Alexa fluor 647 donkey anti rabbit

To confirm that eGFP was expressed in CRH neurons in the CRH-eGFP transgenic mice, immunohistochemistry was performed on brains of four 5-week-old CRH-eGFP mice. Each mouse received an intracerebroventricular stereotaxic injection of colchicine (20 μg in 2 μL saline, Cayman Chemical Company) into the lateral ventricle under isoflurane anesthesia 24 h before sacrifice. Mice were then anesthetized with ketamine/xylazine and transcardially perfused with ice-cold phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS). Brains were dissected from the cranium and post-fixed with 4% PFA at 4°C overnight and then submerged in 30% sucrose/4% PFA/PBS. Forty μm-thick coronal sections of the hypothalamus containing the PVN were cut on a freezing microtome. The sections were pre-incubated in a blocking solution containing 1.5% BSA and 0.4% Triton X-100 in PBS for 1 h and then incubated with rabbit anti-human/rat CRF primary antibody (1:20,000, #PBL rC68; RRID:AB_2650435, Salk Institute for Biological Studies) at 4°C overnight followed by incubation in a secondary antibody Alexa Fluor 647 donkey anti-rabbit (1:500, Abcam) at room temperature for 1 h. Sections were mounted on glass slides, coverslipped, and imaged on a laser scanning confocal microscope (Nikon A1).

Brains were washed in 0.2% Triton X-100 (Sigma T8787) in PBS, followed by 20% DMSO (Fisher Scientific D128) + 0.3 M glycine (Sigma 410225) + 0.2% Triton X-100 in PBS at 37°C for 2 d. Brains were then washed in 10% DMSO + 6% normal donkey serum (NDS; EMD Millipore S30) + 0.2% Triton X-100 in PBS at 37°C for 2–3 d to block non-specific antibody binding. Brains were then twice washed for 1 h at 37°C in 0.2% Tween-20 (Sigma P9416) + 10 mg/ml heparin in PBS (PTwH solution) followed by incubation with primary antibody solution (rabbit anti-Fos, 1:1000; Synaptic Systems 226008) in 5% DMSO + 3% NDS + PTwH at 37°C for 7 d. Brains were then washed in PTwH 6× for increasing durations (10 min, 15 min, 30 min, 1 h, 2 h, overnight) followed by incubation with secondary antibody solution (Alexa Fluor 647 donkey anti-rabbit, 1:200; Abcam ab150075) in 3% NDS + PTwH at 37°C for 7 d. Brains were then washed in PTwH 6× for increasing durations again (10 min, 15 min, 30 min, 1 h, 2 h, overnight).
CGRP stim timepoint samples also received primary (chicken anti-GFP, 1:500; Aves GFP-1020) and secondary (Alexa Fluor 594 donkey anti-chicken, 1:500; Jackson Immuno 703-585-155) antibodies for ChR2-YFP immunolabeling during the above protocol.