Alexa fluor 568 labeled anti goat igg

All animal experiments were performed in accordance with the Animal Experimentation Committee of Kyoto University and Tanabe R&D Service CO Ltd (Osaka, Japan). Mitochondria and foot process morphology in mouse podocytes were ultrastructurally analyzed by transmission electron microscopy (Hitachi S4700, Tokyo, Japan) at a magnification of ×4000. At least total 280 mitochondria and 160 foot processes were examined in three 20-week-old male C57Bl6 wild type mice and three 20-week-old male eNOS knockout mice (BKS.129P2(Cg)-Nos3/J) (Jackson Laboratories, Bar Harbor, ME, USA). The longest mitochondrial diameter and the shortest FP length were measured. For immunofluorescence for nephrin and PFK-L, the tissue samples were incubated with 10 mM citrate buffer (pH 6.0) at 50–90 °C for 30 min for retrievals and subsequently incubated overnight at 4 °C with primary antibodies, including goat anti-Nephrin (R&D Systems, Minneapolis, MN) and rabbit anti-PFKL. Then, sections were incubated with Alexa Fluor 488-labeled anti-rabbit IgG (Invitrogen) and Alexa Fluor 568-labeled anti-goat IgG (Invitrogen) for 1 h at room temperature, mounted with Vectashield anti-fade mounting medium (Vector Labs, Burlingame, CA) and finally examined by confocal microscopy (Leica TCS SP8; Leica Microsystems, Tokyo, Japan).

Immunohistochemical staining was performed for all samples of ectopic models using Ki-67 and TdT-mediated dUTP nick-end labeling (TUNEL). For double immunofluorescence of CD31 and α-smooth muscle actin (α-SMA), sections from all samples were incubated at 4°C overnight with a rabbit anti-human CD31 antibody (1:200; Abcam, Cambridge, UK) and goat anti-human α-SMA antibody (1:200; Abcam), then with fluorescein isothiocyanate-conjugated anti-rabbit IgG (1:200; Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 568-labeled anti-goat IgG (1:200; Invitrogen) for 1 h. Nuclei were counterstained with TO-PRO-3 iodine (1:1000, Invitrogen). Imaging was performed in a six-border zone region for each sample (All-in-one Fluorescence Microscope BZ-X700; Keyence, Osaka, Japan) and analyzed with a BZ-X Analyzer BZ-H3A. The number of analyzed samples was six, eight, nine and seven for the control, SQAP, RT and SQAP + RT groups, respectively. We observed 10 visual fields at 100× magnification and counted the number of positive cells in the evaluation of all immunostaining.