Picrosirius Red Polarization Staining: A Technique To Detect Fibrosis in Cardiac Tissue Sections Using Bright-Field Microscopy

Fibrosis is an increased deposition of fibrillar collagen in the extracellular matrix of the damaged tissue. Structurally, it comprises triple-helix collagen polypeptide chains.

To stain collagen with picrosirius red dye, take a paraformaldehyde-fixed, paraffin-embedded, cardiac tissue section. Immerse the tissue section in xylene - an organic solvent - to dissolve the surrounding wax.

Sequentially treat the deparaffinized tissue with decreasing concentrations of alcohol to replace the xylene. Submerge the tissue in water to rehydrate it for better stain visualization in the subsequent steps.

Dip the tissue into picrosirius red solution containing Sirius red - an anionic azo dye - and picric acid.

Sirius red contains acidic sulfonic groups that interact with the basic amino groups of lysine, hydroxylysine, and arginine present in the collagen. The picric acid helps maintain the acidic pH, facilitating the binding of dye molecules in a parallel orientation with the collagen.

Rinse the slides in an acidified destaining solution to remove excess stain. Put a drop of mounting medium on the slide and view it under a bright-field microscope.

The collagen-containing fibrotic area appears bright red compared to the non-fibrotic, yellow-colored regions.