Immunofluorescence Assay for Protein Localization

For immunofluorescence analysis, samples were fixed with 4% paraformaldehyde for 1 h [28 (link)], embedded in paraffin, and serial-sectioned (5 μm) with a Leica Jung Multicut 2045 Microtome (Leica). After deparaffinization and rehydration through a graded ethanol scale, sections were immersed in 10 mM sodium citrate buffer (pH 6.0) for 10 min in a microwave oven for antigen retrieval and then incubated for 30 min with a blocking solution (2% bovine serum albumin (BSA) and 0.1% Tween20 in phosphate-buffered saline (PBS), 138 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH 7.4). Sections were then incubated for 1 h at 37 °C [44 (link)] with primary antibodies (Alomone Labs, Jerusalem, Israel) all diluted 1:200 in blocking solution. The primary antibodies used are presented in Supplementary Table 1.
After washing with PBS, the specimens were incubated for 1 h at room temperature with goat anti-rabbit Cy3-conjugated antibodies (Abcam, Cambridge, UK, excitation 562 nm, emission 576 nm), diluted 1:250 in blocking solution. Nuclei were stained by incubating for 15 min with 49.6-diamidino-2-phenylindole (DAPI) 100 ng/ml in PBS (Sigma-Aldrich, Milan, Italy). Slides were mounted with CitiFluor (CitiFluor Ltd., UK) and examined with the Nikon Eclipse Ni fluorescence microscope (Nikon, Tokyo, Japan).