Masson Goldner Trichrome Staining: A Technique to Visualize Collagen in Heart Sections for Cardiovascular Fibrosis Detection

Stress-activated cardiac fibroblasts deposit excessive collagen in injured regions, a condition called fibrosis, which impairs heart function.

To visualize the extent of fibrosis, first, section paraffin-embedded heart blocks. Place the sections on a slide. Heat the slide to melt the paraffin, allowing sections to adhere.

Dip the slide in xylol to deparaffinize the tissue. Treat the sections with descending alcohol strengths, to rehydrate the sections, and aid subsequent binding of aqueous stain components.

Incubate the sections in an acidic fixative that cross-links proteins, stabilizing the tissue structure. Rinse with water to remove residual fixative.

Immerse the slide in hematoxylin solution. The cationic dye binds the negatively-charged DNA, staining the nucleus blue. Rinse with water to remove excess stain.

Incubate the slide in acid Fuchsin-Ponceau, a small-molecule anionic dye that interacts with positively-charged cytoplasmic proteins and collagen, staining them red. Treat with diluted acetic acid, to remove excess stain.

Immerse the slide in phosphomolybdic acid-Orange G solution. Orange G, a small-molecule dye, stains erythrocytes orange-red. Phosphomolybdic acid, a large-molecule acid, expels small-molecule dyes from collagen, rendering it colorless. 

Incubate the sections in light-green dye, whose molecules bind collagen fibers, coloring them green. Under a microscope, the fibrotic regions appear green indicating excessive collagen deposition.