During the sixties and seventies the HPS stain was our routine diagnostic stain. We never had problems with the saffron, but we did take care keeping it free from all moisture, including the alcohols prior to and right after the staining with saffron. Method below.
HPS – hematoxylin, phloxine, saffron
Any fixative. Paraffin.
1. Deparaffinize and bring to running water. Wash for 5 minutes.
2. Stain in Harris Hematoxylin for 5 minutes - stains nuclei
3. Wash in running water for 5 minutes.
4. Differentiate in 1% acid alcohol – removes excess hematoxylin
5. Wash in running water for 5 minutes.
6. Blue in ammonia water – blues nuclei
7. Wash in running water for 5 minutes.
8. Stain in 2% Phloxine B for 5 minutes – stains cytoplasm and connective tissue
9. Wash in running water for 5 minutes – removes excess phloxine from connective tissue
10. Differentiate through 1 change of 95% alcohol – finishes removal of phloxine from connective tissue
11. Continue dehydration through 4 changes of 100% alcohol – saffron is hydrophobic so all water must be removed from the section
12. Stain in Saffron for 5 – 10 minutes – stains collagen and connective tissue
13. Remove excess saffron through 4 changes of 100% alcohol.
14. Clear through 4 changes of xylene.
15. Mount sections with permount.
Nuclei – dark blue
Cytoplasm – shades of red
Connective tissue – yellow
Distilled water - 500ml
Potassium aluminum sulphate - 60gm
Hematein - 1.25gm
Glacial acetic acid - 5ml
Make up potassium alum by adding 60gm to 500ml of distilled water. Heat and let come to a boil. Add 1.25gm of hematein (remove from flame when adding) and continue boiling gently for 10 minutes. Cool, while shaking occasionally. Add 5 ml acetic acid when cool. Filter and add 1 crystal of thymol to prevent bacteria. Store in a dark bottle.
2% Phloxine B
Distilled water – 500ml
Phloxine B - 10gm
Add Phloxine to water and dissolve. Add 1 crystal of thymol to prevent bacteria. Store in a dark bottle.
Saffran du Gatinais
Absolute alcohol - 1000ml
Saffron du Gatinais – 30gm
(or dried Spanish saffron in small amounts)
Add 30gm of saffron to 500ml of absolute alcohol and let ripen in a 60ºC oven for 48 hours. Pour off the alcohol and store in a dark bottle. Add another 500ml of absolute alcohol to the saffron and again let ripen for 48 hours in a 60ºC oven. Add to the first batch obtained. Keep away from all moisture.
From: firstname.lastname@example.org [mailto:email@example.com] On Behalf Of Sharon Allen
Sent: Monday, February 09, 2009 9:56 AM
Subject: [Histonet] HPS stain
I would like to know if there are many labs out there doing the "Haematoxylin, Phloxine, Saffron stain, routinely & how they like it.
We have been doing it & it is lovely when it works but can be very unpredictable. I am aware of its hydrophobic properties & do what I can to accommodate this trait. It is mainly the saffron part that is tem