RE: saffron and humidity - re-submitted

From:Hermina Borgerink

During the sixties and early seventies, at Queen's University, Kingston, Ontario, and later at the University of Western Ontario in London,  Ontario, I used the HPS stain exclusively for all my routine staining.   It was particularly useful for research applications, and primarily for aortae because of the excellent contrast between elastic fibes, cytoplasm and connective tissues.  At the time we used Spanish saffron, derived from the crocus flower.  As expensive as it was, we needed only a little and it went a long way.  A synthetic, Saffran du Gatinais, is now available.


 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.


RESULTS:
Nuclei - dark blue
Cytoplasm - shades of red
Connective tissue - yellow

Hematoxylin
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.

Hermina

-----Original Message-----
From: RSRICHMOND@aol.com [mailto:RSRICHMOND@aol.com] 
Sent: Wednesday, June 11, 2003 12:39 PM
To: histonet@pathology.swmed.edu
Subject: Re: saffron and humidity - re-submitted


Patrick Paulusse at Pembroke General Hospital in Ontario, Canada writes

I am running an HPS stain as my routine stain on a Leica Autostainer.<< He 
has a trouble-shooting problem that I can't offer any advice about.

I didn't know that the HPS (hematoxylin-phloxine-saffron Masson trichrome) 
stain was still in use as a routine stain anywhere. The stain originated in 
Canada - in Montreal, I think - and was in use at Columbia University in NYC in 
the 1960's. It is used as a general oversight stain, instead of H & E - many 
pathologists favored this approach in the 1930's, including Chandler Foote at New 
York Hospital (Cornell University Medical Center) in NYC.

Saffron (not safranin) is a natural dye also used as a spice - it flavors 
paella, for instance. It's extremely expensive, and (unless it's come along 
recently) there is no synthetic substitute for it. (It's prepared from the flowers 
- specifically the pistils - of Crocus sativus and is grown in Spain and the 
Middle East - Penzey's offers a wide selection of saffrons.) For histologic 
use, the dye is extracted in alcohol - some methods specify prolonged boiling of 
the dye in alcohol, using a reflux condenser. 

Patrick Paulusse, would you bring us up to date on this interesting old 
technique?

Bob Richmond
Samurai Pathologist
Knoxville TN




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