Alexa fluor 488 and 546 conjugated antibodies

The human monoclonal 828B3B3 and polyclonal anti-EMILIN-2 antibodies were produced in our laboratories as previously described [15 (link),17 (link)]. The anti-histidine antibody was from Abgent (San Diego, CA, USA). Ni-NTA agarose was purchased from QIAGEN (Milan, Italy). The secondary Alexa Fluor 488- and 546-conjugated antibodies and TO-PRO- 3 were from Invitrogen (Milan, Italy). The monoclonal anti-human CD31 antibody was from Invitrogen (Milan, Italy), the anti-αSMA antibody was purchased from Abcam (ab5694, Abcam, UK) and the anti-GLUT1 from Merck Millipore (Milan, Italy). The FuGene6 reagent was purchased from Promega (Milan, Italy). The in vivo anti-mouse PD-L1 antibody and the rat IgG2bisotype control were from BioXcell (Lebanon, NH, USA).

Kidneys were prepared from E16.5 embryos, newborn (NB) and adult mice (4–5 months old), fixed in 4 % paraformaldehyde and processed for tissue sections as described in [13 (link)]. Immunohistochemistry with the anti-Wnt11 antibody (Abcam) was performed using the tyramid signal amplification (TSA) kit (Perkin Elmer) as described in [13 (link)]. The Apoptosis TUNEL assays (Promega) were performed according to the manufacturer’s instruction as reported earlier [14 (link)]. Aquaporin-1 (AQP-1, Millipore), Aquaporin-2 (AQP-2, Sigma-Aldrich), thiazide-sensitive NaCl co-transporter (NCC, Millipore), acetylated α-tubulin (AT, Sigma-Aldrich), Phospho-Histone H3 (P-H3, Millipore) primary antibodies and Lotus Tetragonolobus Lectin (LTL, fluorescein labeled, Vector Laboratories), Dolichos Biflorus Agglutinin (DBA, rhodamine labeled, Vector Laboratories) lectins were used according to the manufacturers’ recommendations. Alexa Fluor 488 and 546-conjugated antibodies (Invitrogen) served as the secondary antibodies. DAPI (Sigma Pharmaceuticals) was used to stain the nuclei of the cells in the tissue sections. Electron microscopy samples were prepared as previously described [15 (link)] and examined using Phillips CM100 transmission electron microscope.