Zenon alexa fluor 488 mouse igg1 labeling kit

Sixteen weeks after the transplantation, the mice were euthanized via CO₂ inhalation. The gastrocnemius skeletal muscles were collected and fixed with 4% paraformaldehyde. The muscles were embedded in a specific immersing solution (SCMM) in liquid nitrogen and cooled isopentane. Afterwards, the frozen samples were embedded in a mounting medium and cut into 5 µm sections using adhesive film. Then, the sections were incubated with primary antibodies (anti-human nuclei antibody; Millipore). Human nuclei were confirmed using the Zenon Alexa Fluor 488 Mouse IgG1 Labeling Kit (Thermo Fisher Scientific, Waltham, MA). The sections were observed using fluorescence microscopy (Leica AF6000LX, Leica Microsystems, Wetzlar, Germany).

Sections were rehydrated and permeabilized with 0.5% Triton X-100 in PBS for 5 min. Samples were blocked with 10% normal goat serum in PBS and incubated with primary antibodies, as listed in Supplementary Table 1. Samples were then incubated with secondary antibodies, anti-rabbit or -rat Alexa Fluor 488-conjugate (Thermo Fisher Scientific, Waltham, MA), for 1 h. When the mouse antibody was used as the primary antibody, we used the Zenon Alexa Fluor 488 Mouse IgG1 Labeling Kit (Z25002: Thermo Fisher Scientific, Waltham, MA) in order to avoid cross-reactivity of the secondary antibody against the resident mouse IgG in the tissue. Nuclear counterstaining was performed using DAPI or DRAQ5 (DR50050; BioStatus; 1:1000), followed by mounting in Prolong-Gold (Thermo Fisher Scientific). Images were obtained by confocal microscopy (FV1000; Olympus, Tokyo, Japan). pATM+ tubular epithelia among tdTomato+ cells were quantified from five of 25 consecutive non-overlapping cortical fields in each kidney under high magnification (n = 5). These five fields were randomly selected in a blinded manner.