2.3. c-Fos and CRF Immunohistochemistry

After paraformaldehyde fixation, brains were kept in 30% sucrose dissolved in PBS for at least three days, until fully dehydrated. Brains were sectioned on a cryostat at 30 μm sections and kept in cryoprotectant until staining for immunohistochemistry as previously described [30,31]. Brain sections were washed with PBS, then incubated with rabbit anti-c-Fos primary antibody (SySy, Göttingen, Germany, 1:2000) dissolved in PBS diluent. A donkey anti-rabbit biotinylated secondary antibody (Vector Labs, Burlingame, CA, USA; 1:10,000) was used along with A + B Vector staining and DAB to visualize c-Fos expression. A guinea pig anti-CRF primary antibody (Peninsula, San Carlos, CA, USA; 1:1000) was then used along with a goat anti-guinea pig AlexaFluor 488 secondary antibody (Fisher, Hampton, NH, USA; 1:10,000) to confirm the presence and LPS-mediated activation of CRF-secreting parvocellular neurons within the paraventricular nuclei. Cells were imaged on an Olympus fluorescence microscope and counted manually on NIH ImageJ software [30].