Cholera toxin b

Primary neurons were grown in NGF containing media for 3 days, NGF was then removed and replaced with DMEM containing 1 mg/ml BSA and 12.5 mM KCl for serum deprivation. After 1 hour, anti-p75ECD antibody tagged with FITC (1:50, Santa cruz biotechnology) was added for 1 hour at 4°C. Neurons were then left in KCl or treat ed with 200 ng/ml BDNF +/− 5μM MS275 for 4 hrs at 37°C, rinsed and incubated at 4°C with Chole ra toxin B (1:500, Invitrogen) for 20 min to label the cell surface. Thereafter, the neurons were fixed in 4% paraformaldehyde and processed for TUJ1 immunostaining as described above. The labelled cells were examined and imaged by using a Zeiss LSM 710 confocal microscope with a Plan apochromat 63×/1.4 oil differential interference contrast (DIC) objective. FIJI was used to quantify the cell-associated fluorescence. The total cellular fluorescence was calculated after subtracting the non-specific fluorescence from images of untreated cells obtained with the same illumination and exposure conditions. Internalized p75NTR ECD was determined as the ratio of the internalized receptor (intracellular fluorescence) versus the cell surface-associated receptor (cell-surface-associated fluorescence) and expressed as a percentage of internalized fluorescence (intracellular p75NTR), considering 100% to be the total cell-associated fluorescence.