Cy2 conjugated streptavidin

Cells grown in 3.5-cm dishes were incubated with 50 μM biotinylated peptides for 30 min. Proteins were then collected in 1 ml of lysis buffer (20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 1% IGEPAL, 1 mM PMSF, protease cocktail (1:100; Cocktail III, Calbiochem), 1 mM NaF and 0.1 mM Na₃VO₄). Lysates were centrifuged at 11000 × g for 10 min at 4°C, and the supernatants were recovered. A 25-μl aliquot of each lysate was used to analyze the protein content, and the remaining lysate was incubated with NeutrAvidin-Agarose (Thermo Scientific, Rockford, IL, USA; Ref. 29200) for 12 h at 4°C with gentle shaking. The avidin beads bound with the peptides were collected by centrifugation (3000 × g for 1 min at 4°C). The beads were then washed five times with lysis buffer, and the bound proteins were eluted and analyzed by Western blotting. To detect biotinylated peptides, the membranes were incubated with HRP-conjugated streptavidin in TTBS (1:40000, Ref. 434323, Life Technologies) and then developed with a chemiluminescent substrate.
In parallel, the cells were incubated with 50 μM biotinylated peptides for 30 min. The cells were then washed with PBS at 4°C and fixed with 4% paraformaldehyde for 20 min. After washing, the cells were incubated with Cy2-conjugated streptavidin (1:500; Jackson ImmunoResearch, Baltimore, USA; Ref. 016-220-084) for 1 h, mounted and visualized as described previously.

The cell labeling protocol has been previously described57 and details are provided in the Supplementary Information. Briefly, to morphologically identify the recorded cells, some neurons were filled with 2% biocytin (Sigma-Aldrich, Saint Louis, MO, USA) through the recording pipette. Immediately after recording, slices containing biocytin-loaded cells were incubated with Cy2-conjugated Streptavidin (1:200; Jackson Immunoresearch Laboratories, PA, USA). To assess the cytoarchitectonic areas and layers, slices were counterstained with NeuroTrace® 640⁄660 deep-red Fluorescent Nissl Stain (Invitrogen) and mounted using an anti-fade medium (Fluoromount; Sigma-Aldrich, Saint Louis, MO, USA). Neurons of interest were identified using a 10× objective (Plan-Apochromat, Zeiss, Germany) and captured on a 40X oil immersion objective through a confocal laser-scanning microscope (CLSM700; Zeiss, Germany).

For immunohistochemical detection of GABA, sections were incubated overnight with a mouse monoclonal anti-GABA antibody (1:5000; mAb 3A12; kindly donated by Dr. Peter Streit, Brain Research Institute, University of Zürich, Zürich, Switzerland). The sections were subsequently incubated for 2 hr at room temperature with a Cy3-conjugated donkey anti-mouse IgG (1:500; Jackson ImmunoResearch) and, in those cases in which the cells were filled with neurobiotin, also with Cy2-conjugated streptavidin (1:1000; Jackson ImmunoResearch). All primary and secondary antibodies were diluted in 1% BSA and 0.3% Triton-X 100 in 0.1 M PB.