Muscle fibers are components of muscle tissue and are composed of muscle cells. According to their morphological and functional characteristics, muscle fibers can be classified into smooth muscle, skeletal muscle, and cardiac muscle (note: smooth muscle is not striated muscle; only skeletal and cardiac muscle are striated). There are many staining methods for muscle fibers, such as Ponceau staining, Aniline Blue staining, and Phosphotungstic Acid Hematoxylin staining. The Tannic Acid‑Azophloxine method is a differential staining method using two acidic dyes applied sequentially.
The staining principle of Azophloxine Staining Solution is that tannic acid readily penetrates highly permeable collagen fibers, which stain yellow; azophloxine preferentially enters less permeable muscle fibers, which stain red. Azophloxine Staining Solution is used to distinguish muscle fibers from collagen fibers with clear contrast and good color fastness. It can also demonstrate myoepithelial cells and is used in the diagnosis of myoepithelial tumors of the breast, skin, and other tissues. This reagent is for research use only and not for clinical diagnosis or other purposes.
Protocol (for reference only):
1. Fix tissues routinely in Bouin’s fixative (Carnoy’s fixative or neutral formalin may also be used) for 2–3 hours.
2. Transfer directly into 95% ethanol for routine dehydration and embedding.
3. Prepare 4‑μm paraffin sections, dewax with xylene or dewaxing clearing agent, and hydrate to water; rinse under running water for 1 minute.
4. Treat with Phosphomolybdic Acid Differentiation Solution for 10 minutes.
5. Stain in Azophloxine Staining Solution for 10–15 minutes.
6. Differentiate in Acid Alcohol Differentiation Solution for 3–5 seconds, then dehydrate repeatedly in absolute ethanol several times.
7. Complete routine dehydration, clear with xylene or dewaxing clearing agent, and mount with neutral balsam.
Staining Results:
Muscle fibers and myoepithelial cells: Rose red
Collagen fibers: Yellow
Precautions:
1. Use Bouin’s fixative whenever possible. Other fixatives may result in weaker staining. 10% neutral formalin may be used as an alternative but yields inferior results compared with Bouin’s fixative.
2. Acid Alcohol Differentiation Solution is critical. Differentiate until excess red dye is removed and muscle fibers appear bright red.
3. For your safety and health, wear a lab coat and disposable gloves during operation.
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