Rhodamine conjugated goat anti rabbit igg

Normal mice were anesthetized by chloral hydrate and were fixed with PFA solution (4% paraformaldehyde in 0.1 M phosphate buffer, pH = 7.4) through heart perfusion. The brains were removed and post-fixed in the PFA solution for 6 h and then cryoprotected in 20 and 30% sucrose at 4°C, respectively. After the dehydration, each brain containing the substantia nigra was cut into 20-μm-thick coronal sections on a freezing microtome (CM 1950, Leica, Germany). Non-specific binding was blocked with 5% BSA in PBS for 1 h. The sections were then incubated overnight with primary antibodies consisting of chicken anti-TH antibody (Abcam, 1:2,000) and rabbit anti-OX1R (Abcam, 1:100) or OX2R (Bioss, 1:200) antibody at 4°C for 36 h in a humidified chamber. Fluorescent isothiocyanate-conjugated goat anti-chicken IgG (Invitrogen, 1:500) and rhodamine-conjugated goat anti-rabbit IgG (Abcam, 1:500) were used as secondary antibodies. Finally, the sections were mounted on polylysine-coated slides and examined using an inverted fluorescent microscope (Axio Observer.A1, Zeiss, Germany). Negative control tests were run without primary or secondary antibodies.

Cryosections (5 mm thick) of the aorta were dried, fixed in cold acetone for 15 min, air-dried, and rinsed twice with PBS. Slides were blocked with 5% normal goat serum for 1 h at room temperature, incubated with antibody against HMGB1 (1/200, Abcam) overnight at room temperature. After three washes in PBS, sections were incubated with rhodamine-conjugated goat anti-rabbit IgG (1/100, Abcam) for 1 h. DAPI solution was added to stain the nuclei. Sections were visualized using a Laser-Scanning Confocal Microscope and imaged (Olympus, Tokyo, Japan).