Tissue Analysis of Collagen Deposition

Formalin-fixed, paraffin-embedded tissue sections were analyzed by Masson’s trichrome, hematoxylin/eosin (H&E) staining or by immunofluorescence (IF) as described [30 (link),35 (link),36 (link),37 (link)]. Collagen deposition was quantified using the Sirius Red/Fast Green kit (Chondrex, Woodinville, WA, USA; #9046). Cultured cells were fixed with 4% paraformaldehyde. Upon blocking, the following primary antibodies (4 °C, 12 h) and secondary antibodies (RT, 1 h) diluted in phosphate-buffered saline (PBS) with 0.05% Tween 20 were used: goat anti-DCN (R&D Systems BAF1060 at 1:200); rabbit anti-Col6A1 (Abcam ab182744 at 1:75; and ab6588 at 1:100); rabbit anti-Co1A1 (Abcam ab34710 at 1:75); Donkey Alexa 488-conjugated (1:200) IgG from Invitrogen; and Cy3-conjugated (1:300) IgG from Jackson ImmunoResearch, West Grove, PA, USA. Nuclei were stained with Hoechst 33258 (Invitrogen Waltham, MA, USA; H3569). IF images were acquired with a Carl Zeiss upright Apotome Axio Imager Z1/ZEN2 Core Imaging software. Quantification was done using NIH ImageJ software by counts in 10 separate 10× fields. Amira 5.4 software (VSG) was used for data capture and analysis. Confocal images were acquired with TCS SP5/LAS AF software (Leica, Buffalo, Grove, IL, USA).