Masson Staining Kit 

In a narrow sense, connective tissue mainly consists of three types of fibrous components: collagen fibers, reticular fibers, and elastic fibers. Among these, collagen fibers are the most widely distributed and abundant, playing an essential role in maintaining tissue structure and mechanical strength.

Masson staining is one of the most widely used and classical methods for connective tissue staining and is considered a standard technique for the visualization and identification of collagen fibers. By using a combination of different dyes, this method simultaneously stains cell nuclei, collagen fibers, and muscle fibers within the same tissue section, thereby enabling clear differentiation of various tissue components.

The principle of Masson staining is mainly related to the molecular size of anionic dyes and differences in tissue permeability. Generally, dyes with smaller molecular weights can more easily penetrate dense tissues with low permeability, whereas dyes with larger molecular weights preferentially enter tissues with looser structures and higher permeability. In this staining system, dyes such as Light Green or Aniline Blue, which have relatively large molecular weights, preferentially bind to collagen fibers, while muscle fibers are mainly stained red by acid fuchsin–type dyes. Therefore, after Masson staining, muscle fibers typically appear red, whereas collagen fibers appear green (Light Green) or blue (Aniline Blue), allowing effective differentiation between collagen fibers and muscle fibers.

MCE Masson Staining Kit features low toxicity, environmental friendliness, simple operation, and stable performance. The staining results show clear coloration and high contrast. The stained sections can be stored for long periods with minimal fading, facilitating long-term preservation and image analysis. This kit is widely used in studies of connective tissue, muscle tissue, and collagen fibers, and is suitable for histological observation and related pathological analyses.

RT, 1 year. Protected from light.

Reagents Preparation

1. Fixative: It is recommended to use Formalin Mercury Solution or Formalin Salt Solution for tissue fixation to preserve tissue morphology and ensure optimal results in subsequent staining procedures.

2. Other Reagents: Xylene, neutral mounting medium or mounting reagent, graded ethanol solutions, distilled water, etc.

Procedure

Note: It is recommended to perform a pre-test before formal staining to determine the optimal staining conditions for tissue sections.

1. Deparaffinization and Hydration: Deparaffinize paraffin-embedded tissue sections using standard procedures and rehydrate to water. Set aside for staining.

2. Weigert Staining

2.1 Preparation of Weigert working solution: Mix Weigert Solution A and Weigert Solution B at a ratio of 1:1 (v/v) to prepare the Weigert working staining solution.

Note: The Weigert working solution should be prepared freshly before use and used on the same day. The staining performance decreases significantly after 24 h, and further use is not recommended.

2.2 Weigert staining: Immerse sections in the freshly prepared Weigert working solution for 5–10 min, then rinse thoroughly with running tap water.

3. Differentiation: Place the sections in Acidic Ethanol Differentiation Solution for approximately 2–10 s, followed by rinsing in distilled water.

4. Masson Bluing: Immerse the sections in Masson bluing solution to achieve nuclear blue coloration. Rinse, then wash in distilled water for 1 min.

5. Acid Fuchsin–Ponceau Staining: Stain sections in Acid Fuchsin–Ponceau Staining Solution for 5–10 min.

6. Weak Acid Treatment: Prepare a working solution of weak acid by mixing distilled water and weak acid solution at a 2:1 ratio. Wash the sections in this working solution for 1 min.

7. Phosphomolybdic Acid Treatment: Immerse the sections in phosphomolybdic acid solution for 1–2 min, followed by washing with the prepared weak acid working solution for 1 min.

8. Aniline Blue Staining: Place sections directly into Aniline Blue Staining Solution for 1–2 min, then rinse with weak acid working solution for 1 min.

9. Dehydration: Quickly dehydrate sections in 95% ethanol, followed by absolute ethanol three times, each for 5–10 s.

10. Clearing and Mounting: Clear the sections in xylene or a suitable clearing agent, then mount with neutral mounting medium. Observe under a microscope and acquire images as needed.

Interpretation of Staining Results

1. Cell nuclei, collagen fibers, and proteins: Blue.

2. Cytoplasm, muscle, and erythrocytes: Red.

1. This staining solution can be applied using dropwise staining or immersion staining; for a small number of sections, dropwise staining is recommended.

2. Weigert staining solution should not be prepared in advance for long-term storage. It should be freshly prepared according to the specified ratio before use. The prepared working solution generally loses its staining capability significantly after 24 h, and continued use is not recommended.

3. Tissue sections should be thoroughly deparaffinized, as fixation plays a critical role. Using different types of fixatives may extend or shorten the staining time.

4. For frozen sections, fixation can be performed with ether-ethanol mixture or formalin-based fixative for 10 s to 3 min, then wash with water before staining.

5. Differentiation in acidified ethanol should be adjusted according to section thickness, tissue type, and the age of the section.

6. This staining method uses Weigert iron hematoxylin to stain nuclei, but Harris hematoxylin can also be used. If nuclear staining is not vivid, it is acceptable because the primary purpose of this method is to distinguish collagen fibers from muscle fibers; only clear differentiation between these fibers is required, nuclear staining is optional.

7. In the staining result: nuclei are blue (hematoxylin), muscle fibers are red (Acid Fuchsin–Ponceau), and collagen fibers are blue (Aniline Blue) or green (Fast Green). Using Fast Green allows trichrome staining, whereas Aniline Blue provides two-color differentiation.

8. Weak acid solution can enhance color contrast and vividness. For large volumes, a 0.1–0.3% acetic acid solution may be used as a substitute.

9. Phosphomolybdic acid serves to differentiate red-stained collagen fibers to colorless or light red, while muscle fibers and cellulose remain red. In addition, it acts as a mordant for collagen fibers, promoting uniform binding with high molecular weight