Axiovert 200 imaging microscope

Cerebellar sections were stained using the In situ cell death detection kit, TMR Red (Roche, Mannheim, Germany) following the supplier's manual, which detects apoptotic cell death. The images were acquired digitally using a 40× objective on a Zeiss Axiovert 200 imaging microscope. All photographs for comparison were taken under identical image acquisition conditions and uniform adjustments of brightness and contrast were made to all images.

The immunohistochemical procedure initiated with a 30 min dehydratation at 37°C followed by a 30 min hydration in 0.1 PBS and 1 h blocking in a 0.3% triton in 0.1 PBS with 10% normal goat serum both at room temperature (RT). The following primary antibodies diluted in a blocking solution with 0.1% triton were used: mouse monoclonal anti-ataxin-3 (1H9, Chemicon, Temecula, CA, USA; 1∶5000; 48 h, 4°C), rabbit polyclonal anti-β-galactosidase (Molecular Probes, Invitrogen; 1∶1000; 48 h, 4°C), rabbit polyclonal anti-DARPP-32 (Chemicon,Temecula, CA, USA; 1∶5000, 48 h, 4°C), rabbit polyclonal anti-calbindin D-28K (Chemicon,Temecula, CA, USA; 1∶1000, 48 h, 4°C). Sections were then incubated in secondary antibody, goat-anti rabbit and/or mouse conjugated to alexa 488 or 594 (Invitrogen) for 2 h/RT e and then mounted in mowiol (Sigma) with 4′,6′-diamidino-2-phenylindole.Fluorescence images were acquired with a Zeiss Axiovert 200 imaging microscope or LSM Zeiss microscope for double staining experiments.

To assess dopaminergic neuronal survival, we performed TH-positive (TH⁺) cell counting as described previously [27 (link), 29 (link)]. First, the sections were incubated with a blocking solution (0.1% Triton X-100 and 10% FBS in PBS), then the endogenous peroxidases were inhibited with H₂O₂ and later incubated overnight at 4 °C with the primary antibody mouse anti-TH (1:500, Transduction Laboratories). After several rinses, the slices were incubated with biotinylated goat anti-mouse secondary antibody (1:200, Vector Laboratories) for 1 h at room temperature. Subsequently, the Vectastain ABC kit was added, and the resulting product was visualized by adding DAB to the slices until color developed (5–10 min). Afterward, the sections were counterstained with Nissl (0.25% Cresyl Violet dissolved in acetate buffer) for 4 min, washed in tap water, air-dried, cleaned with xylene, and mounted with Entellan™ (Merck).
Quantification of the TH⁺ neurons in the mice SN was performed in five consecutive coronal sections separated by 240 µm. The SN was carefully delineated, and the number of TH⁺ cells in each condition was counted. Images were acquired under the magnification of 10 × at the Zeiss Axiovert 200 imaging microscope (Axiobserver Z1, Zeiss), and the number of TH⁺ cells was counted using the ImageJ program.