Modified 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 Modified Masson Staining Kit employs Celestine Blue hematoxylin for light nuclear staining, offering shorter differentiation time compared to conventional methods. It 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.

Celestine Blue Staining Solution, Mayer Hematoxylin Solution: 4°C, 1 year. Protected from light.

Bouin Fixative Solution, Acidic Ethanol Differentiation Solution, Ponceau–Acid Fuchsin staining solution, Phosphomolybdic Acid Solution, Aniline Blue Staining Solution, Weak Acid Solution: RT, 1 year. Protected from light.

Reagents Preparation

1. Fixative: 10% formalin.

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

操作步骤

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. Bouin Mordant Treatment: Place the sections in Bouin Fixative Solution and incubate overnight at room temperature, or incubate in a 37°C incubator for approximately 2 h for mordanting. Subsequently rinse under running tap water until the yellow coloration disappears.

3. Celestine Blue Staining: Immerse the sections in Celestine Blue staining solution for 2–3 min, followed by a brief rinse with water.

4. Mayer Hematoxylin Staining: Stain the sections in Mayer hematoxylin solution for 2–3 min, and then rinse gently with water.

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

6. Acid Fuchsin–Ponceau Staining: Stain the sections in Ponceau–Acid Fuchsin staining solution for 5–10 min, and then rinse gently with distilled water.

7. Phosphomolybdic Acid Treatment: Immerse the sections in phosphomolybdic acid solution for 2 min.

8. Aniline Blue Staining: Without rinsing, directly transfer the sections into Aniline Blue Staining Solution for 5 min, then rinse with weak acid working solution for 2 min.

9. Weak Acid Treatment: Treat the sections with weak acid solution for approximately 2 min to enhance staining contrast.

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

11. 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. Collagen fibers: Blue.

2. Muscle fibers, cytoplasm, cellulose, keratin, and erythrocytes: Red.

3. Cell nuclei: Blue-brown.

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

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

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

4. 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.

5. 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 dyes.

6. After Aniline Blue staining, the sections should be treated with a weak acid solution to remove excess blue dye from the cytoplasm, thereby enhancing the clarity and contrast of the staining results. For tissues fixed with Zenker fixative, the treatment time with the weak acid solution may be extended to approximately 5 min.

7. This product is for R&D use only, not for drug, household, or other uses.

8. For your safety and health, please wear a lab coat and disposable gloves to operate.