Fitc conjugated chicken anti rabbit secondary antibody

Primary gastric epithelial cells and fibroblasts were placed on 96-well culture plates (Thermo Fisher Scientific, Waltam, MA, USA) at a density of 1 × 10⁵ cells/well (volume 100 µl) in DMEM:F12 or RPMI-1640 medium, respectively (37 °C, 5% CO₂). Unstimulated (control) or H. pylori GE-stimulated cells (concentration 10 μg/mL) were incubated for 1, 3, 6, 24, and 48 h, fixed with 4% formaldehyde solution for 20 min, and then washed 3 times in PBS, followed by incubation with 0.02% TritonX-100 for 10 min to permeabilize the membranes. Next, the cells were incubated with 3% BSA in PBS to block nonspecific binding and with primary rabbit anti-phospho-Erk 1/2 (Thr202/Tyr204)antibody(Cell Signaling Technology, Danvers, MA, USA) ) at a 1:200 dilution, followed by incubation with FITC-conjugated chicken anti-rabbit secondary antibody (Thermo Fisher Scientific, Waltam, MA, USA). Three independent experiments were performed in triplicate. Erk activation was assessed quantitatively based on the green fluorescence intensity that was measured using a multifunctional Victor 2 reader (Wallac, Oy, Turku, Finland) at the following wavelengths for FITC: 495 nm (excitation) and 519 nm (emission).

Primary vascular endothelial cells were cultured on glass inserts placed in 6-well culture plate (Thermo Fisher Scientific, Waltam, MA, USA) at a density of 5 × 10⁵ cells/well (volume 0.5 mL) in EGM-2 complete medium (37 °C, 5% CO₂). After 24h of cell exposure on H. pylori GE (10 ug/mL and 1 ug/mL), H. pylori LPS (25 ng/mL and 1 ng/mL) and MMP-9 (75ug/mL) cells were fixed with 4% formaldehyde solution for 20 min, washed 3 times with PBS, followed by incubation with 0.02% TritonX-100 for 10 min to permeabilize the membranes. Next, cells were incubated with 5% bovine serum albumin (BSA) in phosphate buffered saline (PBS) to block unspecific binding and then with primary rabbit anti-phospho-Erk 1/2 (Thr202/Tyr204) antibody (Cell Signaling Technology, Danvers, MA, USA) followed by FITC-conjugated chicken anti-rabbit secondary antibody (Thermo Fisher Scientific, Waltam, MA, USA). Activation of Erk was visualized using fluorescence microscope (Zeiss, AxioScope, A1, Jena, Germany) at a wavelength of 550 nm (excitation) and 590 nm (emission).