Ab5694

Immunohistochemistry was performed using anti-αSMA (Abcam, ab5694, 1:200), anti-MMP2 (R and D Systems, AF1488, 1:400), and anti-phospho-NFκB p65 (Ser536) (Cell Signaling (93H1), #3033, 1:20) antibodies. Briefly, slides of knee joints from 4 month wildtype and Crtap^-/- mice were deparaffinized and treated with 3% hydrogen peroxide and proteinase K solution (20 mg/ml) for 10 min. Slides were blocked using 5% normal donkey serum and incubated for 1 hr. Primary antibodies were diluted in blocking solution and incubated overnight at 4°C. Slides were washed with PBS and incubated in anti-rabbit-biotin (Jackson Immunoresearch, 711-065-152, 1:400 for αSMA or 1:100 for phospho-NFκB) or anti-goat-biotin (Jackson Immunoresearch, 705-065-147, 1:500 for MMP2) for 1 hr. Following PBS wash, slides were incubated in streptavidin-HRP (Jackson Immunoresearch, 016-030-084, 1:400–500 for αSMA and MMP2 or 1:100 for phosho-NFκB) for 30 min then washed with PBS. Slides were incubated with DAB substrate (Vector Laboratories, SK-4100) for approximately 2 min, counterstained with hematoxylin, then cleared and mounted with a coverslip.

A hindgut was dissected from chicken embryo and fixed in 4% (w/v) paraformaldehyde (PFA)/phosphate buffered saline (PBS: 0.14 M NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄–12H₂O, 1.8 mM KH₂PO₄) for 10 min at room temperature (RT). The specimen was washed in PBS twice for 5 min each at RT, and embedded in FSC 22 Clear frozen section compound (Leica, 3801480). Cryostat sections of 20 μm were prepared (Thermo Scientific, Cryostar NX70). Following drying on the hotplate at 37°C, the sections were re-fixed in 4% PFA for 5 min at RT. After washing three times in PBS for 5 min each at RT, the sections were incubated with 0.5% blocking reagent (Roche, 1096176)/PBS for 1 h at RT, followed by primary antibodies; 1:300 dilution of anti-αSMA (abcam, ab5694) and 1:300 dilution of Tuj1 (R & D systems, MAB1195) overnight at 4°C. Following three times washing in PBS for 5 min each at RT, they were incubated with Alexa 488-conjugated second antibodies; 1:300 dilution of anti-rabbit IgG (H + L) (donkey; Invitrogen, A21206), 1:300 dilution of anti-mouse IgG2a (goat; Invitrogen, A21131) and 1:2000 dilution of DAPI (Nacalai Tesque, 11034–56) for 1.5 h at RT. After washing three times for 10 min each at RT, the specimens were sealed with Fluoromount (Diagnostic BioSystems). Fluorescent images were obtained using a Nikon A1R confocal microscope.