Sirius red stain

Sections were fixed for 24 h in PFA/BSA 4% at RT, washed in PBS two times for 5 min, and stained with hematoxylin for 8 min at RT. Slides were then washed twice in lukewarm tap water for 5 min and stained for 1 h at RT with Picro-Sirius Red solution (0.1% [m/v] Sirius Red [Sigma-Aldrich] in saturated aqueous solution of picric acid [LabChem]). Slides were washed two times for 5 min with “acidified water” (acetic acid 0.5%) and dehydrated in 100% EtOH three times for 2 min. Finally, the slides were cleared in Histo-Clear (National Diagnostics) for 5 min and mounted with VectaMount (Vector Labs).