Ab199475

Immunofluorescence was performed as previously described [18 (link)]. The paraffin-embedded sections were deparaffinized with xylene and treated with heat-mediated antigen unmasking solution. Then, the sections were permeabilized in 0.25% Triton X-100 for 15 min. After being blocked with 20% donkey serum for 30 min., the sections were incubated with the primary antibodies anti-Sema3A antibody (1:100, ab199475, Abcam), anti-CD31 antibody (1:50, AF3628, R&D Systems, RRID: AB_2161028), anti-α-SMA antibody (1:100, ab5694, Abcam, RRID: AB_2223021) and anti-α-SMA antibody (1:100, ab21027, Abcam, RRID: AB_1951138) at 4 °C overnight, then incubated with the corresponding fluorescein-conjugated secondary antibodies (1:100 dilution; Life Technologies). Nuclei were stained with DAPI for 10 min. All sections were observed under a confocal microscope (Nikon, Tokyo, Japan).

Tissue and cell samples were lysed in RIPA buffer (Beyotime Biotechnology, China). An equal amount (50 μg) of protein in each group was loaded and separated on 8–12% SDS-PAGE, then transferred onto 0.4 mm PVDF membranes (EMD Millipore, Billerica, MA, USA). After blocking in 5% skim milk for 1 h (h), membranes were incubated at 4 °C overnight with primary antibodies against Sema3A (ab199475, Abcam), Sema3A (ab23393, Abcam) neuropilin-1 (ab81321, Abcam), plexin-A1 (ab92346, Abcam), cyclinD1 (ab16663, Abcam), PCNA (ab29, Abcam, RRID: AB_303394), PDGFRβ (ab32570, Abcam, RRID: AB_777165), p-PDGFRβ (3173, Cell Signalling, RRID: AB_2252179), p53 (2524, Cell Signalling, RRID: AB_331743) and β-actin (A01010, Abbkine, RRID: AB_2737288). Then, membranes were incubated with secondary antibodies for 1 h at room temperature. All protein bands were visualized using ECL solution on a BioSpectrum Imaging System (UVP, Upland, CA, USA). The intensities of the bands were analysed using the ImageJ software package (National Institutes of Health, Bethesda, MD, USA). For immunoprecipitation, an equal amount (500 μg) of protein in each group was incubated with specific antibodies at 4 °C overnight with gentle rotation. The complexes were precipitated with Protein A/G agarose beads and analysed by western blotting.