Rabbit anti neun antibody

Cultured cells were fixed in a PBS solution containing 4% paraformaldehyde for 15 min and washed 3 times with PBS. A PBS solution containing 0.25% Triton was added to the cells for 15 min at room temperature and then incubated with 5% bovine serum albumin for 1 h. After removing this blocking reagent, cells were incubated in a humidified chamber at 4°C overnight with primary antibodies: mouse anti-BrdU antibody (1 : 1400; Cell Signaling Technology; Cat. No. #5292S), rabbit anti-NeuN antibody (1 : 50; Cell Signaling Technology; Cat. No. #24307S), and rabbit anti-NeuN antibody (1 : 50; Cell Signaling Technology; Cat. No. #24307S) diluted in blocking reagent. Then, cells were washed 3 times with PBS and incubated for 1 h in the dark at room temperature in the presence of the fluorescent secondary antibodies: goat anti-mouse IgG-FITC (1 : 100; Absin; Cat. No. #10) and goat anti-rabbit IgG/Cy3 (1 : 100; Absin; Cat. No. #AG04017512). Finally, the coverslips were mounted onto slides in PBS. The preparations were analysed under a fluorescent microscope (Olympus FV1200).

Hippocampal slices were washed 3 times with 0.01 M phosphate buffer saline (PBS) and fixed in 4% paraformaldehyde (PFA) for 1 h at room temperature (RT). At the end of fixation, the slices were washed 3 times with PBS and then stored at 4 °C in PBS containing 0.05% sodium azide until use. Prior to immunolabelling, slices were washed 3 times with PBS for 10 min, then incubated overnight with 1% Triton X-100 in PBS at 4 °C, followed by a blocking stage using 20% bovine serum albumin (BSA) diluted in PBS (0.01 M) and containing 0.1% Triton (PBS-T) for 3 h at RT. Slices were then incubated overnight with primary antibody diluted in PBS-T and 1% normal goat serum (NGS): rabbit anti-NeuN antibody (1: 200, Cell Signalling Technology, D4G4O # 24,307), guinea pig anti-GLT1 antibody (1:5000, Merck Millipore Chemicon International, ab1783), rabbit anti-GLAST antibody (1: 150, Abcam, ab416), or rabbit anti-GS (1:5000, Abcam, ab49873). Following washes in PBS-T, slices were incubated in appropriate secondary antibodies for 3 h at RT, namely goat anti-guinea pig Alexa-fluor 568 or goat anti-rabbit Alexa-Fluor 647 (both at 1:500, Invitrogen), in addition to the nuclear chromatin dye Hoechst 33,342 (1:500, Fisher, 11,544,876). Lastly, slices were washed in PBS-T (0.1%) and mounted in Fluoromount G (Invitrogen—REF 00–4958-02).

A TUNEL assay was performed according to the manufacturer’s instructions (Roche Molecular Biochemicals, Inc., Mannheim, Germany). Sections were incubated with rabbit anti-NeuN antibody (Cell Signaling Technology, Danvers, MA, USA) in PBS/0.2% TX-100 and then incubated with the TUNEL reaction mixture to verify the neuronal identity of the TUNEL-positive cells. Finally, 4′,6-diamidino-2-phenylindole (DAPI) was added. The total number of TUNEL-positive neurons was counted by an investigator who was blinded to the study protocol.