PicroSirius red staining was performed using Kiernan protocol.
5mm sections were deparaffinized in xylene 10min, treated with a graded series of alcohol (100%, twice 5min, 95% twice 4min, 70% twice 3min), rehydrated in PBS. Nuclei were stained with a Wiegert’s hematoxylin kit (reference sigma 1.15973.0002) following the manufacturer’s protocol. Tissues were then stained for one hour at room temperature with picroSirius red solution (Sirius red, reference sigma 365548 (0.5g diluted in 500ml of picric acid (reference sigma P6744–1GA)). Tissues were then washed twice in acidified water (0.5% glacial acetic acid diluted in distilled water). Tissues were dehydrated with 3 changes of 100% ethanol, washed in xylene, and mounted on slides using Richard Allan™ Scientific Mounting medium (ThermoFisher, reference # 4112).
5mm sections were deparaffinized in xylene 10min, treated with a graded series of alcohol (100%, twice 5min, 95% twice 4min, 70% twice 3min), rehydrated in PBS. Nuclei were stained with a Wiegert’s hematoxylin kit (reference sigma 1.15973.0002) following the manufacturer’s protocol. Tissues were then stained for one hour at room temperature with picroSirius red solution (Sirius red, reference sigma 365548 (0.5g diluted in 500ml of picric acid (reference sigma P6744–1GA)). Tissues were then washed twice in acidified water (0.5% glacial acetic acid diluted in distilled water). Tissues were dehydrated with 3 changes of 100% ethanol, washed in xylene, and mounted on slides using Richard Allan™ Scientific Mounting medium (ThermoFisher, reference # 4112).