Vectastain avidin biotin peroxidase kit

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 ×.

Perfused, fresh spinal cord tissues were homogenized in 50 mM Hepes pH 7.5, 100% glycerol, 10 mM NaCl, 10 mM dithiothreitol, 1% SDS, 5 mM EDTA and protease inhibitors (Sigma Aldrich, Milan, Italy). Samples were loaded on a 0.75 mm SDS polyacrylamide minigel (10%, 12%) that was electrophoresed at 150 V for 90 min. The proteins were transferred to nitrocellulose membranes overnight at 30 V and 4 °C. After blocking of non-specific sites with 5% milk, 20 mM Tris HCl (pH 7.4) and 0.2% Tween 20 (TBST), membranes were incubated overnight with primary antibody anti-EAAC1 (1:400), anti-vimentin (1:250) and β-actin (1:2000). After being washed in TBST, membranes were incubated with the appropriate biotinylated secondary antibody (Vector Labs Inc., Burlingame, CA, USA, 1:200) in blocking solution for 60 min at room temperature. Subsequently, they were washed in TBS and processed using the Vectastain avidin–biotin peroxidase kit (Vector Labs Inc., Burlingame, CA, USA) for 30 min at RT. Bands were revealed by reacting with 3,3-diaminobenzidine tetrahydrochloride (DAB) (Sigma Aldrich, Milan, Italy) 0.5 mg/mL Tris-HCl and 0.01% hydrogen peroxide.
The density of each band was measured with a computer-assisted imaging analysis system (MCID 7.1; Imaging Res. Inc., Ontario, CA, USA) and normalized with the corresponding β-actin band used as the internal loading control.