5.6. Tyrosine Hydroxylase Immunohistochemistry

Rat brains were sliced coronally into 50 µm thick sections, using a sliding microtome (SM2010 R, Leica Microsystems, Wetzler, Germany) as previously described [83]. Sections containing the substantia nigra (from bregma: approximately −4.8 to −6.1 mm) were incubated in rabbit anti-TH antibody (1:500; Pel-Freez Biologicals, Rogers, AR, USA) for 24 h followed by a 2-h incubation with biotinylated goat-anti-rabbit secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA). Sections were then incubated with avidin/biotinylated complex (ABC; Vector Laboratories), and bound complexes were visualized using 3,3′-diaminobenzidine and hydrogen peroxide tablets as previously described [56]. The number of TH-positive neurons was estimated by stereological means (Stereo Investigator, MBF Biosciences, Williston, VT, USA). Briefly, the SNc region in 6 coronal sections (collected at 200 µm intervals) was carefully outlined under 4× magnification using a rat brain atlas [78]. TH+ cells from the SNc on the ipsilateral (lesioned) and contralateral (intact) sides were counted at 100× magnification. Cells were only included if the nucleus and soma were visible and under focus. The extent of the lesion was then calculated as the number of TH+ cells on the lesioned side relative to that on the intact side.