Sirius Red Staining Kit 

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

Sirius Red is a strongly acidic anionic dye that binds specifically to collagen fibers under acidic conditions (pH 2–3) through interactions with basic amino acid residues in collagen molecules. As a result, collagen fibers are selectively stained red. Under polarized light microscopy, different types of collagen fibers exhibit birefringence due to differences in molecular alignment and structure. Typically, type I collagen fibers appear orange-yellow to bright red, whereas type III collagen fibers appear green, enabling visualization of collagen distribution and, to some extent, differentiation of collagen subtypes.

Although immunohistochemical methods can also be used to detect type I and type III collagen fibers, the required antibodies are relatively expensive and the procedures are time-consuming and complex. In comparison, Sirius Red staining is cost-effective, simple to perform, and highly reproducible, making it widely used in histological and pathological studies.

MCE Sirius Red Staining Kit consists of hematoxylin staining solution and Sirius Red staining solution and is mainly used to observe abnormal collagen deposition or fibrosis in various pathological tissues. Under a conventional light microscope, collagen fibers in tissues such as the heart and blood vessels appear red. Under polarized light microscopy, this method is useful for the classification and evaluation of different types of fibrotic lesions.

Hematoxylin Solution: RT, 1 year. Protected from light.

Sirius Red Staining Solution: 4°C, 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.

3. Instrument: Conventional light microscopy or polarized light microscopy.

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 3–8 μm, and deparaffinize using standard procedures followed by rehydration to water.

3. Hematoxylin Staining: Apply hematoxylin staining solution to the sections for 3–10 min, followed by gentle rinsing with water for 30–120 s.

4. Sirius Red Staining: Stain the sections with Sirius Red staining solution for 10–60 min. For tissues that stain easily, the staining time can be reduced to 10–20 min.

5. Rinsing: Gently rinse the sections with running tap water to remove excess staining solution from the surface.

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

7. 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

Conventional Light Microscopy

1. Collagen fibers: Red.

2. Muscle fibers and blood cells: Yellow.

3. Cell nuclei: Blue-brown to black.

Polarized Light Microscopy

1. Type I collagen fibers: Strong orange or bright red birefringence.

2. Type III collagen fibers: Green birefringence.

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

2. To obtain clearer results under polarized light microscopy, it is recommended that the section thickness be 6–7 μm when preparing samples for polarized light observation.

3. If hematoxylin staining is omitted, the cell nuclei will appear light red, and the contrast between collagen fibers and muscle fibers will be more pronounced.

4. After Sirius Red staining, the sections should not be treated with low-concentration ethanol for prolonged periods, as this may cause the yellow coloration of muscle fibers to fade.

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

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