To analyze peroxynitrite protein damage in ECs, indicated by the formation and presence of protein nitrotyrosine residues (3NT), an immunohistochemical nitrotyrosine staining was performed on HUVECs and paraffin-embedded RCA rings. With regard to the in vitro staining, HUVECs were cultured in Nunc Lab-Tek chamber slides (BD Biosciences) and exposed to secondary CPPs for 24 hours. After the stimulation, cells were fixed in 2% PFA and permeabilized with 0.5% Triton-X-100. Next, cells were blocked in 5% BSA-PBS and 0.1% H2O2 for 30 minutes at RT, followed by incubation with the primary anti-3NT antibody (#39B6, 1:50, Santa Cruz Biotechnologies) in 1% BSA-PBS at 4 °C overnight. Cells were blocked with both avidin and biotin for 15 minutes at RT (#SP-2001, Avidin-biotin kit, Vector Laboratories). For detection of the primary antibody, an isotype-specific IgG2a secondary antibody (#1081-08, 1:100, Southern Biotech, goat anti-mouse Biotin) and streptavidin-HRP conjugate diluted in 1% serum-PBS were used (#P039701-2, DAKO). Thereafter, tissue sections were incubated with 3’-diaminobenzidine (DAB) and counterstained with hematoxylin. Finally, stained HUVECs were mounted with Kaiser’s glycerol gelatin. Stained sections were scanned with the Hamamatsu slide scanner (Hamamatsu, Japan) and analyzed with the ImageJ Software.42 (link) Graphs showed the mean pixel intensity expressed as delta (Δ) to the experimental control.
For staining the RCA rings, a similar protocol was followed with a few modifications. Briefly, the tissue sections were deparaffinized, followed by overnight antigen retrieval in 10 mmol/L Tris-HCl (pH=9) at 80 °C. Tissue sections were blocked in 5% BSA-PBS and 0.1% H2O2 for 30 minutes at RT and incubated with the primary anti-3NT antibody (#39B6, 1:50, Santa Cruz Biotechnologies) in 1% BSA and 5% serum-PBS for 3 hours at RT. For the detection of the primary antibody, a donkey anti-mouse Alexa Fluor 647-conjugated secondary antibody (#A-31571, Invitrogen) was used. Additionally, tissue sections were incubated with Lycopersicon esculentum Lectin (LEA; #FL-1171, 1:100, Vector Laboratories) to visualize the endothelial glycocalyx. Nuclei were visualized with DAPI and sections were mounted with CitiFluor mounting medium. Stained sections were scanned the Olympus slide viewer VS200 (Olympus Nederland B.V., the Netherlands) and analyzed with the ImageJ Software afterward.42 (link) For analysis, 5 random LEA positive areas were selected, in which the mean fluorescent intensity of the 3NT positive pixels were quantified per sample.
For staining the RCA rings, a similar protocol was followed with a few modifications. Briefly, the tissue sections were deparaffinized, followed by overnight antigen retrieval in 10 mmol/L Tris-HCl (pH=9) at 80 °C. Tissue sections were blocked in 5% BSA-PBS and 0.1% H2O2 for 30 minutes at RT and incubated with the primary anti-3NT antibody (#39B6, 1:50, Santa Cruz Biotechnologies) in 1% BSA and 5% serum-PBS for 3 hours at RT. For the detection of the primary antibody, a donkey anti-mouse Alexa Fluor 647-conjugated secondary antibody (#A-31571, Invitrogen) was used. Additionally, tissue sections were incubated with Lycopersicon esculentum Lectin (LEA; #FL-1171, 1:100, Vector Laboratories) to visualize the endothelial glycocalyx. Nuclei were visualized with DAPI and sections were mounted with CitiFluor mounting medium. Stained sections were scanned the Olympus slide viewer VS200 (Olympus Nederland B.V., the Netherlands) and analyzed with the ImageJ Software afterward.42 (link) For analysis, 5 random LEA positive areas were selected, in which the mean fluorescent intensity of the 3NT positive pixels were quantified per sample.
