In situ cell death detection kit reagents

Sagittal kidney tissue sections (4 μm thick) and HK-2 cells seeded on chamber slides (Thermo Scientific) and incubated with treatments as described previously were prepared for immunofluorescence staining and in situ fluorescent TUNEL staining. First, the sections and cells were fixed with 4% paraformaldehyde (Sigma-Aldrich), followed by permeabilization in 0.1% Triton X-100 and incubated with 5% BSA (Sigma-Aldrich). The slides and cells were then incubated with anti-RIP3 monoclonal antibody (#95702, Cell Signaling Technologies, Danvers, MA, USA; 1:100 dilution) or anti-RIP3 polyclonal antibody (ab152130, Abcam, Cambridge, MA, USA; 1:100 dilution) overnight at 4°C and then incubated with Alexa Fluor 555-conjugated goat anti-rabbit IgG (H+L) (#4413, Cell Signaling Technologies, Danvers, MA, USA; 1:200 dilution). After rinsing 3 times in 0.1 M PBS (pH 7.4), the samples were incubated with in situ cell death detection kit reagents (Fluorescein, Roche, Basel, Switzerland) according to the manufacturer’s instructions and counterstained with 4',6-diamidino-2-phenylindole (DAPI). Finally, the images were captured by confocal microscopy (LEICA TCS SP2, Wetzlar, Germany), and the cell counting was performed by a pathologist blinded to the experimental conditions.

Sagittal kidney tissue sections (4-µm-thick) and HK-2 cells seeded on chamber slides (Thermo Scientific, USA) and incubated with the previously described treatments were prepared for RIP3 immunofluorescence staining and in situ fluorescent TUNEL staining. First, the sections and cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, USA), followed by permeabilization in 0.1% Triton X−100 and incubation with 5% BSA (Sigma-Aldrich, USA). The slides and cells were incubated with an anti-RIP3 monoclonal antibody (#95702, Cell Signaling Technologies, Danvers, MA, USA) or anti-RIP3 polyclonal antibody (ab152130, Abcam, Cambridge, MA, USA) overnight at 4 °C and then with Alexa Fluor 594-conjugated goat anti-rabbit IgG (H + L) (#4413, Cell Signaling Technologies). After rinsing 3 times with 0.1 M PBS (pH 7.4), the samples were incubated with in situ cell death detection kit reagents (Fluorescein, Roche, Basel, Switzerland) according to the manufacturer’s instructions and counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Finally, the images were captured by confocal microscopy (LEICA TCS SP2, Wetzlar, Germany), and cell counting was performed by a pathologist blinded to the experimental conditions.