Anti mouse igg h l dylight 680 conjugate

75,000 cells/mL were seeded on cover glasses and incubated at 37° C and 5% CO₂ for 48 h before staining. Thereafter, cells were washed twice for 1 min with PBS and fixed with 4% paraformaldehyde/PBS for 10 min. Fixed cells were washed for 3 times with PBS and blocked in 2% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) including 0.2% Triton X-100 for 30 min. Thereafter, cells were incubated with MOR-1-antibody (D12, Santa Cruz Biotechnology, TX, USA) in 2% BSA (1:50) at 4° C overnight. The next day cells were washed incubated with antimouse IgG (H + L) DyLight™ 680 Conjugate (Cell Signalling Technology, Danvers, MA, USA) in 2% BSA (1:500) for 1 h. Finally, stained cells were washed for 3 times with PBS and covered with a coverslip using 1 drop of mounting medium. Cells stained with secondary antibody only were used as negative control. MOR expression was visualized using a Zeiss LSM 780 microscope (Carl Zeiss AG, Oberkochen, Germany) and 40 × magnification.

After sample preparation, protein lysates were obtained by lysing cells with RIPA Buffer (Thermo Scientific). Next protein level were quantitated by using Pierce Bicinchoninic Acid (BCA) PROTEIN Assay Kit (Thermo scientific) according to the manufacturer’s protocol. Based on the concentration measurements, equal amounts of protein were taken from each sample and prepared for standard SDS-PAGE polyacrylamide gel electrophoresis and western blotting, based on Bio-Rad Western Blotting Protocols (Bio-Rad). The membranes were imaged with a Li-COR Odyssey Infrared Imaging System to evaluate the protein expression. For western blotting, the primary antibodies used in this study were: HES5 (EPR15578) rabbit, NOTCH1/N1-TMICD (D1E11 XP(R)) rabbit mAb, NOTCH3/N3-TMICD (D11B8) rabbit mAb, HES1 (D6P2U) rabbit, JAG1 ((D4Y1R) XPCR) rabbit mAb. HES5 Ab was purchased from Abcam, (Cambrige, MA, USA), all other primary antibodies were purchased from Cell Signalling (Beverly, MA, USA). β-actin (C4) mouse monoclonal IgG1 (Santa Cruz Biotechnology) was used as house-keeping gene. The secondary antibodies used were anti-rabbit-IR800 (Li-Cor Biosciences, Lincoln, NE) and anti-mouse IgG (H + L) (DyLight 680 conjugate) (Cell Signalling Technology).