Ab100787

Gastric VEGF level was estimated using a commercially available ELISA kit (catalog # ab100787, Abcam, MA, USA). Additionally, gastric tissue was lysed and phospho-NFκB p65(Ser536)/total NF-κB p65 content, a critical regulator of immune and inflammatory responses particularly implicated in gastric ulcer pathogenesis, was estimated using an ELISA kit (catalog # PEL-NFKBP65-S536-T) obtained from Ray Biotech (GA, USA) [55 (link)].

A colorimetric method of Abcam was used for the VEGF (ab100787, Abcam) assessment. A 96-well plate containing 100 μl of all standards and samples was covered and incubated for 2.5 hours at 4°C with gentle shaking. Each well was washed 4 times with 300 μl 1x wash buffer. To remove excess liquid, the plate was inverted and blotted against clean paper towels after the last wash. The administration of 100 μl of 1x biotinylated VEGF detection antibody to each well was performed and incubated for 1 hour at room temperature with gentle shaking. The solution was discarded and repeated the wash. Then, 100 μl of 1x HRP-streptavidin solution was added and incubated for 45 minutes at room temperature with gentle shaking. Then, 100 μl of TMB one-step substrate reagent was added to each well after the repeated wash and incubated for 30 minutes at room temperature in the dark with gentle shaking. Finally, 50 μl of stop solution was added and the absorbance at 450 nm was immediately read.