Anti rabbit antibody conjugated with alexa fluor 488

SVGA cells were seeded at 0.8 × 10⁶ on 1.5mm cover slips followed by treatment with cocaine as before. After termination of treatment, the cells were fixed with 1: 1 ice-cold methanol and acetone solution for 20 minutes at −20°C. The wells were air dried followed by blocking and permeabilization with 1% BSA in PBS with 0.1% Triton for 30 minutes. After blocking, the cells were incubated with a cocktail of rabbit anti-LC3II antibody (1:2000) and a mouse anti-GFAP antibody (1:1500) (Abcam, Cambridge, MA) overnight in a humidified chamber. After 3 washes with 0.1% Triton in PBS, the cells were incubated in the dark chamber for 1 hour with an anti-mouse antibody conjugated with Alexa Fluor 555 (1:2000) and an anti-rabbit antibody conjugated with Alexa Fluor 488 (1:2000) (Cell Signaling, Beverly, MA) followed by 3 washes with 0.1% Triton in PBS. All the antibodies were diluted in 1% BSA in PBS. Finally, the cover slips were transferred onto glass slides with 10μl of Vectashield mounting reagent with DAPI. The fluorescence microscopy was performed using a Leica TCS SP5 II Laser Scanning Confocal microscope. The images were captured using a 40X zoom lens and ImageJ software was used to analyze the images and calculate the intensity values by using GFAP as housekeeping protein.