Anti th

After culturing the cells as described above, were fixed in 4% PFA in PBS for 15 min and permeabilized in CH₃OH for 7 minutes at −20° C. Cells were incubated in 4% BSA (Sigma Aldrich, USA) for 30 minutes then with the subsequent primary overnight at 4° C: rabbit polyclonal anti-β-Tubulin III (1:1000 Abcam, Cambridge, UK), rabbit polyclonal anti-TH (1:200, Novus Biologicals, Centennial, USA), mouse monoclonal anti-GAP43 (1:200, Abcam, Cambridge, UK). After several washings, coverslips were incubated with secondary antibodies, goat anti-mouse or anti-goat IgG Alexafluor 488 or 633 or 546 (1:2000 Life Technologies, California, USA), for 1h at RT. After different washes, Vectashield mounting with DAPI (Vector Laboratories Burlingame, USA) were used. All the samples were observed using confocal laser microscope (Leica, Wetzlar, Germany).

For stereological assessment [47 (link)], mice were perfused with PBS followed by 4% paraformaldehyde. After post-fixed with 4% paraformaldehyde for 12 h, the tissue samples were cryoprotected with 30% sucrose, and processed for immunohistochemistry. 50 μm coronal sections were cut throughout the brain including substantia nigra and every 4th section was used for analysis. The rabbit polyclonal anti-TH (1:1000; Novus) was incubated in blocking solution. The signals were visualized using DAB kit (Vector Laboratories) followed by incubation with biotinylated secondary antibodies and streptavidin-conjugated horseradish peroxidase (HRP) (Vectastain ABC kit, Vector Laboratories). The stained tissue sections were mounted onto slides and counterstained with thionin for Nissl substance. The total number of TH-, and Nissl-positive neurons in the SNpc was counted using Optical Fractionator probe of Stereo Investigator software (MicroBrightfield).