Dako pt link instrument

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.

Antigen retrieval was performed on 4 μm thick sections using Dako Target Retrieval Solution (S1699; Dako, Glostrup, Denmark) on a Dako PT Link Instrument (PT10126; Dako) for 30 minutes at 98 °C. Sections were then allowed to cool. A standard immunofluorescence protocol was performed as previously reported41 (link). Briefly, sections were incubated in primary and secondary antibodies for 1 hour at room temperature with five washes with DakoWash Buffer (K8007; Dako) between each incubation step. In order to assess glomerular and podocyte morphology we used a polyclonal rabbit anti–mouse p57 (1:200; SC8298; Santa Cruz Biotechnology, Santa Cruz, CA) and a goat anti–mouse SNP (1:400; SC21537; Santa Cruz Biotechnology) as primary antibodies and a polyclonal donkey anti–rabbit Alexa Fluor 555 (1:200; A31572; Life Technologies) and a polyclonal chicken anti–goat Alexa Fluor 488 (1:400; A2467; Life Technologies) as secondary antibodies. After incubation with secondary antibodies, sections were washed five more times with DakoWash Buffer, and DAPI (1:10,000; D1306; Life Technologies, Carlsbad, CA) was added for 20 minutes. Five final washes were done before coverslipping each section with Prolong Gold (P36934; Invitrogen, Carlsbad, CA).