Alexa fluor 488 goat anti mouse rabbit

Mice were anesthetized with pentobarbitone (50 mg, intraperitoneally) and transcardially perfused with 4% paraformaldehyde (0.1 mol/L PBS). A vibrating microtome (Leica VT1200) was used to obtain 80-μm sections of brain, spinal cord, and liver. Sections were dehydrated by incubation in 50% ethanol in distilled water (v/v) for 30 min and then were washed three times in 0.3 mol/L PBS, followed by incubation in 10 mM sodium citrate (pH 6, 85°C, 30 min) for antigen retrieval. Sections were then incubated in the blocking solution (5% normal goat serum in 0.3mol/L PBS with 0.3% Triton X-100) for 1 hr at room temperature. Samples then were incubated for 48 hr on a shaker at 4°C with the following primary antibodies: rabbit anti-Myc (Abcam, ab9106), mouse monoclonal anti-MeCP2 (Sigma, WH0004204M1), and chicken anti-GFP (Abcam, ab13970). The primary antibodies were then washed off (3× 0.3 mol/L PBST), and secondary antibodies were applied to the sections overnight at 4°C: Alexa Fluor 488 goat anti-mouse/rabbit (Invitrogen; 1/500), Alexa Fluor 546 goat anti-mouse/rabbit (Invitrogen; 1/500), Alexa Fluor 649, goat anti-mouse (Jackson ImmunoResearch Laboratories, 112-495-003JIR). Finally, sections were incubated with DAPI nuclear stain (Sigma; 1/1,000) for 30 min at room temperature before mounting with Vectashield (Vector Laboratories).