Goat anti mouse polyclonal igg h l alexa fluor 488

Antiserum staining of cell lines MCF7, T47D and HEK293T (negative control) was investigated by confocal microscopy. To this end, 30 000 cells per well were seeded in 8-well μ-Slide Ibidi Plates and grown for 24 h. The cells were then incubated with CellMask Deep Red membrane dye (1 : 1000) for 10 min at 37 °C, followed by a fixation step with 4% paraformaldehyde in PBS for 10 min at 37 °C. The cells were then washed with PBS and permeabilized with 1% Triton X-100 in PBS for 15 min at rt. The cells were then incubated with the mice sera (1 : 100 dilution) at 4 °C overnight. The cells were washed with PBS and incubated with 200 μL per well of goat anti-mouse polyclonal IgG H&L Alexa Fluor 488 (1 : 2000) secondary antibody from Abcam, for 2 h at rt. Finally, the cells were washed and incubated with Hoechst (1 μg mL⁻¹) for 10 min at rt to stain the nuclei and analyzed with a Zeiss LSM 710 confocal laser point-scanning microscope with a 40× oil objective and numerical aperture = 1.3. The secondary antibody was visualized with an argon laser source (488 nm, emission 500–550 nm), while CellMask Deep Red stained membranes were visualized upon excitation with a DPSS 561-10 laser (561 nm, emission 570–640 nm), and nuclei were visualized with a diode 405-30 laser (450 nm, emission 420–470 nm).

The reactivity of the antibodies elicited by KLH-3a towards breast cancer cell lines was determined by staining the cells with the antisera followed by flow cytometry analysis. For this purpose, MCF7 and T47D cells (with high expression of TA-MUC1) and HEK293T cells (with no expression of TA-MUC1) were fixed with an ice-cold solution of 4% paraformaldehyde in PBS (100 000 cells per FACS tube) for 10 min. After fixation, the cells were subjected to a permeabilization step with 0.1% Triton-X100 in PBS for 15 min, followed by a blocking step with 10% FBS in PBS for 30 min. The cells were then incubated with 50 μL of 1 : 50 dilution of mice sera. After 1 h of incubation, cells were incubated with 50 μL per·well of goat anti-mouse polyclonal IgG H&L Alexa Fluor 488 (1 : 2000) secondary antibody from Abcam, for an additional 1 h. All the incubation periods were completed at rt and were followed by washing steps with PBS (with 4 min centrifugation at 4000 rpm to remove the supernatant). Acquisition was done with a BD LSR Fortessa setup with a 488 nm laser and a 530/30 nm band-pass filter (combination used for Alexa488 detection). Data analysis was done with FlowJo (version 6.3.4, FlowJo) software.