Anti crf

Serial hippocampal or hypothalamus sections (40 μm) were made on an oscillating tissue slicer. For c-FOS and CRF immunofluorescence, the sections were incubated with rabbit anti-c-FOS antibody (1:400; Synaptic Systems) and anti-CRF (1:200; Santa Cruz Biotechnology) in 0.1 M PBS with 3% goat serum and 0.3% Triton X-100, and binding was visualized with a Cy3-conjugated secondary antibody (1:200; Jackson immunoresearch). Images of immunostained neurons in all groups were captured with a Zeiss Axio Cam MRC 5(D) camera mounted on Carl Zeiss Axio Observer A1 microscope under same conditions. An experimenter coded all slides from the experiments before quantitative analysis. All CRF-positive cells and c-FOS-positive in the PVN were counted in each section by another experimenter blinded to the study code.

Rats from the PAC1R KD experiment were euthanized 24 h after the last (10th) CSDS session. After transcardial perfusion, coronal 30-μm sections were cut on a cryostat, collected, and stored in cryoprotectant at −20°C. Every sixth section (180 μm apart) of the CeA (bregma range: −2.0 to −3.0 mm) were processed for IHC. Slices were pretreated with 100 mm urea (pH 9.5) for 10 min at 95°C followed by 10 min in an iced water bath. Sections were placed for 1 h in blocking solution (3% normal donkey serum, 0.4% Triton X-100) and subsequently incubated overnight at room temperature with a cocktail of two primary antibodies in blocking solution, an anti-CRF (1:200, Santa Cruz) and an anti-GFP (1:1500, Abcam). Sections were then incubated with the secondary antibodies donkey anti-rabbit Alexa Fluor 488 and donkey anti-goat Cy3 (Jackson ImmunoResearch) 1:400 in blocking solution for 2 h at room temperature. Sections were mounted onto glass slides, coverslipped with Vectashield mounting medium (Vector Laboratories), and stored at 4°C.