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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

    ReagentTimeTechnical Notes
    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 wash5 minutesEnsure that ALL traces of the reagent have been removed.
    Lugol's iodine 5 minutes
    Running water wash5 minutesEnsure that ALL traces of the reagent have been removed.
    5% hypo solution (sodium thiosulfate)
    1 to 2 minutes
    Running water wash 10 minutes
    0.25% potassium permanganate
    5 minutesOxidizes
    Running water wash 5 minutes
    5% oxalic acid
    Bleach until tissue is colorless

    Running water wash5 minutes
    Distilled water 3 changesDiscard solution after use.
    PTAH staining solutionOvernight at room temperature

    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