Lmw ta

Conventional TEM was performed as described previously (Ichimura et al., 2009 (link), 2010 (link)). In brief, the slices of fixed kidney cortex were successively immersed in 0.4% osmium tetroxide (OsO₄) in 0.1 M PB for 1 h, 2% low-molecular-weight tannic acid (LMW-TA, Electron Microscopy Sciences, Hatfield, PA, United States) in 0.05 M maleate buffer for 4 h, and 1% uranyl acetate in 0.05 M maleate buffer for 3 h. The slices were then dehydrated with a graded series of ethanol and embedded in Oken Epok 812 epoxy resin (Oken-shoji, Tokyo, Japan). Ultrathin sections (90–100 nm thickness) were produced with an ultra 45° diamond knife (Diatome, Biel, Switzerland) and transferred to 50-mesh copper grids coated with a Formvar membrane. The ultrathin sections stained with lead citrate and uranyl acetate were digitally photographed with an H-7100 transmission electron microscope (Hitachi High-Technologies, Tokyo, Japan), which was equipped with a C4742-95 CCD camera (Hamamatsu Photonics, Shizuoka, Japan).

Conventional SEM was performed as described previously (Dong et al., 2010 (link); Miyaki et al., 2020a (link)). Small cubes of the fixed kidney cortex (approximately 4 × 4 × 2 mm) were processed with conductive staining. First, the cubes were immersed in 1% OsO₄ in 0.1 M PB for 30 min at 24°C, washed with 0.1 M PB for 5 min three times, and then immersed in 1% LMW-TA (Electron Microscopy Sciences) in distilled water (DW) for 2 h at RT. After the cubes were washed three times with DW for 5 min, the same staining procedure was repeated twice. However, OsO₄ was diluted with DW.
The stained samples were dehydrated with a graded series of ethanol and then immersed in t-butyl alcohol. The samples were freeze-dried using an ES-2030 freeze dryer (Hitachi High-Technologies, Tokyo, Japan). The dried specimens were mounted on aluminum stubs with carbon paste (Pelco Colloidal Graphite, Ted Pella, Inc., Redding, CA, United States). The mounted specimens were coated with osmium with an OPC80T osmium plasma coater (Filgen, Inc., Nagoya, Japan). The samples were observed with an S-4800 field emission-SEM (Hitachi High-Technologies). Regions of interest were imaged using a backscattered electron detector with an acceleration voltage of 3 kV. Individual podocytes were denoted with different transparent colors using Adobe Illustrator.