Confocal Imaging and Tracing of Interneurons

Slices were fixed in 4% paraformaldehyde (Boston BioProducts) and incubated with streptavidin Alexa Fluor 633 (Life Technologies, Invitrogen) in phosphate buffered saline (PBS) with Triton-X 100 as previously described (Bell et al., 2011 (link)). Processed slices were then reconstructed using a Zeiss LSM 710 confocal microscope (Carl Zeiss, Jena, Germany). Alexa Fluor 633 was excited with the 633 nm line of a HeNe 5 mW laser and cells were visualized using a 20× dry lens (0.8 N.A., voxel dimensions 0.2 × 0.2 × 1.1 µm). The imaged interneurons were traced using the Autoneuron module within the Neurolucida program (MBP, Burlington, VT). For amplification of YFP-labeled interneurons, 1:200 dilution of rabbit anti-GFP conjugated to Alexa Fluor 488 (Life Technologies, Invitrogen) in goat blocking buffer (10% normal serum, 2% bovine serum albumin, 0.4% Triton-X 100 in 0.1 M phosphate buffer) was added to fixed and washed slices for overnight incubation. Before and after primary and secondary antibody incubations, slices were washed in PBS. Slices were mounted in either Prolong Gold® (Life Technologies, Invitrogen) or VECTASHIELD® hard mount (Vector Laboratories).