Phosphotungstic Acid-Hematoxylin (PTAH) Staining - Staining Protocol
Sample type required: Deparaffinized and rehydrated tissue section (3-5 microns) on positively (+) charged slides
Preferred fixative: Zenkers; however 10% neutral buffered formalin (NBF) may be used as well
Control: Skeletal or cardiac muscle
| Zenker's fixative | 5 minutes (in heated solution)
| Used as both a fixative and mordant. Microwave solution for 45 seconds on high power before placing slides inside. |
| Running water wash | 5 minutes | Ensure that ALL traces of the reagent have been removed.
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| Lugol's iodine | 5 minutes |
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| Running water wash | 5 minutes | Ensure that ALL traces of the reagent have been removed.
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5% hypo solution (sodium thiosulfate)
| 1 to 2 minutes |
|
| Running water wash | 10 minutes |
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0.25% potassium permanganate
| 5 minutes | Oxidizes |
| Running water wash | 5 minutes |
|
5% oxalic acid
| Bleach until tissue is colorless
|
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| Running water wash | 5 minutes |
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| Distilled water | 3 changes | Discard solution after use. |
| PTAH staining solution | Overnight at room temperature |
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Post staining procedure: Dehydrate quickly through two changes each of 95% and 100% alcohol, along with two changes of xylene, then cover-slip.
Expected results:
- Cross striations - Blue
- Nuclei - Blue
- Collagen - Red-brown
- Elastic fibers - Purplish
