Rabbit anti kcc2

Antibodies used for Western blotting and co-immunoprecipitation experiments were as follows, with dilutions used for Western blotting: rabbit anti-KCC2, Millipore, 1:1000; mouse anti-α-tubulin, Cedarlane Laboratories, 1:10,000; rabbit anti-β1 catalogue #13950, Cell Signaling Technologies, 1:1000; rabbit anti-NKCC1, Cell Signaling Technologies 1:1000; mouse anti-NKCC1 clone T4, Developmental Hybridoma Studies Bank, 1:500. For Western blotting experiments, secondary antibodies were HRP-conjugated goat anti-rabbit or goat anti-mouse as appropriate, ThermoFisher Scientific, 1:500. For immunofluorescent experiments, primary antibodies were rabbit anti-KCC2, Millipore, 1:500, and guinea pig anti-NeuN, Millipore, 1:1000; secondary antibodies were AlexaFluor 488 goat anti-rabbit and AlexaFluor 568 goat anti-guinea pig, Molecular Probes.

The various human KCC2 constructs were transfected into HEK293 cells using the MaxCyte STX electroporation. Expression of human KCC2 was confirmed by Western-blot using Rabbit anti-KCC2 as primary antibody (Sigma/Millipore #07-432). Culture media was DMEM (Sigma, D6546) with 10% FBS and 1% GlutaMAX (Gibco, 35050-038). For large scale production of protein for binding studies, suspension Expi293F cells were cultured in Expi293F media and transfected at high density with KCC2 plasmid DNA diluted to 1 mg/ml using PEIMax at 1 mg/ml^{38 (link)}. Transfection was carried out on 250 ml of suspension cells at 4 × 10⁶/ml in vented 2 L capacity roller bottles shaken in an atmosphere of 5% CO₂ at 37 °C and 140 rpm in an orbital incubator (25 mm orbit). DNA and PEIMax were pre-incubated in E293F media for 15 minutes before adding to the cells. Twenty-four hours post transfection, cultures were fed with 50% media making 500 ml total volume per roller bottle and incubated for a further 48hrs. Cell pellets were harvested 72hrs post transfection by centrifugation at 10,000 × g for 20 min and stored at −80 °C prior to purification. Cells and media were purchased from ThermoScientific; 40 kDa linear PEI Max as hydrochloride salt (catalogue #24765) from Polysciences. Mouse KCC2 was expressed in HEK293 and N2a cells as previously described^{5 (link),39 (link)–41 (link)}.

For blocking and permeabilization in 40-μm-thick sections, we used “staining buffer” containing 0.05 M Tris, 0.9% NaCl, 0.25% gelatin, and 0.5% Triton X-100, pH 7.4. Primary antibodies, rabbit anti-KCC2 (1:500; Sigma-Aldrich) were diluted in staining buffer and incubated overnight at 4 °C. The next day, the sections were washed in PBS and incubated with donkey anti-rabbit FITC antibody (1:500; Sigma-Aldrich). After a final three washes, sections were mounted in antifade mounting medium. Immunofluorescence-labeled cells were observed using a confocal laser-scanning microscope (Leica). KCC2 staining intensities in the CA1 region were measured using NIH Image J software. A box was drawn in the pyramidal cell body layer of CA1 field was quantified. Three independent background fluorescence intensity measurements were averaged and subtracted from the fluorescence intensity of each cell. KCC2 expression was estimated by calculating the relative mean fluorescence intensities per unit area.