Fig. 7. Surface biotinylation reveals the endocytic nature of BAD-LAMP-containing vesicles. Cortical neurons were surface biotinylated for 15 minutes at 4°C, prior to warming at 37°C for different times, fixation and visualization by confocal microscopy. (A) Prior to warming, no significant co-localization of biotinylated proteins with BAD-LAMP was observed. Co-localization was evaluated using the Image J image analysis software. A low Pearson's coefficient and strong negative pixel shift are both indicative of the absence of staining overlap (right). (B) After 5 minutes of endocytosis at 37°C, extensive co-localization of biotinylated proteins (green) was observed with BAD-LAMP (red) in neurites (arrowheads), as also shown by a higher Pearson's coefficient and the absence of pixel shift (right). (C) After 45 minutes of endocytosis co-localization of BAD-LAMP with biotin is decreased as shown by a decreased Pearson's coefficient and negative pixel shift (right). Bar, 10 µm.