Donkey anti rabbit conjugated to alexa fluor 488

Experiments were performed on hippocampal slices incubated in DMSO (0.1% v/v), CTZ (50 μM), FUR (100 μM), or CTZ (50 μM) + FUR (100 μM) for 2 h. Slices were fixed with 4% (v/v) paraformaldehyde (PFA; pH 7.4) at 4 °C overnight. After dehydration in 30% (w/v) sucrose, the slices were cut into 30-µm sections that were thoroughly rinsed in Tris-buffered saline (TBS), permeabilized, and blocked for 2 h in 0.2% (v/v) Triton X-100% and 10% (v/v) normal donkey serum (NDS) in TBS at RT. The sections were incubated with primary antibody (anti-KCC2, 1:300; EMD Millipore, Billerica, MA, USA) diluted in 10% (v/v) NDS overnight. After several rinses in TBS, the sections were incubated with secondary antibodies for 2 h (donkey anti-rabbit conjugated to Alexa Fluor 488; Molecular Probes, Eugene, OR, USA) diluted in 10% (v/v) NDS at RT. The sections were then rinsed several times in TBS for ≥ 30 min each time and mounted on slides using the Fluoromount (Sigma-Aldrich Corp.). The sections were then viewed under an Olympus FV1000 confocal microscope with 60 × oil immersion objective, and the images were analyzed with Olympus Fluoview v.1.6a (Olympus Corp., Tokyo, Japan).

Neurons (DIV 11) were incubated in DMSO (0.1% v/v) control, CTZ (5 μM), FUR (100 μM), or FUR (100 μM) + CTZ (5 μM) for 48 h. They were then rinsed once with TBS and fixed with 4% (v/v) PFA in 0.1 M phosphate buffer (pH 7.4) for 10–12 min. After several rinses in TBS, the cells were permeabilized and blocked for 2 h in 0.2% (v/v) Triton X-100 (Sigma-Aldrich Corp.) and 10% (v/v) NDS (EMD Millipore) in TBS (pH 7.4) at RT. The neurons were incubated with primary antibody (rabbit anti-KCC2, 1:300; EMD Millipore) diluted in 10% (v/v) NDS at 4 °C overnight. After several rinses in TBS, the neurons were incubated with the corresponding secondary antibodies (donkey anti-rabbit conjugated to Alexa Fluor 488, 1:300; Molecular Probes) diluted in 10% (v/v) NDS at RT. The neurons were then rinsed several times in TBS for ≥ 30 min and mounted on slides with coverslips using ProLong Gold antifade reagent (Molecular Probes). The slides were viewed under an Olympus FV1000 confocal microscope with 60 × oil immersion objective, and the images were analyzed with Olympus Fluoview v.1.6a (Olympus Corp.).