Mouse anti gfp antibody

The Hi5 cells were seeded into 6-wells culture plate and transfected with plasmids pHaABCC2-GFP, pmHaABCC2-GFP and pGFP as described above, respectively. Cells were harvested by centrifugation and subjected to protein extraction at 24 h post transfection. The proteins were separated on 8% SDS-PAGE gel. After electrophoresis, the proteins were transferred onto PVDF membrane (Millipore Corporation, Billerica, MA, USA). The membrane was blocked with 5% non-fat milk in TBS-T buffer for 2 h at room temperature, and then incubated with mouse anti-GFP antibody (Abcam, Cambridge, UK) 1: 3000 dilution in TBS for 2 h at room temperature. The membranes were then washed for three times with TBS-T and then incubated with fluorescent secondary antibody 1:5000 dilution (Earthox, San Francisco, CA, USA). Finally, the membranes were washed for three times with TBS-T and bands were visualized using the Odyssey system (LI-COR Bioscience, Lincoln, NE, USA).

For double immunofluorescence labeling, six adult GAD67-GFP mice were perfused transcardially with 0.1 M phosphate buffer (PB; pH 7.4) containing 4% paraformaldehyde. Lumbar segments 3–5 of the spinal cord were obtained and postfixed with the same fixative for 4 h, placed in 30% (w/v) sucrose solution in 0.05 M PB solution (PBS; pH 7.4) overnight at 48°C and cut into 25-μm thick sections on a freezing microtome. The sections were incubated overnight at 4°C with a mixture of mouse anti-GFP antibody (1:1,000; Abcam) and rabbit anti-NT antibody (1:500; Abcam). The incubation medium was prepared by 0.01 M PBS (pH 7.4) containing 0.3% (v/v) Triton X-100, 0.12% (w/v) carrageenan, 1% (v/v) normal donkey serum, and 0.02% (w/v) sodium azide (PBS-XCD). After a rinse with PBS, the sections were incubated for 3 h at room temperature in PBS-XCD with a mixture of Alexa488-conjugated donkey anti-mouse antibody (1:500; Invitrogen) and Alexa594-conjugated donkey anti-rabbit antibody (1:500; Invitrogen). The sections were mounted onto gelatin-coated glass slides and cover-slipped with 50% (v/v) glycerol and 2.5% (w/v) triethylenediamine (antifading reagent) in 0.01 M PBS. The sections were observed under a confocal laser scanning microscope (FV-1000, Olympus, Japan) with a confocal depth of 1.0 mm.