Picrosirius red collagen staining

Collagen visualization on PFA fixed, paraffin embedded tissue sections was carried out by Picrosirius red staining. Briefly, sections were dewaxed with following incubation steps: 10 minutes in xylene 100%, 10 minutes in xylene:ethanol 1:1 V/V, 10 minutes in ethanol 100%, 5 minutes in ethanol:distilled H₂O 1:1 V/V. Nuclei were stained in Weigert’s iron hematoxylin for (Sigma, HT1079) according to manufacturer’s instructions; sections were washed 10 minutes in running tap water and collagen was stained in Picrosirius red solution (0,5 g of Direct Red 80 (Sigma, 365548) into 500 ml of Picric Acid Solution 1,3% (Sigma, P6744) for one hour. Following collagen staining, sections were washed twice in acidified water (glacial acetic acid 1:200 V/V) and dehydrated in ethanol 100%, cleared in xylene and mounted with Eukitt Quick-hardening mounting medium (Sigma, 03989). Bright field images and collagen birefringence⁵³ images were collected with a Leica 5000B microscope under parallel and orthogonal polarized light, respectively, and quantified using ImageJ software; signal threshold was maintained equal for all images across all conditions.