Peroxidase substrate

Total protein concentration was determined with the Bradford method [18 (link)]. Proteins were separated on a polyacrylamide gel (6 for proteolysis markers, 12 for protein oxidation and 10 % for others) and transferred onto a polyvinyldifluoride membrane. Ponceau S staining was used to ensure equal loading and proper transfer of the proteins. Blots were incubated overnight at 4 °C with appropriate primary antibody and subsequently with the suitable secondary antibody. Proteins were visualized with chemiluminescent Peroxidase Substrate (Sigma-Aldrich, Bornem, Belgium) and analysed with the software package (Bio 1D) of the imaging system (Photo print, Vilber, France).

The specimen sections were analyzed for apoptosis by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. TUNEL activity was measured using in situ cell death detection kits (Roche Diagnostic, Penzberg, Germany), according to the manufacturer’s instructions, to identify apoptotic cells in the tissue [32 (link)]. The specimens were incubated with peroxidase-conjugated anti-digoxigenin antibody (Roche Diagnostics, Penzberg, Germany). Staining was performed, and a peroxidase substrate (Sigma-Aldrich, Saint Louis, MO, USA) was used to present the color of the TUNEL reaction.

The IL-1 alpha, IL-6, IL-12, IFN-gamma, and TNF-alpha levels in the sera of immune-treated dogs at 3, 8, 12, and 24 h after the last treatment dose were measured by ELISA using commercial kits (PeproTech, NJ, USA) in accordance with the manufacturer's instructions as follows. The capture antibody was diluted to 1.0 μg/mL with PBS, and 100 μL/well was added to the 96 MaxiSorp plates (Nunc) and incubated overnight at room temperature. The plates were washed four times with 215 μL PBS-T, blocked with 200 μL of blocking buffer per well for 1 h at room temperature, and washed four times. The standard sample was prepared for each cytokine, and 100 μL/well was added; then, 100 μL of the immune-treated dog serum was added to each well and incubated 2 h at room temperature. The plates were washed four times, and then, 100 μL of the previously diluted detection antibody was added and incubated at room temperature for 1.5 h. The plates were washed, and then, 100 μL of previously diluted avidin was added and incubated 30 min at room temperature. The plates were washed again, and then, 100 μL of peroxidase substrate OPD (Sigma-Aldrich) in citrate buffer at pH 4.5-0.03% H₂O₂ was added. The plates were incubated 15 min at room temperature, and the reaction was stopped with 50 μL of 5 N H₂SO₄ per well. The reading was performed at 490 nm using a Microplate Reader (Bio-Rad, Model 550).

Ig-fusion protein of sRAGE in a concentration of 5 mg/mL was bound to protein A (1 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) coated wells (Maxisorb ELISA plates, Sigma-Aldrich). Unbound proteins were washed away, and the wells were blocked with BSA (Sigma-Aldrich). Biotinylated amyloid β 1–42 peptide (20 nM, American Peptide, Sunnyvale, CA, USA) or biotinylated HMGB1 (20 nM) were bound to the wells in the presence or absence of inhibitors, and unbound ligands were washed away. Bound biotinylated ligands were detected with horse radish peroxidase conjugated streptavidin (Sigma-Aldrich) and peroxidase substrate (Sigma-Aldrich). The color was developed using o-phenylenediamine dihydrochloride peroxidase substrate (Sigma-Aldrich). Absorbance at 490 nm was measured.

For immunohistochemical and immunofluorescence stainings cryosections from Optimal Cutting Temperature compound (OCT, Tissue Tek) embedded tissues were fixed (4% PFA or in methanol), blocked (10% normal goat serum in PBS) and incubated with primary antibodies (diluted in blocking buffer) over night at 4 °C (ref. 60 (link)). Bound primary antibody was detected by incubation with peroxidase-conjugated (EnVision System, Dako) secondary antibody, followed by incubation with peroxidase substrate (Sigma), or Alexa-Fluor 488- or Alexa Fluor 594-conjugated antibodies (Invitrogen). Nuclei were counterstained with hematoxylin or 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen). After washing slides were mounted in mounting medium. Images were taken with a Zeiss Meta 710 Confocal Microscope. Used primary antibodies and their dilutions (Supplementary Table 1): mTOR (Cell Signaling Technology, CST), S6-pS240/244 (CST); Akt-pS473 (CST); K14 (PROGEN Biotechnik); K10 (Covance); K14 (Covance); K15 (Covance); loricrin (Covance); filaggrin (Covance); p63 (Santa Cruz Biotech); P-cadherin (Zymed); active Caspase3 (CST); Survivin (CST), K8/18 (PROGEN Biotechnik); Par3 (EMD Millipore); LGN (EMD Millipore) and β4-integrin (BD Biosciences).

ECM glycoproteins (Fn, Ln, Cn-I, and Cn-IV) and Mu were applied at a concentration of 10 mg/mL/well in triplicate to ELISA plates in a coating carbonate buffer (pH 9.8) at 4 °C for 18 h. Then, the plates were washed five times with PBS–0.01% Tween (PBST) and 1 × 10⁸ bacteria in 100 μL of bacterial cultures (wild-type EPEC strain E2348/69, ΔecpA or ΔecpD mutants) were added for 1 h at room temperature. After washing three times with PBST, an antibody against LPS O127 was added at 1:3000 dilution for 1 h followed by a washing step and the addition of an anti-rabbit IgG–peroxidase conjugate (1:3000) for an additional hour. After washing, the peroxidase substrate (Sigma) was added and the color developed was read at 595 nm using a spectrophotometer. In addition, colony-forming units (CFUs) were also quantitated from replica plates. BSA was used as a negative control.

Briefly, 1x10⁶ responder cells from spleen were incubated with 3x10⁵ antigen-presenting cells in complete medium (1% NEAA, 1% L-glutamine, 1% vitamins and 1% pyruvate, 0,1% 2-ME, 10% FBS (HyClone), 20 U/mL mouse recombinant IL-2 (SIGMA) and on a plate previously coated with capture antibody those cells were incubated in the presence or absence of 10 μM of peptide TEWETGQI. After 24 hours the plates were washed with PBS and PBS-Tween 20 (0.05% Tween). Each well received 2 μg/mL of biotinylated anti-mouse monoclonal antibody (clone XMG1.2, Pharmingen). The plates were incubated with streptavidin-peroxidase (BD) and developed by adding peroxidase substrate (50mM Tris-HCl, pH 7.5, containing 1 mg/ml DAB and 1 μL/ml 30% hydrogen peroxide, both from SIGMA). The number of IFN-γ-producing cells was determined using a stereoscope.