Cy3 conjugated goat anti rabbit immunoglobulin g igg h l

Mice were transcardially perfused with 4% paraformaldehyde (PFA) in phosphate buffer. Kidneys were removed and were further fixed with 4% PFA at 4 °C overnight. The kidneys were then placed in 15% sucrose in PBS at 4 °C for 4 hrs; this was then exchanged for 30% sucrose in PBS, and incubation continued at 4 °C overnight. The kidneys were frozen in optimum cutting temperature (OCT) compound and sectioned serially into 7-μm-thick sections using a cryostat. Samples were kept frozen at −80 °C until used. For immunofluorescence, sections were stained using rabbit polyclonal anti-lysosome-associated membrane protein type 1 (LAMP1) (Abcam, Cambridge, UK; 1:1000). Cy3-conjugated goat anti-rabbit immunoglobulin G (IgG) (H + L) was used as a secondary antibody (Jackson Immuno Research; 1:1000 dilution). All fluorescence images were digitally acquired at 400× magnification with KEYENCE Fluorescence Microscope BZ-X700 (KEYENCE Co., Osaka, Japan).