Biotinylated anti mouse igg

Hela or JR1 cells were plated onto 12-well Costar culture plates on cover slips with 50,000 cells per well. Transient transfection was done on the second day of the seeding by polyethylenimine (PEI, Sigma) with a total of 4 μg of DNA per well. Cells were fixed 24 h post-transfection using 4% paraformaldehyde/PBS for 20 min and washed again with PBS. The cells were then blocked with 3% BSA/PBS solution for 1 hr followed by the primary antibody (rabbit anti-HA, sc-805, or mouse anti-Flag, sc-166355, Santa Cruz Biotechnology) diluted (1:200) in BSA/PBT and added to the cells with an overnight incubation at 4 °C. The cells were then washed with PBT 3 times, before adding the secondary antibody (donkey anti-rabbit IgG biotinylated, RPN1004V, or anti-mouse IgG biotinylated, GE Healthcare) with a dilution of 1:250 in BSA/PBT for 1 hour at RT with shaking. After washing 3 times with PBT, cells were incubated with Chromeo^TM 488 Streptavidin (Santa Cruz) for 1 hour at RT with shaking. Nuclei staining was also performed by applying Hoechst, diluted 1:30 in water, for 30 minutes. The cells were then washed with PBS and mounted on a rectangular slide containing an anti-fading agent DABCO (Sigma–Aldrich). The slides were examined using the Olympus BH-2 microscope at the molecular core facility in the faculty of medicine.

NCI-H1299 cells were plated onto 12-well Costar culture plates on coverslips at sub-confluency ~60% per well. Transient transfection was done on the 2nd day of the seeding using 2 μl Lipofectamine 2000 (Life Technologies) and a total of 2 μg of DNA per well. Cells were fixed 24 h post-transfection using 4% paraformaldehyde/PBS for 20 min and washed again with PBS. The cells were then blocked with 3% BSA/PBS solution for 1 h. followed by two primary antibodies (rabbit anti-HA or mouse anti-Flag and anti-mouse Ki-67 or rabbit Ki-67, respectively) diluted (1:300) in BSA/PBT and added to the cells with an overnight incubation at 4°C. The cells were then washed with PBT 3 times, before adding the secondary antibody (donkey anti-rabbit IgG biotinylated, RPN1004V, or anti-mouse IgG biotinylated, GE Healthcare) with a dilution of 1:250 in BSA/PBT for 1 h at RT with shaking. After washing 3 times with PBT, cells were incubated with ChromeoTM 488 Streptavidin (Santa Cruz), and Alexa Fluor 555 for 1 h at RT with shaking. Nuclei staining was also performed by applying Hoechst, diluted 1:30 in water, for 30 min. The cells were then washed with PBS and mounted on a rectangular slide containing an anti-fading agent DABCO (Sigma–Aldrich). The slides were examined using the Olympus BH-2 microscope at the molecular core facility at AUB.

For confocal microscopy, anti-neurofascin antibody was applied at a dilution of 1∶100 at 4°C in a wet chamber over night. After washing with phosphate buffered saline (PBS) biotinylated anti-mouse IgG (GE Healthcare, Buckinghamshire, UK) was applied for 1 hour at room temperature at a dilution of 1∶200, followed by Avidin HRP (1∶100; Sigma, St. Louis, USA) for one 1 hour. After enhancement with 0,1% CSA in PBS/0.03% H₂O₂ for 20 min at room temperature (protocol as described in [30] (link)) signal was visualized by Avidin-Cy2 (Jackson ImmunoResearch, West Grove, PA, USA) at 1∶75 for one hour at room temperature. For detection of bound anti-neurofascin antibody in fetal tissue slides were subjected to the protocol as described above but without applying the anti- neurofascin antibody in the first step. Fluorescent preparations were examined using a Leica TCS SP5 laser scanning microscope with LAS AF software (Leica Microsystems, CMS GmbH, Germany). An argon laser was used for detection of Cy2 (488 nm excitation) signal.