Cultured glial cells on coverslips were pre-extracted with PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, and 2 mM MgCl2, pH 6.8) containing 0.025% saponin for 2 min, then washed twice for 2 min with PHEM buffer containing 0.025% saponin and 8% sucrose. The cells were fixed with a solution of 4% PFA and 8% sucrose in PBS for 30 min at room temperature and blocked with a solution of 1% BSA and 0.025% saponin in PBS for 1 hr. Primary antibodies were diluted in 1% BSA and incubated with the cells for 1 hr. Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen) and Alexa Fluor 568 (Invitrogen) goat anti-mouse IgG were used at a 1:1000 dilution for 30 min. Cells were mounted onto slides using Dako Fluorescent Mounting Medium. Slides were imaged on a Zeiss LSM510-Meta confocal microscope. Fractional colocalization was determined from Mander’s coefficient, which measures the direct overlap of green and red pixels in the confocal section. The value range is from 0–1 (0, no colocalization; 1, all pixels colocalize). The Mander’s coefficient is independent of differences in signal intensity between the two channels.
