Ab43841

Drd1a-ChR2 mice were administered a single injection of colchicine (2.5 mM; Sigma, St. Louis, MO) in the lateral ventricle. ~24 hours later, colchicine-injected mice were deeply anesthetized and transcardially perfused with 4% (w/v) paraformaldehyde (PFA; Sigma). Brains were removed and post-fixed with 4% PFA overnight, and cryoprotected in 20% sucrose in PBS. A cryostat was used to obtain 40 μm coronal slices containing the SCN. Slices were then labeled for AVP using rabbit polyclonal anti-vasopressin (1:5000, ab1565, Millipore, Billerica, MA) or VIP using rabbit polyclonal anti-VIP (1:2500, ab43841, Abcam, Cambridge, MA). For cFos experiments, membrane-attached organotypic SCN cultures were fixed for 1 hour in 4% PFA post-ChR2 stimulation and labeled for cFos using rabbit polyclonal anti-cFos (1:1000, ab7963, Abcam). For visualization, slices were incubated with Alexa Fluor 488 goat anti-rabbit IgG (1:500, Invitrogen). Slices were examined under a confocal microscope (LSM510; Zeiss; Thornwood, NY) at 488 nm for Alexa Fluor 488 and 543 nm for ChR2-tdTomato. Colocalization of Drd1a-ChR2 and AVP or VIP was determined manually using ImageJ and was defined as an AVP or VIP-positive (i.e., green fluorescent) neuron completely surrounded by red tdTomato membrane-bound fluorescence. ImageJ was also used to perform manual quantification of cFos-positive cell numbers.