Ht770 transmission electron microscope

Renal cortical tissues were fixed in 2% glutaraldehyde in phosphate-buffered solution (pH 7.4). Samples were further incubated with 2% osmium tetroxide in phosphate-buffered solution (pH 7.4) for 2 h at 4 °C. Ultrathin sections were stained with lead citrate and uranyl acetate and viewed on a HT770 transmission electron microscope (Hitachi, Japan) at an accelerating voltage of 80 kV. ImageJ 1.51k software (National Institutes of Health, rsb.info.nih.gov) was used to measure the glomerular membrane thickness. After separating out the various segments and leaving only the GBM, we used BoneJ, an ImageJ plugin for bone image analysis, to measure the GBM thickness as previously described^{8 (link)}.

The EDTA blood was processed for transmission electron microscopy (TEM) as per previous reports [11 (link),12 (link)]. In brief, buffy coats from centrifuged capillary tubes (5 capillary tubes) were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer at 4 °C for 24 h. Then, the fixed buffy coats were processed post-fixed with 1% osmium tetroxide for two hours and embedded in Spurr’s epoxy resin. Ultrathin sectioning was performed, stained with uranyl acetate and lead citrate. The stained sections were used for observation of the ultrastructure using the JEM1230 transmission electron microscope (JEOL, Tokyo, Japan) or the HT770 transmission electron microscope (Hitachi, Tokyo, Japan). This depended on the availability of the transmission electron microscope. Identification of blood cells was based on the number, shape, distribution of granules, and the nuclear appearance.