2.5% aniline blue in 2% acetic acid
3. Green dye: 2% light green in 0.8% acetic acid

• Bouins or mercury fixation is best.
• Green dye gives best results on skin and C.N.S.. Use the blue dye for all other tissues.
• It is important to check and adjust the staining at each step as the end result is dependant on tissue type and fixation.
• Prolonged formalin fixation can cause problems with this stain.
• If a tissue will not stain well try pre-treating in Bouins for 30 minutes before staining.

NB This method is fine-tuned for tissues processed using our protocols as outlined here. If other methods for fixation and/or processing are used the timings may have to be changed; specifically the differentiation of the red dye and staining time of the blue/green dye. This may require an amount of trial and error before the correct timings are achieved.

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