This article discusses the process of staining collagen in cardiac tissue using picrosirius red dye to visualize fibrotic areas. The methodology includes steps for tissue preparation, staining, and imaging under a microscope.
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.
After fixing the heart tissue, place it on a tissue sectioning device, cut it into 2-millimeter-thick slices, and place the sections in an embedding cassette.
Following paraffin embedding and additional sectioning according to the text protocol, deparaffinize the slides by placing them into a tank with xylene for 30 minutes. Then, rehydrate the tissue in a sequentially diluted alcohol series, followed by distilled water.
Next, use adequate picrosirius red solution to completely cover the tissue sections, and incubate for one hour. Then, use a 0.5% acetic acid solution to rinse the slides two times, and rinse the samples twice in absolute alcohol.
Air-dry the samples, and mount them in synthetic resin with a coverslip. Then, use visible light under a microscope at 200X magnification to image the slides.
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