Rats were anesthetized with a combination of ketamine and xylazine, euthanized by cardiac puncture, and transcardially perfused for 5 min with 1XPBS (75 mL), followed by 15 min of 4% paraformaldehyde in 1XPBS (225 mL). Brains were then frozen with dry ice; using a microtome (Leica SM 2010R), 40 μm coronal slices containing the LH were collected and stored in vials of 0.01% sodium azide in 1XPBS at 4 °C until use. Immunohistochemistry staining for GFP was conducted as previously described [10 (link),12 (link)]. Slices were mounted onto Superfrost Plus microscope slides and allowed to dry overnight. Slides were rehydrated and cover-slipped according to previously published protocols [10 (link),12 (link)]. Targeting for each rat was assessed by examining immune-enhanced GFP expression under a confocal microscope. All images for targeting were captured using Leica True Confocal Scanner SPE and Leica Application Suite Advanced software (Leica Microsystems, Germany) [12 (link)].
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