Alexa fluor 488 or 555 or 647 conjugated secondary antibodies

Hippocampal cell culture was fixed with 4% PFA/4% sucrose/PBS, pH7, for 5min. The cells were permeabilized with block solution (PBS, 10% horse serum, and 0.5% BSA, 0.2% Triton) for 10min. The cells were incubated with primary antibodies including, anti-GLT1 (1:500, Alomone), anti-EEA1 (1:500, BD Bioscience, Cat. No: 610456), anti-Map2 (1:1000) for 1hr in block solution, followed by incubation with Alexa Fluor-488 or 555 or 647-conjugated secondary antibodies (1:1000, Invitrogen) for 1hr. After extensive washing, coverslips were mounted on microscope slides using ProLong Gold antifade reagent (Invitrogen) and sealed with nail varnish. Images were attained using Zeiss LSM 700 upright confocal microscope with an Apochromat 63× oil immersion lens with 1.4 numerical aperture. Images were digitally captured using ZEN software with excitation at 488nm for GFP and Alexa-Fluor 488, 555nm
for Alexa-Fluor 555 and 633nm for Alexa-Fluor 647 and conjugated secondary antibodies.
Pinholes were set to 1 Airy unit creating an optical slice of 0.8μm. For measuring colocalization, the colocalization plugin of the Metamorph software was used. Fluorescence intensity was measured using the ImageJ software.