Af4076

Mouse embryonic kidney rudiments were fixed overnight in 4% PFA at 4 °C, embedded in gelatin for cryosection or in wax for paraffin section. Cryo-samples and paraffin samples were sectioned at 8 μm and 10 μm intervals, respectively. The sections were
blocked and penetrated for 1 h at room temperature in 3% FBS, 10% blood serum, and 0.2% triton-X100 followed by primary antibodies incubation overnight at 4 °C. Sections were immunostained using the following antibodies: CALB1 (1:400, C9848, Sigma), SIX2 (1:200, 11562-1-AP, Proteintech), GFP (1:200, ab6556, Abcam), Integrin α8 (1:400, AF4076, R&D), BrdU (1:100, #555627, BD), ETV5 (1:200, 13011-1-AP, Proteintech), phosphorylated-ERK (1:100, #4370, CST), cleaved-Caspase3 (1:200, #9661, CST), EphrinB1 (1:400, AF473, R&D), pFAK (1:300, #611722, BD), act Integrin β1 (1:100, #553715, BD) and N-cad (1:300, #610920, BD), Alexa Fluor 568 donkey anti mouse IgG (1:500, Invitrogen, A10037), Alexa Fluor 488 donkey anti rabbit IgG (1:500, Invitrogen, A32790), Alexa Fluor 594 donkey anti goat IgG (1:500, Invitrogen, A32758), Alexa Fluor 647 donkey anti rabbit IgG (1:500, Invitrogen, A31573) and Alexa Fluor 594 donkey anti rat IgG (1:500, Invitrogen, A21209). Sections were mounted with anti-fade mountant with DAPI (Invitrogene, S36938) and imaged under an Olympus BX-53 microscope or Zeiss LSM 800 confocal microscope.

Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and electrophoretically transferred to PVDF membrane. Membranes were blocked with 5% nonfat dry milk/0.05% Tween-20/PBS for 1hr at room temperature, incubated with primary antibodies (goat anti-ITGA8 antibody, 1:500, R&D Systems AF4076, Minneapolis, MN; mouse anti-GAPDH, 1:10,000, Millipore Sigma CB1001, Billerica, MA) overnight at 4°C, washed with 0.1% Tween-20/PBS, incubated with horseradish peroxidase-conjugated secondary antibodies (rabbit anti-goat IgG HRP, 1:10,000, Thermo Fisher Scientific #31402; goat anti-mouse IgG HRP, 1:10,000, Thermo Fisher Scientific #62–6520) for 1 hr, washed with 0.1% Tween-20/PBS and then developed with enhanced chemiluminescence (Pierce^TM ECL 2 Western Blotting Substrate, Thermo Scientific #80196).

Cryosections of embryonic kidney rudiments were processed for proximity ligation assay as previously described^{66 (link)}. For the PLA labelling, probe anti-goat PLUS (Sigma, Duolink DUO92003), probe anti-rabbit MINUS (Sigma, Duolink DUO92004), and Duolink in situ detection reagent red (Sigma, Duolink DUO92008) were used. The following primary antibodies were used: rabbit anti-Ret antibody (1:200, Abcam, ab134100) goat anti-GDNF (1:400, R&D, AF212), rabbit anti-Ism1 antibody (1:300, Genescript) and goat anti-Integrin α8 (1:400, AF4076, R&D). Sections were mounted in the mounting medium with DAPI and subject to photograph using Zeiss LSM 800 confocal microscope.