Virginie Orgogozo
Muscle dye staining protocols
Toluidine Blue and Thionine
These closely related dyes offer a good and extremely rapid staining
reaction for diagnostic work. The staining pattern is particularly
useful for lymph node and brain evaluation due to the metachromatic
qualities of the staining solutions.
REAGENTS REQUIRED
1 Toluidine Blue or Thionine 0.5 g
2 Ethanol 20 ml
3 Distilled water 80 ml
4 Phenol 0.5 g
METHOD
1 Use fixed or air dried sections.
2 Apply stain to the section for 20 seconds.
3 Rinse in water.
4 Mount in water or an aqueous mounting medium. (For
permanent mount, dehydrate in acetone, clear in acetone-xylene, then in
xylene and mount).
RESULTS
Nuclei - Blue
Cytoplasm, muscle and connective tissue - pink to purple
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Alcoholic Pinacyanide
This metachromatic stain is particularly recommended for staining thyroid sections.
REAGENTS REQUIRED
1 Pinacyanole 0.5 gm
2 70% ethyl or methyl alcohol 100 ml
METHOD
1 Use fixed or air dried section.
2 Apply stain to the section for 5 - 15 seconds.
3 Rinse in water.
4 Mount in water or aqueous mounting medium.
RESULTS
Nuclei - blue
Cytoplasm, collagen - pink
Muscle and elastic tissue - violet
Plasma cells - red
Haemosiderin - orange
Thyroid and pituitary colloid and amyloid - bright red
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Phloxine - Methylene Blue - Azure B
This method gives a permanent stain with a similar appearance to
H&E. It is recommended particularly for unfixed tissue where it
gives good nuclear definition and clear staining.
REAGENTS REQUIRED
1 Solution A
Phloxine 0.5 g
Acetic acid 0.2 ml
Distilled water 100 ml
2 Solution B
Methylene Blue 0.25 g
Azure B 0.25 g
Borax 0.25 g
Distilled water 100 ml
3 0.2% acetic acid
METHOD
1 Rinse section briefly in water and drain.
2 Apply staining solution A for 1 minute.
3 Wash in water for 10 seconds and drain.
4 Apply staining solution B for 30 seconds.
5 Remove excess stain in 0.2% acetic acid in
distilled water. Agitate the slide gently until stain ceases to flow
from the section (about 20-30 seconds).
6 Differentiate in three washes of 95% ethanol.
7 Dehydrate in two changes of absolute ethanol, clear
in two changes of xylene and mount in synthetic mounting medium.
RESULTS
Nuclei and bacteria - blue
Collagen and muscle - bright rose to red
Erythrocytes - scarlet