Jeol1400 transmission electron microscopy

A549 cells were seeded (3 × 10⁵ cells per well) in 6-well plates, grown for 24 h to 70–80% confluence and starved for 18 h. Media was replaced to antibiotic free medium prior to infection and after incubation with either Kp52145 or PBS (non-infected control) for 3 hours, medium was removed, and the cells were gently washed twice with PBS. Cells were then fixed by the addition of 2.5% (v/v) glutaraldehyde + 1.5% (v/v) paraformaldehyde in PHEM buffer (Electron Microscopy Sciences, catalogue number 11165) for 1 hour at room temperature. Cells were them scraped and collected into a microcentrifuge tube. Cell suspension was pelleted at 800 g for 15 minutes and embedded in agarose (2%, w/v, in PHEM buffer). Cells were then post fixed with 2% osmium tetroxide (v/v in PHEM) and immersed in Spurr replacement (Low viscosity resin kit, TAAB, T262) and polymerised at 60 °C for 48 hours. The block was trimmed, thin sectioned (90–100 nm thick) and then applied to copper grids and air dried. The grid was loaded in a JEOL1400 transmission electron microscopy (Jeol). At least 10 randomly selected electron micrographs from each sample were examined.

To confirm induction of LMP by malathion, lysosomal morphological analysis was performed based on the method reported by Kang et al.^{41 (link)} To examine lysosomal integrity, the N2a cells were treated as described previously. At the end of the treatment period, cells were immersed in fixative (PBS-buffered 2.5% glutaraldehyde) and incubated overnight at 4°C. In addition, specimens were dehydrated by drenching in buffered 2% osmium tetroxide for 2 h at 4°C then embedded in resin followed by 50–60 nm thin sections were taken and stained with uranyl acetate and lead citrate. Lysosomal morphology was examined using Jeol 1400 transmission electron microscopy (Jeol Ltd, Tokyo, Japan). Sample preparation was done and the TEM instrument facility was provided by the Korean Basic Science Institute (KBSI, Chungbuk Ochang Center, Ochang, South Korea).

Tissue samples were fixed in buffered formaldehyde solution, dehydrated, and embedded in paraffin wax. Two μm sections were cut and stained with hematoxylin–eosin, periodic‐acid–shiffs reagent (PAS), or with immunohistochemistry reagents as described below. Histopathological assessment of mesangial matrix accumulation (PAS), podocyte markers (nephrin and WT1), and arteriolar wall thickening (α‐SMA) was performed, and the sections were scored semiquantitatively on a scale of 0 (normal) to 5 (severe).
Glomerular basal membrane (GBM) thickness was assessed using electron microscopy (Jeol 1400 Transmission Electron Microscopy) over three glomeruli per kidney, images (n = 10–14) taken at 12,000× magnification and mean basal membrane thickness (nm) calculated.