Eukit

Paraformaldehyde (PFA)  fixed brains (4% in 0.1 M PBS) were washed in 0.1 M PBS, dehydrated in ethanol solutions with increasing concentrations (70, 80, 90, 96, 100%, three times, 30 min each), cleared in xylol (three times, 15 min and overnight), incubated (twice at 60 °C, overnight and 2 h), and finally embedded in paraffin. Sagittal brain slices (8 μm) were mounted on poly-l-lysine-coated glass slides. Paraffin was removed by incubation of the slices with RotI-histol (2 × 10 min, Carl Roth GMBH) and subsequent treatment with ethanol 100, 96, and 70% (3 min, each) and rinsing in H₂O. Staining was performed in 0.1% cresyl violet solution for 5–10 min followed by short rinsing in H₂O and two brief washes (in 96% ethanol). After dehydration in 100% ethanol (twice, 3 min each) slices were cleared in RotI-histol (twice, 3 min each) and mounted in a permanent mounting medium (Eukit, Sigma-Aldrich).

Cells on coverslips were fixed in 4% paraformaldehyde, permeabilized in PBS/0.3% triton blocked with 10% FBS in PBS and incubated with the primary (anti-GFP-FITC goat polyclonal-ABCAM ab6662 and anti-cardiac troponin T [1C11] mouse monoclonal-ABCAM ab8295) and secondary (ALEXA FLUOR 568 goat anti-mouse ABCAM ab175473) antibodies diluted in blocking solution (1:500). Nuclei were marked using DAPI (Sigma), and the coverslips mounted on slides using Eukit (Sigma).

Immunohistochemistry was performed in twelve coronal sections covering the extent of the mice striata (25 μm-thick sections at 200 μm intervals). After the blockage of endogenous peroxidases with phenylhidrazyne/phosphate solution, free-floating sections were washed with PBS 0.1 M and blocked for 1 h at room temperature in blocking solution (0.1% Triton X-100 in PBS 0.1 M supplemented with 10% normal goat serum). Sections were processed overnight at 4 °C in blocking solution with the following primary antibodies: a polyclonal rabbit anti-ubiquitin antibody (1:300; Enzo Life Sciences) and a polyclonal rabbit anti–DARPP-32 antibody (1:1000; Merck Millipore), followed by 2-h incubation at room temperature with the respective biotinylated goat anti-mouse or anti-rabbit antibodies (1:200; Vector Laboratoires). Bound antibodies were visualized using the Vectastain ABC kit, with 3,30-diaminobenzidine tetrahydrochloride (DAB metal concentrate, Pierce) as substrate. Dry sections were mounted in gelatin-coated slides, dehydrated with ethanol solutions and xylene and mounted in Eukit (Sigma-Aldrich).