Musto's Hematoxylin
Scarlet-Saffron for Elastic

Solutions

Stock hematoxylin
Hematoxylin 2 g
Ethanol, 95% 100 mL
Stock ferric chloride
Ferric chloride, hexahydrate 12.4 g
Distilled water 500 mL
Hydrochloric acid, concentrated 5 mL
Stock iodine
Potassium iodide 4 g
Iodine 2 g
Distilled water 100 mL
Working solution
Stock hematoxylin 30 mL
Stock ferric chloride 20 mL
Stock iodine 10 mL
Acetic acid
Acetic acid, glacial 1 mL
Distilled water 99 mL
Woodstain scarlet
Woodstain scarlet, 1% aqu. 50 mL
Tungstophosphoric acid 2 g
Prepare fresh
Tungstophosphoric acid solution
Tungstophosphoric acid 5 g
Distilled water 100 mL
Alcoholic saffron solution
Saffron 1 g Extract the saffron in ethanol at 56°C for 24 hours,
shaking occasionally. Cool and filter.
Ethanol, absolute 100 mL

Tissue sample
A 10% Formalin variant is suitable.
Paraffin sections at 5µ are preferred.

Method

  1. Bring sections to water via xylene and ethanol.
  2. Place sections in Bouin's fluid at 60°C for 1 hour.
  3. Remove the yellow colouration with running tap water.
  4. Place in working solution for 45 minutes.
  5. Wash well with tap water, then rinse with distilled water.
  6. Place into the woodstain scarlet solution for 5 minutes.
  7. Rinse well with distilled water.
  8. Differentiate in tungstophosphoric acid solution for 5 minutes.
  9. Transfer directly to acetic acid solution for 5 minutes.
  10. Dehydrate with 95% ethanol for 1 minute.
  11. Place in absolute ethanol, two changes, for 1 minute each.
  12. Place in alcoholic saffron solution for 5-10 minutes.
  13. Place in absolute ethanol, two changes, for 1 minute each.
  14. Clear with xylene and mount with a resinous medium

Expected results

Notes

  1. The original paper also gave a procedure using a Van Gieson counterstain.
  2. This is a modification of Verhöeff's hematoxylin which does not require differentiating.

 

Reference

Musto, Linda, (1981),
Improved iron-hematoxylin stain for elastic fibers.,
Stain Technology, v 56, page 185-7.

 


 

 

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