Modified Van Gieson Staining Kit 

Van Gieson staining (Van Gieson Staining, VG staining) is a classical method for connective tissue staining and is widely used for the visualization and identification of collagen fibers. The staining system typically consists of Ponceau and Picric Acid, which produces a clear color contrast between collagen fibers and muscle fibers within the same tissue section, enabling effective differentiation of connective tissue components. Therefore, this method is commonly applied in histological observation and pathological studies of collagen distribution, connective tissue structure, and tissue fibrosis.

The principle of Van Gieson staining is mainly related to the differences in molecular size of anionic dyes and the permeability of tissue structures. Generally, dyes with smaller molecular weights penetrate dense tissues with lower permeability more easily, whereas dyes with larger molecular weights preferentially bind to tissues with looser structures and higher permeability. In the VG staining system, Picric Acid (PA) has a relatively small molecular weight, while Ponceau or acid fuchsin dyes have comparatively larger molecular weights. During the staining process, the smaller dye molecules preferentially penetrate structures such as muscle fibers, whereas collagen fibers bind more readily to the larger dye molecules. As a result, after Van Gieson staining, collagen fibers typically appear red, while muscle fibers and cytoplasm appear yellow, allowing clear differentiation between collagen fibers and muscle tissue structures.

MCE Modified Van Gieson Staining Kit employs Celestine Blue and Mayer’s hematoxylin for nuclear staining, providing clearer and more stable nuclear visualization while facilitating longer preservation of stained sections. Ponceau S is used for collagen fiber staining, offering stable coloration and strong resistance to fading. This method enables effective differentiation between collagen fibers and muscle fibers, and can assist in distinguishing collagen fiber–derived tumors from myogenic tumors to a certain extent. It is also suitable for observing tissue or organ injury, repair processes, and the degree of fibrosis.

This product can be used for research on connective tissues and collagen fibers and is suitable for histological observation and related pathological analyses.

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

Mayer Hematoxylin Solution, Acidic Ethanol Differentiation Solution, Ponceau S Staining Solution, Saturated Picric Acid Solution: RT, 1 year.

Reagents Preparation

1. Fixative: 10% formalin.

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. Sample Fixation and Embedding: Tissues are fixed in 10% formalin, followed by routine dehydration and paraffin embedding.

2. Deparaffinization and Hydration: Cut tissue sections to a thickness of 4–5 μm, and deparaffinize using standard procedures followed by rehydration to water.

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

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

5. Differentiation: Place the sections in acid alcohol differentiation solution for approximately 1–2 s, followed by rinsing under running water for 10 min.

Note: The typical differentiation time is 1–2 s. After differentiation and washing, the sections may be examined under a microscope: If nuclear staining is too intense, perform an additional 1–2 s differentiation. If nuclear staining is too weak, restain with Celestine Blue and Mayer hematoxylin, followed by acid alcohol differentiation.

6. Van Gieson Staining

6.1 Preparation of Van Gieson staining solution: Mix Ponceau S staining solution and saturated Picric Acid solution at a ratio of 1:9 (v/v).

Note: It is recommended to prepare the Van Gieson staining solution fresh before use. For tissues containing low levels of collagen, the ratio may be adjusted to 1:7.

6.2 Van Gieson staining: Apply the Van Gieson staining solution to the sections for 1–2 min, followed by a quick rinse with water.

Note: a. Rinsing may be omitted after Van Gieson staining; however, a brief rinse is recommended to avoid uneven differentiation.

b. After Van Gieson staining, the water rinse and subsequent dehydration in 95% ethanol should be carried out rapidly to avoid the removal of Ponceau S and Picric Acid.

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

8. 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: Bright red.

2. Muscle Fibers, Cytoplasm, and Erythrocytes: Yellow.

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

3. Reagents should be used as soon as possible after opening to avoid potential effects on experimental results.

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

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