Pvc elisa microplate

Indirect Enzyme-Linked Immunosorbent Assay (ELISA) assay was used to test the levels of pro-inflammatory cytokines in the total protein samples from hippocampal CA1 3 days after GCI [31 (link)]. In brief, 30 µg protein samples from each animal were diluted to 50 µL with bicarbonate/carbonate coating buffer (Sigma-Aldrich). The samples were loaded in PVC ELISA microplates (Corning) and incubated overnight at 4°C. After washing, the plate wells were blocked for 2 h at room temperature using 1% BSA blocking buffer. Then, specific antibodies against IL-1β, IL-18, TNF-α and IL-6 were added and incubated for 2 h at 37°C. Plate wells were then washed and incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. Finally, the wells were washed three times and developed by incubation with TMB (3,3’,5,5’-tetramethylbenzidine, Thermo fisher) for 30 min at room temperature. The reaction was stopped by the addition of sulfuric acid, and optical density was read at 450 nm on a spectrophotometer (Bio-Rad). Data were calculated and expressed as percent change compared with sham control group.

The indirect (ELISA) assays were used in the present study to assay the levels of pro-inflammatory cytokines in AD mice brain. The lysates obtained from cortex and hippocampal CA1 region in 5XFAD mice following four weeks of anti-GMF antibody injection were used for pro-inflammatory cytokines assay (Gan and Patel, 2013 (link); Ahmed et al., 2016 (link)). Briefly, equal amount of (30 μg) protein samples from cortex and hippocampus were diluted up to 50 μl with coating buffer and incubated overnight at 4 °C in PVC ELISA microplates (Corning). Thereafter, following 3 washes, each well was blocked with 1 % BSA (blocking buffer) for 1 h at room temperature. Then, each well was incubated with respective antibodies against TNF-α anti-rat (1:200, R&D Systems, Minneapolis, MN), IL-1β anti-rabbit (1:250, CST) and IL-6 anti-goat (1:200, R&D Systems, Minneapolis, MN) for 2 h at 37 °C. Thereafter, following three washes each well was incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, wells were washed three times and color was developed by TMB solution (3, 3, 5, 5–tetramethyl benzidine, Thermo Fisher) for 30 min at room temperature. The reaction was eventually stopped by using sulfuric acid and the optical density was read at 450 nm on a spectrophotometer (Molecular Devices, Sunnyvale, CA). Data were analyzed as percent change compared with the WT mice.

ELISA analysis was used to determine the levels of pro-inflammatory and anti-inflammatory cytokines in brain homogenates. Briefly, protein samples of different groups containing the same amount of proteins were diluted in carbonate coating buffer (Sigma-Aldrich; St. Louis, MO, USA) to 50 µL. Samples (50 µL) were loaded in PVC ELISA microplate (Corning, New York, NY, USA), then sealed and incubated overnight at 4 °C. After discarding the samples, the remaining protein-binding sites were blocked by blocking buffer (1% BSA in PBS, 0.3% solution of H₂O₂) for 1 h at room temperature. Samples loaded in wells were incubated with 50 µL primary antibodies overnight at 4 °C followed by incubation with HRP conjugated secondary antibodies for 1 h at room temperature. Following three washes, the plates were incubated with TMB (3, 3’, 5, 5’ -tetramethylbenzidine, BD Biosciences; San Jose, CA, USA) substrate reagent for 30 min. Subsequently, 50 µL of sulfuric acid was administered to stop the reaction and the plate was read at 450 nm on a spectrophotometer (Bio-Rad; Hercules, CA, USA). The minimal and maximal absorbance of the low and high standards was in the range of 0.10 to 1.50, and the absorbance values of the samples range from 0.30 to 0.99. The coefficient of variation between all duplicates was lower than 15%.