Slide scanner axio scan

Four-μm-thick paraffin-embedded sections were cut with a microtome (RM2235, Leica Biosystem). Antigen retrieval was performed in a low pH buffer using the PT-Link™ Dako (Agilent Technologies). VCAM-1 (Abcam, ab134047) was stained and revealed with a one-step polymer goat anti-rabbit IgG coupled with horseradish peroxidase (HRP) (Dako Envision™, Agilent Technologies, #K4003). Staining was visualized using diaminobenzidine (DAB). Slides were scanned by Nanozoomer® (Hamamatsu) and analyzed with NDPview2® (Hamamatsu). Periodic acid–Schiff coloration was performed by routine procedures using sections of paraffin-embedded kidneys. Coloration of slides was scanned by Slide Scanner Axio Scan (Zeiss).

Moreover, 3-μm-thick sections of fixed (PFA 4%) frozen kidneys were cut with Cryostat Leica AS-LMD. Heme oxygenase 1 (HO-1) expression was studied using rabbit anti-mouse HO-1 (Abcam, Ab13243) followed by a polymer anti-rabbit IgG-HRP (DAKO, K4003). Staining was revealed with DAB solution. Slides were scanned by Nanozoomer (Hamamatsu). Hematoxylin–Eosin, Perl’s Prussian blue, and PAS coloration were performed by routine procedures using sections of paraffin-embedded kidneys at days 1, 2, and 4. Coloration of slides was scanned by Slide Scanner Axio Scan (Zeiss).

Formalin-fixed tissue was embedded in paraffin and 4 µM sections were cut and stained with hematoxylin and eosin (H/E). Sections were dewaxed, rehydrated, and incubated with permeabilisation solution (0.1% Tx100 + 0.1% NaCitrate) for 8 min at room temperature. Slides were rinsed with bidi, followed by PBS. Next, cell death was analyzed using in situ cell death detection kit (TMR-red, Roche) according to manufacturer’s instructions. Nuclei were stained with 1 µM Hoechst solution for 30 min at room temperature. Images were taken with the Slide Scanner Axio Scan (Zeiss) and images analyzed using the ZEN (blue) software (Zeiss).