Modified Masson Trichrome Staining Solution

Connective tissue, in its narrow sense, consists of three types of fibers: collagen fibers, reticular fibers, and elastic fibers. Among them, collagen fibers are the most abundant and widely distributed. The traditional method of staining connective tissue, known as Masson three-color staining or Masson staining, is considered the authoritative and classic technique for visualizing collagen fibers. This staining method selectively displays collagen fibers and muscle fibers while staining the nucleus. It works based on the size of the anionic dye molecule and the tissue penetration. Smaller molecules can easily penetrate dense and less permeable tissue structures, while larger molecules can only enter loose and highly permeable structures. In Masson staining, light green or aniline blue dyes with large molecular weights are used. As a result, muscle fibers appear red, while collagen fibers appear green (light green) or blue (aniline blue), enabling the differentiation between the two types of fibers.
Our modified Masson tri-color staining technique differs from the conventional method in terms of the staining of cell nuclei using celestine blue hematoxylin. It offers several advantages, including a short differentiation time (1-2 minutes), clear and vibrant colors, wide applicability to staining tissue paraffin sections, frozen sections, etc., long-lasting staining that resists fading, and excellent visibility of collagen fibers (blue), muscle fibers, cytoplasm, cellulose, keratin, and red blood cells (red), and nuclei (blue-brown).
The components required for our modified Masson tri-color staining technique include:
- Preservation reagent (A): Bouin solution (50ml) - Store at room temperature (RT)
- Staining solution (B): Lapis lazuli staining solution (50ml) - Store at RT and avoid light exposure
- Staining solution (C): Mayer hematoxylin staining solution (50ml) - Store at RT and avoid light exposure
- Differentiation solution (D): Acidic Ethanol Differentiation Solution (50ml) - Store at RT
- Staining solution (E): Ponceau Fuchsin Staining Solution (50ml) - Store at RT and avoid light exposure
- Staining solution (F): Phosphomolybdic Acid Solution (50ml) - Store at RT and avoid light exposure
- Staining solution (G): Aniline Blue Staining Solution (50ml) - Store at RT and avoid light exposure
- Staining solution (H): Weak Acid Solution (50ml) - Store at RT.

Bring your own materials:
1. 10% formalin fixative
2. Distilled water
3. Series of ethanol
Operation steps (for reference only):
1. The tissues are fixed in 10% formalin fixative solution, and routinely dehydrated and embedded. The slices are 4μm thick and deparaffinized to water as usual.
2. Put the slices into Bouin solution, leave them at room temperature overnight or put them in a 37°C incubator for 2 hours for mordant dyeing, and then rinse with running water until the yellow color on the slices disappears.
3. Dye with lapis lazuli dyeing solution dropwise for 2~3min, then wash with water.
4. Dye with Mayer hematoxylin staining solution dropwise for 2~3min, then wash with water.
5. The acidic ethanol differentiation solution is differentiated for a few seconds and rinsed with running water for 10 minutes.
6. Dye with Ponceau red fuchsin staining solution for 10 minutes and rinse with distilled water.
7. Treat with phosphomolybdic acid solution for about 10 minutes.
8. Pour off the upper liquid, do not wash the slices with water, and drop directly into the aniline blue staining solution for 5 minutes.
9. Weak acid solution treatment for 2 minutes.
10. 95% ethanol is quickly dehydrated. Anhydrous ethanol is dehydrated 3 times, 5~10s each time.
11. Xylene is transparent 3 times, 1~2min each time. Sealed with neutral gum.
Dyeing result:
Collagen fiber: blue
Muscle fibers, cytoplasm, cellulose, keratin and red blood cells: red
Cell nucleus: blue-brown

1. Deparaffinization of sections should be as clean as possible. The differentiation time of acid ethanol should be determined according to the thickness of the section, the type of tissue and the old and new.

2. The role of phosphomolybdic acid is to make the red-stained collagen fibers differentiate into colorless or light red, while the muscle fiber and cellulose are still bright red; on the other hand, the collagen fibers are mordant to make the collagen fibers. It is easier to combine with aniline blue liquid.

3. After dyeing, the aniline blue solution is treated with a weak acid solution. The purpose is to remove the blue color in the original pulp and make the dyeing bright and clear. Such as Zenker liquid fixed tissue, weak acid solution treatment can be extended to 5min.

4. The weak acid solution can make the color clearer and brighter. If you use a large amount, you can prepare a 0.1-0.3% acetic acid solution to replace it.

Store according to the requirements of each component, and the validity period is 12 months.

Modified Masson's Trichrome Stain Kit