Sc21537

At P21 and 24 weeks, podocytes were counted in 20 whole glomeruli in a single 800‐μm slice from each mouse podocytes were identified by their nuclear expression of p57 (sc‐8298; Santa Cruz Biotechnology, Santa Cruz, CA) and cytoplasmic expression of synaptopodin (sc‐21,537; Santa Cruz Biotechnology). Following immunofluorescence labeling kidney slices were cleared using ethyl cinnamate (ECi; 99% concentration, product number 112372; Sigma‐Aldrich) and then imaged using a Leica SP8 Multiphoton Microscope fitted with a Leica 20× BABB objective lens. Twenty glomeruli per kidney were imaged over the z‐axis at 1‐μm intervals to obtain a stack of optical sections through each whole glomerulus.

For frozen sections, human or mouse kidney tissue was embedded in OCT, snap frozen in liquid nitrogen and stored at −80°C. Tissue sections were cut and fixed in −20°C acetone before immunofluorescence staining. Sections were blocked at room temperature for 1 h and incubated with the primary antibodies – CD45 (anti-mouse: Clone 30-F11, 550539,BD Pharmingen, anti-human: Clone HI30, 14-0459-82, eBioscience), CD11b (anti-mouse and anti-human: Clone M1/70, 101202, Biolegend), CD4 (Clone RM4-5, 5505319, BD Pharmingen), CD8 (Clone YTS.169.4, MBS520216, Mybiosource), F4/80 (Clone CI:A3-1, MCA497GA, AbD Serotec), CD3 (anti-human: Clone OKT3, 70-0037, Tonbo Biosciences), WT1 (polyclonal rabbit, SC-192, Santa Cruz), and synaptopodin (polyclonal goat, SC-21537, Santa Cruz) in blocking buffer at 4°C overnight. Sections were incubated with the appropriate secondary antibody (Invitrogen Life Technologies, Grand Island, NY, USA) and mounted with medium containing DAPI to visualize nuclei (Vector Laboratories, Burlingame, CA, USA). Fluorescence images were acquired using a Zeiss 700 LSM confocal microscope with an iLCI Plan Neofluar 63×/1.3 Oil Imm Corr M27 with a AxioCam camera and analyzed using the Zen software (Carl Zeiss Group, Hartford, CT, USA).

One 800-μm-thick slice from each kidney was immunostained for podometric analysis as previously described (Puelles et al. 2016b (link)). In short, slices were subject to a 1-h antigen retrieval protocol at 98 °C for 1 h using Dako Target Retrieval Solution (S1699; Dako, Glostrup, Denmark) in a Dako PT Link Instrument (PT10126; Dako). Slices were then incubated for 6 days at 37 °C in a primary antibody solution containing polyclonal rabbit anti-mouse p57 (1:200; SC8298; Santa Cruz Biotechnology, Santa Cruz, CA) and polyclonal goat anti-mouse SNP (1:400; SC21537; Santa Cruz Biotechnology). All antibodies were diluted in Dako Antibody Diluent (S0809; Dako). Slices were then washed in 4 ml of Dako Wash Buffer (K8007; Dako) at 37 °C for 24 h. Slices were then immersed in a secondary antibody solution containing polyclonal donkey anti-rabbit Alexa Fluor® 555 (1:200; A31572; Life technologies, Carlsbad, CA) and polyclonal chicken anti-goat Alexa Fluor® 488 (1:200; A21467; Life technologies) and incubated for 6 days at 37 °C, protected from light. Finally, slices were washed in 4 ml of Dako Wash Buffer (K8007; Dako) at 37 °C for 24 h. A full list of antibodies used in this study may be found in Supplementary Table 2.