Pelco colloidal graphite

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.

The scanning electron microscopy (SEM) samples were processed as described in Vizcaíno et al. [55 (link)]. The intestinal samples were fixed for 24 h in phosphate-buffered formalin (4% v/v, pH 7.2). Then, the fixed samples were washed with phosphate buffer and progressively dehydrated in graded ethanol. After this, the samples were dried in a critical point dryer (CDP 030 Critical Point Dryer, Leica Microsystems, Madrid, Spain) using absolute ethanol as the intermediate fluid and CO₂ as the transition fluid. The dried samples were mounted on supports, fixed with graphite (PELCO^® Colloidal Graphite, Ted Pella Inc., Redding, CA, USA), and gold sputter-coated (SCD 005 Sputter Coater, Leica Microsystems). All the samples were screened via high-resolution field emission scanning electron microscopy (FESEM) (Carl Zeiss, Sigma 300 VP, Jena, Germany). All the digital images were analyzed with Image J (National Institutes of Health, USA) software and a morphometric analysis was carried out to determine the enterocyte apical area (EA) [55 (link)].