Fluorescently tagged phalloidin

In situ hybridization was performed at a hybridization temperature of 62 and 65 °C for SSC-washes with riboprobes against xirp2a^{20 (link)} and her7^{10 (link)}. Whole-mount immunofluorescence was used to visualize F-actin with fluorescently tagged phalloidin (Thermo Fisher Scientific, dilution 1:2000) and antibodies against acetylated tubulin (T6793, Sigma Aldrich) and alpha tubulin (GT114, Genetex), each at a dilution of 1:1000.

Cells were seeded onto Matrigel-coated PAGs and glass coverslips at a density of 7–9 × 10⁶ in supplemented media. Media was aspirated and adherent cells were washed with 1 × PBS, fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich) (v/v) for 10 min at room temperature, then washed three times with 1 × PBS and permeabilised for 5 min in permeabilising solution—0.2% Triton X-100 (v/v) (Sigma-Aldrich) in wash buffer (0.5% bovine serum albumin (BSA, ThermoFisher, (w/v) in PBS).Permeabilising solution was aspirated, and the cells were washed three times with wash buffer. For TAZ staining, samples were first blocked for 1 h at room temperature with 5% BSA in normal serum (10% Donkey serum (sigma-Aldrich) (v/v) in 1 × PBS). Cells were then incubated with a primary antibody targeting CTGF (1:1000, Ab6692, Abcam) or TAZ (1:200; Ab110239; Abcam) for 1 h at room temperature followed by washing three times with wash buffer and incubation with fluorescently-tagged secondary antibody (Abcam) diluted at 1:1000 in normal serum for 1 h at room temperature. Cells were counter-stained with DAPI (Sigma-Aldrich) and fluorescently-tagged phalloidin (ThermoFisher) using standard protocols and washed a final three times with wash buffer and twice with distilled water before mounting onto microscope slides.