Vectastain kit

Brains from protein (PM₁₀) treated mice (IP, 200 µg/day×5 consecutive days) after lesioning with MPTP were isolated. Striatum and midbrain were rapidly dissected out, the hemispheres divided and the cortex removed from the surrounding structures. The tissues were fixed with 4% paraformaldehyde for 24 hrs and cryosected (20 µm). Dopaminergic neuronal cell marker in brain - tyrosine hydroxylase (TH) was immunostained with anti-TH (1∶1,000, Millipore, Ramona, CA) monoclonal antibody, followed by biotin-conjugated goat anti-rabbit secondary antibody (1∶500, Santa Cruz Biotechnology, Santa Cruz, CA), and developed with ABC kit (Vectastain kit, Vector Laboratories, Burlingame, CA).

Mice were transcardially perfused with 4% paraformaldehyde (PFA) and postfixed for an additional 24 h. Brains were then transferred to PBS and immersed into 4% agarose before sectioning. 3,3′-Diaminobenzidine (DAB) staining was carried out as before (Engel et al., 2006 (link)). Slices were treated with 1% H₂O₂ for 45 min to inactivate endogenous peroxidases. This was followed by an incubation with blocking solution (10 ml: 8.3 ml 1× PBS, 1 ml 10% BSA, 0.5 ml 5% FBS, 0.2 ml Triton-X-100) for 1 h and primary antibody (AT8, 1:100, Invitrogen, CA, USA) overnight at 4°C. On the following day, tissue sections were washed 3× with PBS for 10 min each and incubated with Vectastain kit (Vector Laboratories, Burlingame, CA, USA; one drop of biotinylated antibody and three drops of horse/donkey serum were mixed in 10 ml of 1% BSA-PBS). This solution was added to the tissue and incubated for 90 min at room temperature. Three washes were performed with PBS for 10 min followed by a 90 min incubation with Avidin (ABC) peroxidase complex at room temperature (two drops of reagent A and reagent B in 10 ml of 1% BSA-PBS). Slices were washed with PBS for 20 min and immersed in DAB solution (Sigma-Aldrich, Arklow, Ireland) for approximately 10 min. Then, slices were mounted using FluorSave^TM. Staining was examined under a light microscope.