Using the previously collected mouse brain samples, 35 µm coronal sections were cut (Leica CM1950), and free floating sections were incubated overnight at room temperature with rabbit anti-TH primary antibody (Novus Cat. NB300-109), followed by incubation for 1 h with secondary antibody using the Elite rabbit IgG kit (vector laboratory PK-6101) [43 (link)]. The DAB kit (Vector laboratory PK-4100) was used for color development.
The number of TH positive dopamine neurons in the SN was estimated using unbiased stereological counting. The SN was delineated from 1.70 to 3.88 mm posterior to bregma using the Allen brain atlas [44 (link)]. A total of 7 coronal sections from the midbrain of each mouse were used for quantification (1 section from every 5 serial coronal sections across the midbrain was used); representative pictures containing TH+ neurons in the SN were taken using the 10 × magnification objective lens of an Olympus IX83 microscope. Scale bar, 500 µm. The number of TH+ neurons in each section was stereologically quantified using Stereologer 2000™, at 63 × magnification. The 7 sections from each mouse were then used to compare the number of TH+ neurons in Tg and NTg mice (n = 5 for Tg, n = 5 for NTg).
The number of TH positive dopamine neurons in the SN was estimated using unbiased stereological counting. The SN was delineated from 1.70 to 3.88 mm posterior to bregma using the Allen brain atlas [44 (link)]. A total of 7 coronal sections from the midbrain of each mouse were used for quantification (1 section from every 5 serial coronal sections across the midbrain was used); representative pictures containing TH+ neurons in the SN were taken using the 10 × magnification objective lens of an Olympus IX83 microscope. Scale bar, 500 µm. The number of TH+ neurons in each section was stereologically quantified using Stereologer 2000™, at 63 × magnification. The 7 sections from each mouse were then used to compare the number of TH+ neurons in Tg and NTg mice (n = 5 for Tg, n = 5 for NTg).