Fluorescent mounting media

After ISH processing, sections were processed for immunofluorescence using standard methods. Antibody diluent was 0.1% (w/v) Triton-X100, 10% (v/v) normal goat serum (NGS; Chemicon, USA), in PBS. Primary antibody (α-βIII-tubulin (TUJ1); mouse monoclonal, 1:2,000 dilution; Covance, USA) incubation was 4°C overnight in a humidified chamber, followed by secondary antibody (α-mouse FITC, goat raised; 1:400 dilution; ICN Cappel, USA) incubation for 2 hours in a dark humidified chamber at room temperature. Slides were coverslipped using fluorescent mounting media (DAKO, Australia).
Cell cultures were stained for βIII-tubulin and F-actin. βIII-tubulin was labelled by IHC according to standard practice, using the same antibody diluent, incubation times, and antibodies as described above. Following immunostaining for βIII-tubulin, cultures were incubated with phalloidin-FITC (Sigma-Aldrich, Australia) diluted 1:1,000 in PBS for 2 hours at room temperature to stain F-actin, then were washed 3 times with PBS, before being coverslipped using DAKO fluorescent mounting media.

Cells were seeded on 10-mm glass coverslips (Thermo Fisher Scientific) that were placed in 48-well plates, and treated with vehicle control or two doses of HtrA4 for 24 h. Cells were fixed with 4% paraformaldehyde (VWR, Radnor, USA) for 10 min and permeabilised with 0.1% Triton X-100 (Sigma-Aldrich) in PBS for 5 min. All procedures from here onwards were carried out at room temperature unless stated otherwise. Cells were blocked with 1% BSA in PBS for 2 h, incubated at 4 °C overnight with an anti-Ki67 rabbit monoclonal antibody diluted 1:250 (cat# ab16667, Abcam, Cambridge, United Kingdom), and then with a donkey anti-rabbit Alexa Fluor 568 antibody diluted 1:200 (cat# A10042, Thermo Fisher Scientific) for 2 h. Nuclei were stained with 5 µg/ml 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich) for 10 min, and coverslips were mounted onto glass slides with fluorescent mounting media (Dako, Glostrup, Denmark). Staining was visualised using an Olympus BX60 fluorescent microscope (Olympus, Notting Hill, Australia) and images were taken at 10x magnification using an Olympus DP70 camera and Olympus CellSens software (Olympus). Three to four images per each treatment were taken randomly. Ki67 positive cells were quantified using FIJI software (NIH, Maryland, USA). Experiments were repeated independently three times.