HPS Variant
General Oversight Stain
This is also known as the Hematoxylin Phloxine Saffron stain.
Materials
- Regressive hemalum
- Solution A
Material Amount Phloxine B 2 g Distilled water 100 mL Dissolve the phloxine B and filter. Preserve with a crystal of thymol.
- Solution B
Material Amount Saffron 30 g Absolute ethanol 1 L Add the saffron to 500ml absolute ethanol and extract at 60°C for 48 hours. Decant, and store in a dark bottle. Repeat with 500 mL fresh absolute ethanol and combine with the first extraction.
Tissue Sample
No fixative was specified, but most methods using acid dyes benefit from picric acid or mercuric chloride fixation. 5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable but results may be improved by refixing in Bouin’s fixative or saturated aqueous picric acid at 56°C for one hour prior to staining, then washing the sections in tap water to remove all yellow discoloration.
Protocol
- Bring sections to water with xylene and ethanol.
- Treat with hemalum for 5 minutes, differentiate and blue.
- Wash with running tap water for 5 minutes.
- Place in solution A for 5 minutes.
- Wash with running tap water for 5 minutes.
- Differentiate phloxine with 95% ethanol.
- Thoroughly dehydrate with absolute ethanol.
- Place into solution B for 5-10 minutes until a suitable red-yellow balance is achieved.
- Dehydrate with 4 changes of absolute ethanol.
- Clear with 4 changes of xylene and mount with a synthetic resinous medium.
Expected Results
- Nuclei – blue
- Cytoplasm – red shades
- Collagen – yellow
Notes
- Harris’ hemalum was named, but the formula given was a variant of Bencosme’s hemalum.
- Saffron is expensive. It may be available in East Indian grocery stores or health food stores as its most common use today is as a spice and food coloring. Usually, whole stigmata are more effective than ground saffron.
- Store and use the saffron solution at room temperature. It has a limited life of about a month and is best when freshly made. It is important that there be no water in the saffron solution and that sections be thoroughly dehydrated before it is applied. If contaminated with moisture the solution must be discarded.
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Histonet posting
