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The page below is a sample from the LabCE course Histology Special Stains: Connective Tissue. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Phosphotungstic Acid-Hematoxylin (PTAH) Stain - 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
    Zinc chloride
    5 minutes (in heated solution)

    Used as a secondary fixative and mordant.
    Microwave solution for 20 seconds on high power before placing slides inside. Microwaved solution should not be reused.
    Running water wash5 minutesEnsure that ALL traces of the reagent have been removed.
    0.25% potassium permanganate
    5 minutes
    Running water wash 5 minutes
    5% oxalic acid 1 minuteTissue should be bleached/colorless after exposure
    Running water wash10 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