Wisteria floribunda lectin

Two-day-old colonies were prepared for thin sectioning as described above. Rehydrated colonies were post-stained in 100 µg/ml fluorescein-labeled Wisteria floribunda lectin (Vector Laboratories (Burlingame, CA) FL-1351) in PBS before being washed twice in PBS, mounted in TRIS-buffered DAPI and overlaid with a coverslip. Fluorescent confocal images were captured using an LSM700 confocal microscope (Zeiss).

The mice were killed by transcardial perfusion with 4% paraformaldehyde and the brains were dissected and sectioned coronally at 50 μm. Sections comprising the somatosensory cortex were incubated in a blocking solution containing 5% donkey serum and 0.2% Triton X-100 in phosphate buffer for 1 h, followed by the incubation with the blocking solution containing mouse anti-parvalbumin (Sigma, 1:1,000) or rat anti-somatostatin (Millipore, 1:200) antibodies or fluorescein-labelled Wisteria floribunda lectin (Vector Laboratories, 1:500), in combinations with rabbit anti-GABA (Sigma, 1:500) or guinea pig anti-type 2 vesicular glutamate transporter (VGluT2, Millipore, 1:1,000) antibodies overnight at 4 °C. Primary antibodies were fluorescently labelled by incubation with appropriate secondary antibodies conjugated with Alexa Fluor-405, -488 and -594 (1:200, Invitrogen). Fluorescence images were taken using LSM710 confocal laser-scanning microscope (Zeiss). VGluT2 staining was used to visualize barrel structures of the somatosensory barrel cortex, based on the dense labelling of axon terminals from thalamocortical projections49 (link). The specific interneuron cell types were counted manually using FIJI image processing software.