Blocker bsa

To test the inhibitory effect of β15–42 on binding of biotinylated NDSK-II or E₁ to the VE-cad(1–5) fragment, wells of Immulon 2HB microtiter plates were coated overnight at 4°C with 2 μg/mL VE-cad(1–5) in coating buffer (0.1M Na₂CO₃, pH 9.5). The wells were then blocked with Blocker BSA in TBS (Tris-buffered saline) (Thermo Scientific) for 1 hour at room temperature. Following washing with the binding buffer (TBS containing 0.05% Tween 20 and 1mM CaCl₂), biotinylated NDSK-II or E₁, each at 1.5 μM in the binding buffer, was pre-incubated with β15–42 at 12 or 120 μM, and with (β15–44)₂ at 12 μM or with mAb T2G1 at 0.5 μM, for 30 min at 37°C, and 100 μL aliquots of the mixtures were added to the wells and incubated overnight at 4°C. Bound NDSK-II or E₁ fragment was detected by the reaction with NeutrAvidin conjugated with HRP (1 hour at 37°C). TMB microwell peroxidase substrate, SureBlue Reserve (KPL), was added to the wells, and the amount of bound E₁ or NDSK-II was measured spectrophotometrically at 450 nm after termination of the reaction with KPL TMB stop solution.

Wells of Immulon 2HB microtiter plates were coated overnight with the (β15–66)₂ fragment at 2 µg/ml in 0.1 M Na₂CO₃, pH 9.5 (coating buffer) at 4 °C. The wells were then blocked with Blocker BSA in TBS (Thermo Scientific, Rockford, IL) for 1 hour at room temperature. Following washing with TBS containing 0.05% Tween 20 and 1 mM CaCl₂ (ELISA-binding buffer), the indicated concentrations of VLDLR fragments in this buffer were added to the wells and also to control wells coated just with Blocker BSA in TBS and incubated for 1 hour at 37 °C. Bound fragments were detected by reaction with anti-His monoclonal antibody conjugated with HRP (1 hour at 37 °C). Alternatively, bound VLDLR fragments were detected by reaction with a mixture of anti-VLDLR mAb 1H5, 5F3, and 1H10 (1 hour at 37 °C) and the HRP-conjugated donkey anti-mouse polyclonal antibodies (1 hour at 37 °C). The peroxidase substrate, SureBlue TMB (KPL, Gaithersburg, MD), was added to the wells and the amount of bound ligand was measured spectrophotometrically at 450 nm. Data were analyzed by nonlinear regression analysis using equation 1:
$$A=A_{\mathit{\text{max}}}/(1+K_d/[L])$$
where A represents the absorbance of the oxidized substrate, which is assumed to be proportional to the amount of ligand bound, A_max is the absorbance at saturation, [L] is the molar concentration of the ligand, and K_d is the equilibrium dissociation constant.

TM4 and human Sertoli cells were seeded in pre-coated 18-well chamber slides (ibidi, Martinsried, Planegg, Germany) for 24 h prior to CBD treatment. After CBD exposure, cells were washed with 1 × PBS and fixed with 4% formaldehyde for 10 min at room temperature. Triton^™X-100 (0.5%) in 1 × PBS was used to permeabilize the cellular membrane and then cells were blocked with 1 × Blocker^™ BSA in PBS (Thermo Fisher Scientific) for 30 min. After blocking, the cells were incubated with DyLight 488-Phalloidin (Thermo Fisher Scientific) in 1 × Blocker^™ BSA in PBS for 30 min. Prior to image acquisition, cell nuclei were stained for 10 min with Hoechst 33342 (Thermo Fisher Scientific). For each sample, the images from at least three randomly selected fields were captured using a Cytation 5 Cell Imaging Reader (BioTek, Winooski, VT).