Toluidine Blue O is a commonly used synthetic dye belonging to the quinone-imine dye class. Dyes of this category mainly contain two chromophores, namely amino groups and quinoid benzene rings, which form chromogens to produce color. The cations in toluidine blue are responsible for the staining effect; acidic substances in tissue cells combine with these cations to be stained. Toluidine blue also contains two auxochromes that can promote the ionization of the dye into salts, helping chromophores exert staining power on tissues and thus coloring the tissue cells on the sections—it can stain cell nuclei to appear blue. In addition, the cytoplasm of mast cells contains metachromatic substances such as heparin and histamine, which will turn metachromatic purple-red when exposed to toluidine blue. Clinically, 1% Toluidine Blue O Stain (phosphate method) is often used to stain chondrocytes and mast cells. This reagent is for research use only and is not suitable for clinical diagnosis or other purposes.
Materials to Be Prepared by the User：
Distilled water or deionized water, graded ethanol solutions, glacial acetic acid, acetone,
xylene or environment-friendly deparaffinizing clearing solution, neutral balsam
Operating Procedures (For Reference Only):
(I) Mast Cell Staining
1. Deparaffinize sections to distilled water.
2. Immerse the sections in toluidine blue staining solution for 5–30 minutes, depending on section thickness and tissue type.
3. Rinse gently with distilled water or deionized water for 3–5 minutes.
4. (Optional) Differentiate with 0.5% glacial acetic acid until cell nuclei and granules are clearly visible.
5. Dehydrate rapidly with 95% ethanol and anhydrous ethanol, clear with xylene or deparaffinizing clearing solution, and mount.
Staining Results: Mast cells appear purple-red; background appears pale blue.
(II) Cartilage Staining
1. Place paraffin sections in xylene twice, 15 minutes each time.
2. Treat with graded ethanol solutions for 1 minute each, then rinse with tap water for 2 minutes.
3. Immerse in Toluidine Blue O Stain for 10–15 minutes.
4. Rinse with tap water for 2 minutes, blot dry with filter paper, and differentiate with acetone until chondrocytes are clearly visible as purple-blue.
5. Dehydrate with graded ethanol solutions step by step, clear with xylene or deparaffinizing clearing solution, and mount with neutral balsam.
Staining Results: Cartilage and osteoblasts appear purple-red; background appears pale blue.
(III) Cell Smear Staining
1. Dilute toluidine blue staining solution with 20% ethanol solution; generally, a dilution to 0.1% is required.
2. Immediately after preparing the cell smear, fix it in 95% ethanol for 15 seconds, then remove and place on absorbent paper.
3. Add 1–2 drops of the diluted toluidine blue staining solution for drop staining, and cover with a coverslip to allow the dye to penetrate the cells.
4. After 10–15 seconds, stand the slide upright and apply slight pressure to absorb excess dye with absorbent paper. Observe under a microscope directly without drying.
Staining Results: Cell nuclei and lymphocytes appear dark blue; nucleoli appear purple-red; red blood cells appear orange-red; cytoplasm and monocytes appear pale blue.
(IV) In Situ Hybridization Staining
1. Dilute the staining solution to the appropriate concentration with distilled water or deionized water; the dilution ratio is generally more than 1:100 based on experience.
2. Briefly immerse the glass slides in the diluted staining solution.
3. Soak the slides in distilled water or deionized water several times.
4. Perform squashing and fixation as required.
Staining Result: Positive signals of in situ hybridization appear dark blue/ violet blue, nuclei are light blue, and the background is clean and colorless.
Precautions:
1. For staining hard-to-stain tissues such as gastric mucosal tissue and cartilage tissue, the immersion time in toluidine blue staining solution should be appropriately prolonged.
2. For your safety and health, wear a lab coat and disposable gloves during operation.
ZJ26F0332679