Toluidine Blue O is a commonly used synthetic dye belonging to the quinone-imine dye class. Dyes of this class mainly contain two chromophores, namely amino groups and quinonoid benzene rings, which form chromogens to produce color. The cations in Toluidine Blue O exert the staining effect, and acidic substances in tissue cells combine with these cations to be stained.
Toluidine Blue O contains not only two chromophores but also two auxochromes. The auxochromes can promote the ionization of the dye to form salts, enhance the staining power of the chromophores on tissues and thus stain the tissue cells on sections, and can stain cell nuclei to show a blue color. The metachromatic substances such as heparin and histamine in the cytoplasm of mast cells will exhibit a metachromatic purplish red color when exposed to Toluidine Blue O.
Operating Procedures (For Reference Only):
(I) Mast Cell Staining
1. Dewax sections to distilled water.
2. Immerse in Toluidine Blue Staining Solution for 10–15 minutes; the exact staining time depends on section thickness and tissue type.
3. Rinse gently with distilled water or deionized water.
4. (Optional) Differentiate with 0.5% glacial acetic acid until cell nuclei and granules are clearly visible.
5. Dehydrate rapidly in 95% ethanol and absolute ethanol.
6. Clear in xylene and mount the sections.
Staining Result: Mast cells appear purplish red; the background appears pale blue.
(II) Cartilage Staining
1. Immerse paraffin sections in xylene twice for 15 minutes each time.
2. Treat with a graded ethanol series for 1 minute each.
3. Rinse with tap water for 2 minutes.
4. Immerse in Toluidine Blue O Stain for 30 minutes.
5. Rinse with tap water for 2 minutes and blot dry with filter paper.
6. Differentiate with acetone until chondrocytes are clearly visible in violet blue.
7. Dehydrate through a graded ethanol series.
8. Clear in xylene and mount with neutral balsam.
Staining Result: Cartilage and osteoblasts appear purplish red; the background appears pale blue.
(III) Cell Smear Staining
1. Dilute Toluidine Blue Staining Solution with 20% ethanol solution, generally to a final concentration of 0.1%.
2. Immediately after preparing the cell smear, fix it in 95% ethanol, then remove and place on absorbent paper.
3. Add the diluted Toluidine Blue Staining Solution dropwise for staining, and cover with a coverslip to allow the dye to penetrate the cells.
4. Stand the glass slide upright and apply slight pressure to absorb excess dye with absorbent paper.
5. Examine under a microscope directly without drying.
Staining Result: Cell nuclei and lymphocytes appear dark blue; nucleoli appear purplish 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 greater 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 squash 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. It is recommended to conduct a pre-experiment with 1–2 samples when using this reagent for the first time.
2. For staining of hard-to-stain tissues such as gastric mucosa and cartilage tissue, the immersion time in Toluidine Blue Staining Solution should be appropriately prolonged.
3. For your safety and health, wear a lab coat and disposable gloves during operation.
4. This product is for research use only and shall not be used for any other purposes.
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