Axioskope 2 light microscope

Slides were imaged with a Zeiss Axioskope 2 light microscope equipped with a high-resolution digital camera (C4742-95, Hamamatsu Photonics, Arese, Milan, Italy) for standard light microscopy. Zeiss LSM 510 Meta laser scanning microscope (Oberkochen, Germany) confocal images of dorsal horns of the lumbar spinal cord were captured at a resolution of 512 × 512 pixels. Argon laser fluorescence was used for visualization of the vGLUT1 and vGAT (excitation wavelength of 488 nm and bandpass emission filter of 505–530 nm), and HeNe laser fluorescence was used for the GAD65/67 signal (excitation wavelength of 546 nm and long-pass emission filter of 560 nm).

Brain sections were blocked in 10% normal serum in 0.01 M PBS containing 15mM NaN3 and 0.25% Triton for 1 h at room temperature (RT). Each primary antibody was diluted in the blocking solution. Following 48-h incubation at 4 °C, sections were washed six times (10 min each) in PBS and incubated with the appropriate biotinylated secondary antibody (Vector Labs Inc., Burlingame, CA, USA; 1:200) for 90 min at RT, washed in PBS, and processed using the Vectastain avidin–biotin-peroxidase kit (Vector Labs Inc., Burlingame, CA, USA) for 90 min, at RT. Sections were washed in 0.05 M Tris–HCl and reacted with 3,3-diaminobenzidine tetrahydrochloride (DAB; Sigma, 0.5 mg/ml in Tris–HCl) and 0.01% hydrogen peroxide. Floating sections were mounted on chrome-alum gelatine-coated slides, dehydrated, and coverslipped. After mounting, serial sections were (counter)stained using Nissl staining (cresyl violet acetate solution) for 45 min, dehydrated, and coverslipped. Slides were imaged with a Zeiss Axioskope 2 light microscope equipped with a high-resolution digital camera (C4742-95, 72 px/inch, Hamamatsu Photonics, Italy), using objective lens spanning from 2.5 × to 40 ×.