Tenascin

Immunostaining was performed as described (McGlinn et al., 2019 (link)). Briefly, embryos were fixed 2 h to overnight in 4% paraformaldehyde at 4°C and embedded in OCT. 8–10 μm sections were cut, tissue was permeabilized and blocked in 5% Normal Donkey Serum/0.3% Triton-X/PBS and incubated in primary antibodies overnight at 4°C, washed in PBS, and incubated for 3 h RT with Alexa-488 or 594-conjugated secondary antibodies (Jackson Immunoresearch, diluted 1:400). Slides were washed in PBS counterstained with DAPI, and mounted in Prolong Diamond. Primary antibodies: HoxA5 (Phillipidou et al., 2012 (link); 1:5000); Sox 9 (Millipore-Sigma AB5535, 1:500 or RnD Systems AF3075-SP 1: 500); RFP (Chromotek 5F8, 1:1000); Ebf3 (RnD Systems AF5166, 1:1000); Tenascin (Sigma-Aldrich T3413, 1: 100); PCNA (Santa Cruz sc-56 1:200), Cleaved Caspase 3 (CST 9661 1:200).

Fibronectin, type I collagen, tenascin and perlecan deposition into the ECM was measured by ECM ELISA using optimised monoclonal mouse-anti human perlecan (2 μg/ml) (ThermoFisher Scientific), type I collagen (2 μg/ml)(Sigma-Aldrich), Fibronectin (0.5 μg/ml) (Merck, Bayswater, Australia) and tenascin (0.5 μg/ml) (Sigma-Aldrich) antibodies as previously described by Kuo et al. (2011) [34 (link)].

Cell lysates were subjected to Western blot analysis using specific antibodies to CD63 (Santa Cruz Biotechnology, Dallas, Tx, USA), TSG101 (Santa Cruz Biotechnology), tenascin (TNC) (Sigma), fibrinogen-β (FGB) (Santa Cruz Biotechnology), phospho-NF-κB p65 (Ser536) (Cell Signaling Technology, CST, Denvers, MA, USA), and NF-κB p65 (CST). The blot was reprobed with actin-horseradish peroxidase (HRP) antibody (Santa Cruz Biotechnology) to compare protein load in each lane. Densitometry analysis was done using ImageJ software (National Institutes of Health, Bethesda, MD).