Anti tenascin

Embryos were fixed in 4% paraformaldehyde (PFA)/PBS overnight at 4°C, rinsed in PBS-T (PBS with 0.05% Tween 20), and exposed to 0.2% Triton X-100/PBS. For whole-mount immunostaining, embryos were incubated in Blocking One solution (Nacalai Tesque), washed with 0.2% Triton X-100/PBS, and stained with the following primary antibodies: anti-Ebf3 (1:1000; R&D Systems, AF5166), anti-Tenascin (1:1000; Millipore, AB19013), anti-MF20 [1:200; Developmental Studies Hybridoma Bank (DSHB)]; Alexa Fluor-conjugated antibodies (1:1000; Thermo Fisher Scientific, A-21203, A-21206) were used as secondary antibodies. For WISH, GFP-marked RNA probes were generated from the cDNA of scxa amplified by reverse transcription PCR (RT-PCR) with the following PCR primers: Scxa forward primer, 5′-ACCGGCTCCGAGCCGATACGTGTA-3′; Scxa reverse primer, 5′-GGTTGCTGAGGCAGAATGTGCAGA-3′.

Both cellularized (with v-HCFs or a-HCFs) and decellularized samples were fixed in paraformaldehyde 4% in PBS (PFA, Alfa Aesar) for 15 min, washed with PBS, and cells were permeabilized with Triton X-100 (Sigma-Aldrich) 0.5% in PBS for 10 min. Samples were then blocked with bovine serum albumin (BSA, Sigma-Aldrich) 2% in PBS for 30 min, followed by staining with Phalloidin-Rhodamine (ThermoFisher) or primary and secondary antibodies, diluted in BSA 2% in PBS. Primary antibodies for fibroblasts staining were: Anti-Actin Smooth Muscle (α-SMA, Sigma Aldrich) and Anti-Discoidin Domain Receptor 2 (DDR2, ThermoFisher). Primary antibodies used for extracellular matrix protein detection were anti-Collagen I, anti-Collagen III, anti-Fibronectin, anti-Laminin, anti-Tenascin, (all purchased from Sigma-Aldrich), and anti-Collagen IV (Abcam). Secondary antibodies used were anti-mouse Alexa Fluor 555 and anti-rabbit Alexa Fluor 488 (both from ThermoFisher). Nuclei were counterstained with DAPI (Sigma-Aldrich). Samples were maintained in PBS during imaging by using Nikon Ti2-E fluorescence microscope (Nikon Instruments). Immunofluorescence experiments were performed in biological triplicate.