The kidney of ACSL4F/F and cdh16-Cre/ACSL4F/F mice were snap-frozen in liquid nitrogen and placed in an optimal cutting temperature embedding matrix. Frozen sections (10 μm) were fixed in icy acetone for 10 min then washed using PBS. Then, sections were stained with ACSL4 (1:250, Abcam, ab155282) antibody or Cytokeratin 18 (1:200, Proteintech, 66187-1-Ig) antibody followed by staining of secondary Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher, A11008), or Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 633 (Thermo Fisher, A21052), respectively. A Carl Zeiss LSM700 laser confocal microscope was used to obtain images.
Kidney cell death was labeled by TUNEL staining (Beyotime, cat number: C1088) as previously described for details [22 (link),33 (link)]. Briefly, the tissue sections were dewaxed using xylene then permeabilized with 0.1% Triton X-100. Sections were then incubated with TUNEL for 1 h at 37 °C, then counterstained with DAPI (Beyotime, cat number: C1005). The FITC-labeled TUNEL-positive cells were imaged under a fluorescent microscope and cells with green fluorescence were defined as tissue cell-death (Carl Zeiss LSM700).
Kidney cell death was labeled by TUNEL staining (Beyotime, cat number: C1088) as previously described for details [22 (link),33 (link)]. Briefly, the tissue sections were dewaxed using xylene then permeabilized with 0.1% Triton X-100. Sections were then incubated with TUNEL for 1 h at 37 °C, then counterstained with DAPI (Beyotime, cat number: C1005). The FITC-labeled TUNEL-positive cells were imaged under a fluorescent microscope and cells with green fluorescence were defined as tissue cell-death (Carl Zeiss LSM700).