Brains from newborn mice were fixed for 8 h in Immunofix and clarified with the X-Clarity system (Logos Biosystem, South Korea). Briefly, whole brains were polymerized with acrylamide “hydrogel solution” for 3 h at 37 °C with a vacuum followed by the passive clarification for 48 h at 37 °C in “clearing solution”.
Brains were then subjected to deep-tissue TH immunolabeling by staining with TH antibody (dilution 1:100, Merck Millipore) in PBS/0.3% Triton for 3 days, 1 day of washing followed by incubation with anti-rabbit Alexa 488 for 3 days (Jackson Immunoresearch).
Finally, brains were observed by a two-photon upright LSM 880 Zeiss microscope equipped by W Plan-Apochromat 20x/1.0 DIC (UV) VIS-IR M27 75 mm objective. The fluorochrome was excited with the IR laser set at 860 nm. Z-stack were set with the height of the sections scanning = ~1 μm. Images were then reconstructed using Zen lite 2.3 (Carl Zeiss).
Brains were then subjected to deep-tissue TH immunolabeling by staining with TH antibody (dilution 1:100, Merck Millipore) in PBS/0.3% Triton for 3 days, 1 day of washing followed by incubation with anti-rabbit Alexa 488 for 3 days (Jackson Immunoresearch).
Finally, brains were observed by a two-photon upright LSM 880 Zeiss microscope equipped by W Plan-Apochromat 20x/1.0 DIC (UV) VIS-IR M27 75 mm objective. The fluorochrome was excited with the IR laser set at 860 nm. Z-stack were set with the height of the sections scanning = ~1 μm. Images were then reconstructed using Zen lite 2.3 (Carl Zeiss).