Toludine Blue
Classification: connective tissue stain
Purpose: stains mast cells
Staining principle: Hydrogen bonding
Mechanism of staining: Metachromasia
Positive controls: small intestine, skin, any tissue containing mast cells
Well Stained Slide
Chromotropes stain purple
All other tissue stains various shades of blue or colorless if rinsed in 1% acetic acid.
REAGENT |
PURPOSE |
MECHANISM OF STAINING |
SOURCE OF ERROR |
Toluidine blue |
Basic primary dye demonstrates carboxylated
and sulphated acid mucopolysaccharides (progressive) |
Attaches to chromotropes by salt linkages. Metachromia occurs. |
Omitted: Mast cells not demonstrated. |
Too short: Will not affect the staining. |
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Too long: Will not affect the staining. |
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1% Acetic acid (optional) |
Differentiator |
|
Omitted: Difficulty seeing mast cells. |
Too short: No effect on the staining. |
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Too long: Other tissue components may be
pale. |
Special Considerations
Some labs use 1% Acetic acid to rinse the slide after Toluidine blue to make the background clear.
In order for merachormasia to occur the tissue’s reactive sites must be negatively charged, evenly spaced, close together, dye must be polymerized and water must be present.
Over powering. Mast cells are not demonstrated.
Treatment: dip in acetic acid, longer time in alcohol.
Well Stained Slide
Mast cells are demonstrated as purple.
Slide has been treated with acetic acid. Cell cytoplasm is clear and nuclei are blue.
References
Officer B. HIML251 Lecture notes: Elastic Stains: Toluidine blue staining method, March 19, 2009