Alkaline phosphatase conjugated goat anti rabbit igg antibody

The titers of antisera were determined by an indirect ELISA. Each well of a 96-well ELISA plate (Corning Inc., USA) was coated with 1 μg of rRHT-D1A dissolved in 100 μL of 50 mM carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at 4 °C. After three washes with phosphate-buffered saline (PBS) Tween buffer (PBST; 0.05% of Tween 20 in PBS, pH 7.4), the wells were blocked with 100 μL of 3% BSA in PBST for 1 h at 37 °C and then washed again twice with PBST. After blocking, 100 μL of serially diluted anti-RHT-D1A serum (1:1000 to 1:128000) was added into the antigen-coated wells. The plate was covered with an adhesive plastic and incubated for 2 h at room temperature and then washed four times with PBST. At the next step, a 1:30,000-diluted alkaline phosphatase–conjugated goat anti-rabbit IgG antibody (Sigma, Canada) was added at 100 μL/well and incubated for 1 h at 37 °C. After a wash, 100 μL of a freshly prepared p-Nitrophenyl phosphate substrate solution was added into each well, and the plate was incubated at room temperature in a dark place. Finally, an absorbance was measured at 405 nm (A₄₀₅) on a multiskan FC (Thermo Scientific, MA, USA). All samples were tested in triplicate, with each plate containing control wells with positive serum samples and control wells with negative reference serum.

Chicken HD11 cells were stimulated with 0.2M lactose in the presence or absence of 2mM butyrate for 6, 12, or 24h, followed by wash with phosphate buffered saline and lysis in the radioimmunoprecipitation (RIPA) lysis buffer (Santa Cruz Biotechnology). Protein concentration was measured using the Bradford Assay (Bio-Rad). To determine the levels of histone H4 acetylation, 20μg proteins were separated in 12.5% SDS-PAGE gels and then transferred to polyvinylidene difluoride (PVDF) membranes. After overnight blocking in the blocking buffer containing 5% dry skim milk in TTBS (0.05% Tween 20, 20mm Tris-HCl, 150mm NaCl, pH 7.5) at 4°C, the membranes were incubated with a primary rabbit antibody against acetyl-histone H4 (Cell Signaling, Danvers, MA, United States) or a rabbit antibody against β-actin (MilliporeSigma) in the blocking buffer for 1h at room temperature. After three washes in TTBS, the membrane was incubated with an alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (MilliporeSigma) for 45min at room temperature. Western blots were visualized with Western Blotting Luminol Reagent (Santa Cruz Biotechnology).

Chicken HTC cells were treated with 2 mM butyrate, 20 μM quercetin, or their combination for 6 or 12 h, followed by lysis in the radioimmunoprecipitation (RIPA) lysis buffer (Santa Cruz Biotechnology). The resulting proteins were quantified using the Bradford Assay (Bio-Rad), followed by loading of 20 µg proteins from each sample in 12.5% SDS-PAGE gels and transferring to Immobilon-P^® polyvinylidene difluoride membranes (MilliporeSigma). After overnight blocking in 5% dry skim milk in TTBS (0.05% Tween 20, 20 mM Tris-HCl, 150 mM NaCl, pH 7.5) at 4°C, the membranes were incubated with a primary rabbit antibody against acetyl-histone H4 (Cell Signaling, Danvers, MA; diluted 1:500) or a rabbit antibody against β-Actin (MilliporeSigma; diluted 1:1000) in the blocking buffer for 1 h at room temperature. After three washes in TTBS, the membranes were incubated with an alkaline phosphatase-conjugated goat anti-rabbit IgG antibody (MilliporeSigma; diluted 1:2,000) for 45 min at room temperature, followed by visualization using enhanced chemiluminescence (ThermoFisher Scientific). The band intensity of acetyl-histone H4 was quantified as the area under the curve using ImageJ (https://imagej.nih.gov/ij/) and further normalized against the band intensity of β-Actin for each sample.