C2 si confocal microscope system

Mice were subjected to flush-perfusion with phosphate-buffered saline (PBS) followed by perfusion-fixation with 4% paraformaldehyde (PFA). The removed brain was immersed in 4% PFA overnight, and subsequently in 30% sucrose for 48 h at 4°C. After the brain had been cut into coronal 30-μm sections, H₂O₂-inactivation of endogenous peroxidase activity was performed for immunohistochemistry, followed by treatment with 2% bovine serum albumin (BSA) in PBS-0.2% Triton X-100 for 60 min at room temperature to block non-specific protein binding. The sections were incubated with a rabbit monoclonal antibody against c-Fos (Cell Signaling Technology, Beverly, MA, United States) and a mouse monoclonal antibody against FOX3 overnight at 4°C. After incubation with a HRP-conjugated secondary antibody, the sections were then treated with 3,3′-diaminobenzidine (DAB) for immunohistochemistry. An Alexa Fluor 594-labeled donkey polyclonal antibody against rabbit IgG (H + L), F (ab’)2 fragment and an Alexa Fluor 488-labeled donkey polyclonal antibody against goat IgG F (ab’)2 fragment were used for immunofluorescence. Fluorescence was observed using a Nikon C2 Si confocal microscope system.

The mouse colon was opened and pinned on a silicone-sheeted glass dish. Each preparation was fixed overnight with 4% PFA, and then the mucosa, submucosa, and circular muscle layer were removed under a dissecting microscope. Preparations were incubated in PBS-1% Triton X-100 for 30 min, followed by incubation with rabbit monoclonal antibody against c-Fos (Cell Signaling Technology, Beverly, MA, United States), goat polyclonal antibody against choline acetyltransferase (ChAT, Merck Millipore, Guyancourt, France), and goat polyclonal antibody against neuronal nitric oxide synthase (nNOS, Abcam, Cambridge, MA, United States) for 24 h at 4 C. And subsequently with Alexa Fluor 594-labeled donkey polyclonal antibody against rabbit IgG (H + L), F (ab’)₂ fragment (Cell Signaling Technology, Beverly, MA, United States) or Alexa Fluor 488-labeled donkey polyclonal antibody against goat IgG F (ab’)₂ fragment (Abcam, Cambridge, MA, United States) for 3 h at 4°C. Nuclear staining was performed with 2′-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5′-bi-1H-benzimidazole (Hoechst 33342, Kumamoto, Japan). Fluorescence was observed using a Nikon C2 Si confocal microscope system.