Alexa488 donkey anti chicken

Mice were administered a xylazine and ketamine cocktail (10 mg and 100 mg per kg body weight, respectively) intraperitoneally and were transcardially perfused with 8–10 ml of 0.9% saline, followed by 8–10 ml of 4% paraformaldehyde in PBS. Following extraction, brains were placed in 4% paraformaldehyde in PBS overnight, before being placed in 30% sucrose in PBS for 5–7 d in preparation for sectioning. Then, 40-μm coronal sections were prepared using a freezing microtome and tissue was labeled with the following antibodies: chicken anti-GFP (1:500 dilution; Aves Labs, GFP-1020), mouse anti-mCherry (1:500 dilution; Clontech, 632543), rabbit anti-cFos (1:500 dilution; Synaptic Systems, 226 003), Alexa-488 donkey anti-chicken (1:250 dilution; Jackson Immuno Research, 703-545-155), Cy3 donkey anti-rabbit (1:250 dilution; Jackson Immuno Research, 711-165-152) and Cy3 donkey anti-mouse (1:250 dilution; Jackson Immuno Research, 715-165-151). For secondary and primary incubations, antibody blocking was performed using normal horse serum (Sigma-Aldrich, H0146). Antibody amplification was used to visualize ChR2-mCherry and ChR2-EYFP. Images were taken at ×20 magnification using an Olympus VS120 slide scanner^{18 (link)}.