Lsm510 imaging system

Mice were deeply anesthetized by pentobarbital and perfused transcardially with PBS followed by ice-cold 4% paraformaldehyde/PBS. Transverse L4 spinal cord sections (30 μm) and L4 DRG sections (15 μm) were incubated for 48 h at 4 °C with primary antibody for CD11b (1:1,000, Serotec, USA), Iba1 (1:2,000, Wako), GFAP (1:500, Chemicon, USA), NeuN (1:200, Chemicon), APC (1:200, Calbiochem, USA), NF200 (neurofilament 200, 1:400), P2X3R (1:2,500, Chemicon), IB4 (isolectin B4)-Alexa488 (1:200, Invitrogen, USA) and PKC-γ (protein kinase C gamma, 1:5,000, Santa Cruz, USA). Tissue sections were incubated with secondary antibodies conjugated to Alexa Fluor 405, 488 or 546 (1:1,000, Molecular Probes) and mounted with VECTASHIELD with or without DAPI (Vector Laboratories, USA). Three to five sections from the L4 spinal cord segments or L4 DRG segments of each mouse were randomly selected and analysed using an LSM510 Imaging System (Carl Zeiss, Japan).

Mice were deeply anaesthetized by pentobarbital and perfused transcardially with PBS followed by ice-cold 4% paraformaldehyde/PBS. The L4 segment of the lumbar spinal cord were removed, post-fixed in the same fixative and placed in 30% sucrose solution for 24 h at 4 °C. Transverse L4 spinal cord sections (30 μm) were incubated for 48 h at 4 °C with primary antibody. Identification of cell types was performed using the following markers: microglia, Cd11b (1:1,000, Serotec), Iba1 (ionized calcium-binding adapter molecule-1, 1:2,000, Wako) and Cd68 (1:1,000, Serotec); astrocytes, GFAP (1:500, Chemicon); neurons, NeuN (Neuronal Nuclei, 1:200, Chemicon). Spinal sections were incubated with secondary antibodies conjugated to Alexa Fluor 488 or 546 (1:1,000, Molecular Probes) and mounted in VECTASHIELD with or without DAPI (Vector Laboratories). Three to five sections from the L4 spinal cord segments of each mouse were randomly selected and analysed using an LSM510 Imaging System (Carl Zeiss, Japan).

Bright-field images were taken using a Zeiss Axioplan 2 + microscope, Nikon Digital Sight DS-F12 camera, and Nikon NIS 4.0 imaging software. Images of immunohistochemistry were obtained using a Zeiss LSM 5 Exciter-AxioImager M1 imaging system and Zeiss LSM510 imaging system. Image stacks were generated by scanning at intervals of 4 μm for lower magnification and at intervals of 0.8 μm for higher magnification using filters of the appropriate wavelengths. The stacks were analyzed, merged, and projected using ImageJ software from the National Institutes of Health. Figure panels were prepared using Adobe Photoshop CS6.