X0907

For immunocytochemical analysis of the human brain endothelial cell culture, cells were fixated with 4% paraformaldehyde, permeabilized with 0,1% Triton in PBS for 10 min and non-specific binding was blocked with 10% normal goat serum (X0907, DAKO, Santa Clara, CA, USA) for 30 min. Subsequently cells were incubated overnight at 4 °C with primary antibodies against CD31 (DAKO, M0823) and HOPX (Invitrogen, PA-72855), SCN3B (Sigma, HPA04707) or DSP (Sdix, 2282.00.02). Secondary antibodies against goat-anti-rabbit Alexa633 (Invitrogen, A-21070) and goat-anti-mouse IgG1 Alexa568 (Invitrogen, A-21124) together with Hoechst for nuclei staining were incubated for 1 h at room temperature. The representative images were taking using a Leica TCS SP8 X microscope (63x objective, Leica Microsystems, Wetzlar, Germany).

Formalin-fixed paraffin-embedded human carotid endarterectomy sections were permeabilized with 0.1% triton X-100 for 10 min, washed 3 times for 5 min before blocking with 10% goat serum (DAKO X0907) for 1 h at room temperature. Sections were incubated with either primary antibodies: p16 (20 µg rabbit polyclonal, ProSci 4211), Smooth muscle cell α-actin-cy³-conjugated (1:1000 mouse monoclonal, Sigma-Aldrich C6198), CD68 (1:100 mouse monoclonal, Thermo 14-0689-82) or isotype control antibodies: rabbit monoclonal IgG isotype control (Abcam ab172730), mouse monoclonal IgG isotype control (Abcam ab37355) diluted in 3% BSA for 1 h at room temperature. After washing 3 times for 5 min at room temperature, sections were incubated with secondary antibodies: goat anti-rabbit Alexa Fluor 647 (1:500, Abcam ab150083) or goat anti-mouse Alexa Fluor 488 (1:800, Invitrogen A-11017) for 1 h at room temperature. After counterstaining with DAPI for 10 min at room temperature and 3× washing for 5 min, sections were mounted in ProLong Gold antifade mountant (Invitrogen P36930). Four different symptomatic human carotid artery sections were analysed.

DU145 cells were incubated with 20 μM sorafenib for 12 and 24 h. After the indicated times, cells were harvested and processed as described [48 (link)]. Micrographs were taken at 2000-2500x magnification. Ultrathin sections were incubated in 8% H₂O₂, washed in dH₂O and incubated in 0.01M PBS pH7.4 and blocked with 5% goat serum (X0907, Dako, Denmark) in PBS for 30 minutes. Sections were then incubated with LC3 mouse monoclonal antibody (MBL, M152-3) in a dilution of 1:10 in PBS containing 1% BSA for 20h at 4°C. Subsequently sections were incubated in 0.05M Tris/HCl pH7.4, 0.05M Tris/HCl pH7.2 : 0.2% BSA (1:1) and finally in 0.05M Tris/HCl pH8.2 : 1% BSA (1:1) followed by incubation with 10nm colloidal gold conjugated secondary antibody in a dilution 1:10 for 1h at RT. They were then washed in 0.05M Tris/HCl pH7.2 : 0.2% BSA (1:1), 0.05M Tris/HCl pH7.4 and dH₂O and dried on blotting paper and stained in 7.5% Uranyl Acetate ethanol solution and then in 0.4% Lead Citrate aqueous solution at RT. Sections were visualized and micrographs were captured on a FEI Morgani 268 Transmission Electron Microscope.

After fixation in acetone, guinea pig lung sections were blocked in 3% hydrogen peroxide (H₂O₂) for 30 min, followed by 1 hour blocking in 10% serum (normal rabbit serum (Dako, X0902) for CBS, normal goat serum (Dako, X0907) for Nrf2). Sections were then incubated with primary antibodies (Santa-Cruz, sc-271886, 1:50 for CBS; Abcam, ab31163, 1:100 for Nrf2) for 1 hour at room temperature. Primary antibodies were visualized using horseradish peroxidase-labelled secondary antibodies (1:100) and diaminobenzidine. Sections were counterstained with haematoxylin (Sigma, GHS3) for 1 min and mounted in Kaiser’s glycerol gelatin.

Donor splenocytes were isolated as above and resuspended in RPMI1640 Medium (Gibco/Thermo Fischer, Waltham, USA) containing 10% inactivated FCS and 1%Penicillin/Streptomycin. Heat-inactivated recipient serum and donor splenocytes (200,000 cells/well) were incubated at 4 °C for 30 min. Rabbit complement (BAG 7018, Lich, Germany) was added and incubated for 2 h at 24 °C. Goat (DAKO X0907, Hamburg, Germany) or rabbit (DAKO X0902, Hamburg, Germany) serum were used as positive control. Cells were washed and stained with propidium iodide (PI) (Invitrogen/Thermo Fischer, Waltham, USA) to distinguish dead cells and then measured by flow cytometry. Complete lysis was measured using FixPerm (Thermo Fischer, Waltham, USA). Percent cytotoxicity was calculated using the formula: (“PI⁺cells in sample” – “PI⁺cells in medium”)/(“PI⁺cells in FixPerm” – “PI⁺cells in Medium”) ×100.