Llewellyn's PATS
(Periodic acid, Thiosemicarbazide, Schmorl)
This technique demonstrates carbohydrates in a manner analogous to that of the periodic acid Schiff reaction, but gives a blue product.
Materials
- Periodic acid, 1% aqueous.
- Thiosemicarbazide, 1% aqueous
- Schmorl’s solution
Material Amount Ferric chloride, 1% aqueous, fresh 30 mL Potassium ferricyanide, 1% aqueous, fresh 4 mL Distilled water 6 mL This solution should be used fresh. Prepare immediately before use.
Tissue Sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.
Protocol
- Bring sections to water via xylene and ethanol.
- Oxidize in 1% periodic acid for 10 minutes or longer.
- Rinse well with water.
- Place into 1% thiosemicarbazide for 5 minutes.
- Wash well with running tap water to remove all traces of thiosemicarbazide.
- Place into freshly made Schmorl’s solution for 10 minutes.
- Wash well with water.
- Counterstain with nuclear fast red.
- Rinse well with water.
- Dehydrate with ethanol, clear with xylene and mount with a resinous medium.
Expected Results
- Oxidizable carbohydrates – blue
- Reducing substances – blue
- Nuclei – red
Human kidney stained with periodic acid, thiosemicarbazide, Schmorl’s reaction 400x magnification.
Human liver stained with periodic acid, thiosemicarbazide, Schmorl’s reaction 400x magnification.
Human intestine stained with periodic acid, thiosemicarbazide, Schmorl’s reaction 100x magnification.
Human intestine stained with periodic acid, thiosemicarbazide, Schmorl’s reaction 400x magnification.
Notes
- It is well known that metallic azides can be explosive. However, thiosemicarbazide is not a simple metallic azide, it is a carbazide (-C=N-) and is not explosive. It is safe to use.
- Reducing substances which may be present are also coloured blue. This includes melanin and enterochromaffin. Others may also be seen.
- If acid hydrolysis is used instead of periodic acid oxidation (as in Feulgen’s nucleal reaction), nuclei are coloured blue.
- The Schmorl’s solution is from Lillie’s modification of Schmorl’s ferricyanide reduction method for tissue reducing substances.
- There is a modification of this technique for fungi.
- Thiosemicarbazide has a hydrazine group at one end of it’s molecule and a thiocarbamyl group at the other. The hydrazine group combines with any aldehydes generated by periodic acid oxidation, and in so doing attaches the thiocarbamyl group to the carbohydrate. The thiocarbamyl group is a more powerful reducing group than aldehydes and rapidly reduces ferricyanide to ferrocyanide, which is immediately trapped by the ferric salt to form prussian blue at the site.
- Thiosemicarbazide: (hydrazine–thiocarbamyl) = H2NNH–CSNH2
Safety Note
Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.
References
- Hayashi, I., Tome, Y. and Shimosato, Y., 1989
Thiosemicarbazide used after periodic acid makes methenamine silver staining of renal glomerular basement membranes faster and cleaner.
Stain Technology, v 64, p 185. - Lillie, R.D., (1954)
Histopathologic technique and practical histochemistry Ed.2
Blakiston, New York, USA. - Llewellyn, B. D., (2014)
Thiosemicarbazide-ferricyanide reduction for the histochemical demonstration of aldehydes in tissue sections.
Biotechnic & Histochemistry, v 89, p 228-31.
