Histopathological Analysis of Kidney Injury

H&E staining of 4-μm-thick formalin-fixed, paraffin embedded kidney tissue sections was performed according to standard protocols. Images were captured by microscopy (Olympus BX53). In each kidney, the numbers of damaged tubules were counted separately in 10 nonoverlapping fields observed at ×400 magnification. Renal tubular injury scores expressed as the percentage of damaged tubules, and the mean values were calculated (GraphPad Prism). For immunofluorescence staining of kidney tissue sections, heat-induced antigen retrieval was performed by heating sections in 1 mM EDTA at 95 °C in a pressure cooker for 20 min and then 20 min of cooling at room temperature. Sections were permeabilized with 0.1% Triton X-100 in 1× PBS. Sections were incubated for 30 min at room temperature with blocking buffer 5% BSA in 1× PBS and subsequently incubated with primary antibodies including anti-dsRNA mAb J2, anti-PNPT1, anti-AQP1, and anti-podocin antibodies for 3 h. Sections were washed in 1× PBS three times and then incubated with secondary antibodies including goat anti-mouse Alexa Fluor 488, donkey anti-rabbit Alexa Fluor594, donkey anti-rabbit Alexa Fluor 488, and goat anti-mouse Alexa Fluor 594 antibodies for 1 h at room temperature followed by 3 washes with 1× PBS. The sections were then mounted in Prolong Diamond Antifade Mountant with DAPI. Confocal images were taken using a confocal microscope (Zeiss LSM 880) with ZEN 3.1 blue edition software. Fluorescence intensities were quantified using ImageJ 1.44p software.