Cartilage tissue is composed of chondrocytes and cartilage matrix; cartilage tissue and the surrounding perichondrium together form cartilage. There are numerous staining methods for cartilage, such as the toluidine blue method, alcian blue method, safranin O method and others. Based on the differences in fibrous components contained in the matrix, cartilage is classified into hyaline cartilage, elastic cartilage and fibrocartilage. Hyaline cartilage is mainly distributed in the upper respiratory tract, the medial ends of ribs, the articular surfaces of short bones and other locations.
Unna Alkaline Methylene Blue Staining Solution, also known as Polychrome Methylene Blue, is an oxidized mixture of methylene blue. It exhibits polychromasia (blue, purple, red and other hues) after preparation via specific processes and has unique applications in microbiology, pathology and materials science. Unna's alkaline methylene blue staining solution can be used in combination with alizarin red for the staining of hyaline cartilage. Bone tissue (sites of calcium salt deposition) combines with alizarin red to appear red, cartilage reacts with polychrome methylene blue to appear purple, cytoplasm appears yellow and cell nuclei appear blue. This method is the optimal staining method for phalanges of 6–8 month-old fetuses. This reagent is for research use only and not intended for clinical diagnosis or any other applications. Composed of potassium carbonate, methylene blue and other components.
Materials to Be Prepared by the User:
1. Graded ethanol, distilled water, xylene or environment-friendly deparaffinization solution, environment-friendly clearing solution, neutral balsam, alizarin red S staining solution
2. Microscope
Operating Procedures (For Reference Only):
1. Fix the tissue in 90% ethanol for 12–24 hours, then perform paraffin embedding and sectioning. Cut sections at a thickness of 3–5 μm, deparaffinize to water and rinse with distilled water.
2. Immerse in alizarin red S staining solution for 5–20 minutes, then rinse briefly with distilled water.
3. Counterstain in Unna's alkaline methylene blue staining solution for 2–6 minutes, then differentiate with 95% ethanol until the staining is distinct.
4. Perform routine dehydration, clear with xylene or environment-friendly clearing solution, and mount with neutral balsam.
Staining Results:
Sites of calcium salt deposition appear red, cartilage matrix appears purple, cytoplasm appears yellow and cell nuclei appear blue.
Precautions:
1. For insoluble calcium staining, the staining time of alizarin red S should be determined according to the content of calcium salts. Observation under a microscope is required until the staining reaction is intense without diffusion.
2. After staining with alizarin red S staining solution, calcium deposits are birefringent.
3. Unna's alkaline methylene blue staining solution is volatile; a cover should be placed over the staining vessel during the staining process.
4. For your safety and health, wear a lab coat and disposable gloves during operation.
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