Rat anti cd86

Nodose ganglia and whole brains (n = 4−5 per group) were immersed in 4% paraformaldehyde/phosphate buffer for 24 h at 4°C, incubated for 24 h in PB containing 20% sucrose, quickly frozen on dry ice and cut into 8-µm slices using a cryostat at −20°C. Sections were blocked for 5 min in protein-block serum-free solution (Dako), and then incubated overnight at 4°C with rabbit anti-Iba1 (1:10,000; Wako Pure Chemicals), rat anti-CD86 (1:100; Abcam), mouse anti-HSP72 (1:50; Enzo Life Sciences, New York, NY, USA) or mouse anti-NeuN (1:200; Millipore, Chemicon International). Immunofluorescence was performed with Alexa Fluor 488-labeled anti-rabbit secondary antibody or Alexa Fluor 594-labeled anti-mouse secondary antibody (both 1:400; Invitrogen). Images were captured on an OLYMPUS AX-7 fluorescence microscope (Olympus). Cells immunoreactive for Iba1, CD86 or HSP72 in the nodose ganglion and hypothalamus of three mice were counted manually in three to five sections per mouse using the cellSens imaging software (Olympus). Quantitation was performed in a blinded fashion.

Nodose ganglia and whole brains (n=4/group) were immersed in 4% paraformaldehyde/PB for 24 h at 4 °C, incubated for 24 h in PB containing 20% sucrose, quickly frozen on dry ice, and cut into 8-μm slices with a cryostat at −20 °C. Sections blocked for 5 min in protein-block serum-free solution (Dako, Carpinteria, CA, USA) were incubated overnight at 4 °C with rabbit anti-IBA1 (1:10 000; Wako Pure Chemicals), rat anti-CD11b (1:50; AbD Serotec, Oxford, UK), and rat anti-CD86 (1:100; Abcam, Cambridge, UK). Immunofluorescence was performed with a combination of Alexa Fluor 488-labeled anti-rabbit secondary antibody or Alexa Fluor 594-labeled anti-rat secondary antibody (both 1:400; Invitrogen). Images were captured on an OLYMPUS AX-7 fluorescence microscope (Olympus). Cells immunostained with IBA1, CD11b, or CD86 antibody were counted manually with Olympus cellSens Imaging Software (Olympus). Quantitation was performed in a blinded fashion.