Non immune horse serum

For immunohistochemistry, sections were washed in PBS and incubated for 1h at room temperature with PBS containing 10% non-immune horse serum (Sigma-Aldrich now Merck, North Ryde NSW Australia) and 0.1% Triton X-100. Sections were washed then incubated for 18–24h at room temperature with the primary antibodies listed in Table 1, diluted in hypertonic PBS (PBS containing 17g NaCl per litre). Slides were washed in PBS prior to being incubated with Alexa Fluor® (AF) secondary antibodies: donkey anti-rabbit AF488 (Jackson; Cat# 711-545-152), donkey anti-goat AF594 (Jackson; Cat# 705-585-003) or donkey anti-chicken AF647 (Jackson; Cat# 703-605-155) diluted 1:1000 in hypertonic PBS, for 2.5h at room temperature. Slides were then washed in PBS, incubated with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich), washed in PBS, and mounted in buffered glycerol. Images were captured with a Zeiss AxioImager M2 microscope and AxioCam MRm camera using Zen software (Carl Zeiss). Where required, minor adjustments were made in images using Adobe Photoshop, to ensure close matching to labelling as viewed directly down the microscope. A minimum of four non-consecutive sections per sample were examined for each antibody.