Right kidney removed at each autopsy was fixed in cold 10%-buffered formalin for 24 h.
Transversally trimmed kidney tissues were submitted to a routine process for paraffin
embedding [14 (link), 15 (link)]. The renal sections were prepared, deparaffined, and stained with
Hematoxylin & Eosin (HE). Anti-nephrin IgG {sc-19000, Santa Cruz Biotechnology (SCB),
Dallas, TX, USA}, anti-WT1 IgG (sc-7385, SCB), anti-synaptopodin (65194, Progene,
Heidelberg, Germany) were used for immunohistochemistry. Anti-phospho-Y1208-nephrin IgG
was produced in our laboratory, as reported [33 (link)].
Immunostaining assays were performed as described previously [14 (link),15 (link),16 (link)]. The sections were dewaxed and then were treated with 0.1 M citrate
buffer at pH 6.0 in an autoclave for five minutes for antigen retrieval. The sections were
incubated at 4°C for overnight with primary antibodies, washed with PBS and incubated with
biotin-labeled anti-mouse and anti-goat antibody (Vector, Burlingame, CA) for 1 hour at
room temperature. Avidin-biotin coupling reaction was performed on the sections using a
kit (Elite®, Vector). All antigens were visualized as brown with 3-3” diamino
benzidine (Nacalai, Kyoto, Japan).
Transversally trimmed kidney tissues were submitted to a routine process for paraffin
embedding [14 (link), 15 (link)]. The renal sections were prepared, deparaffined, and stained with
Hematoxylin & Eosin (HE). Anti-nephrin IgG {sc-19000, Santa Cruz Biotechnology (SCB),
Dallas, TX, USA}, anti-WT1 IgG (sc-7385, SCB), anti-synaptopodin (65194, Progene,
Heidelberg, Germany) were used for immunohistochemistry. Anti-phospho-Y1208-nephrin IgG
was produced in our laboratory, as reported [33 (link)].
Immunostaining assays were performed as described previously [14 (link),15 (link),16 (link)]. The sections were dewaxed and then were treated with 0.1 M citrate
buffer at pH 6.0 in an autoclave for five minutes for antigen retrieval. The sections were
incubated at 4°C for overnight with primary antibodies, washed with PBS and incubated with
biotin-labeled anti-mouse and anti-goat antibody (Vector, Burlingame, CA) for 1 hour at
room temperature. Avidin-biotin coupling reaction was performed on the sections using a
kit (Elite®, Vector). All antigens were visualized as brown with 3-3” diamino
benzidine (Nacalai, Kyoto, Japan).