Immunostaining Striatal Neuron Subtypes

To identify direct- and indirect-pathway SPNs electrodes were loaded with biocytin, as described (Martella et al., 2009 (link)). Briefly, slices were fixed with 4% PFA in 0.12 M PB and 30 μm thick sections were cut from each slice with a freezing microtome, then dehydrated with serial alcohol dilutions to improve antigen retrieval and reduce background (Buchwalow et al., 2011 (link)). We used the following primary antibodies: goat anti-DARPP-32 (1:500 AF6259, R and D system), mouse anti-Enkephalin (1:1000 MAB350, Millipore), and secondary antibodies: anti-goat alexa 647 (Invitrogen), anti-mouse cyanine 3 (Jackson ImmunoResearch) and streptavidin-conjugated alexa 488 (Life Technologies). All sections used for analysis were processed together. Images were acquired with a LSM700 Zeiss confocal laser scanning microscope and analyzed with ImageJ software (NIH; Schneider et al., 2012 (link)). Noise was reduced by applying background subtraction in ImageJ.