Histological Analysis of Lung and Kidney Tissues

Removed lung and kidney tissues were fixed in 10% buffered formalin overnight, and embedded in paraffin. Lung tissues were cut into 5 µm sections, and stained with hematoxylin and eosin (H&E). Paraffin-embedded sections were de-paraffinized in xylene, dehydrated in ethanol and rehydrated in distilled water. To determine GN, mouse renal tissues were analyzed by Periodic acid-Schiff (PAS) staining [26 (link)]. For TUNEL staining, de-paraffinized lung sections were treated by proteinase K to reactivate antigens, re-fixed by 4% formaldehyde, incubated with equilibrate buffer, and finally labelled by TUNEL detection cocktail [5 (link), 26 (link)]. TUNEL-positive cells were determined by averaging the number from 3 fields (× 400) of positively stained cells with the highest density in each section. Cell nuclei were counterstained with DAPI. Fluorescence was detected by confocal microscopy. For detecting the expression of CitH3, de-paraffinized human or mouse lung sections were stained with anti-CitH3 antibodies, followed by Alexa Fluor 488-conjugated antibodies (Thermo Fisher Scientific). Cell nuclei were counterstained with Hoechst 33258. Fluorescence was detected by confocal microscopy.