Rhodamine red or fitc green conjugated secondary antibody

Male C57BL/6 mice, 25–30 g, were administrated P7C3 or MPTP as described in the animal experiments above. Then, the mice were perfused with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). After perfusion, the mice brains were harvested for post-fixation in the same fixing agent overnight at 4 °C, followed by the treatment with the 30% sucrose at 4 °C for another night. Serial 20 μM-thick mouse midbrain slices were prepared with a freezing microtome. Immunohistochemical staining was conducted using anti-TH antibodies (Millipore) against six slices per mouse (120 μm interval). After incubation with primary antibody at room temperature for 6 h, the slices were incubated with rhodamine (red)- or FITC (green)-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) for 2 h. Finally, the slices were stained with DAPI for 5 min and observed using an inverted IX71 microscope system (Olympus). TH⁺ neurons were counted with Image J software (National Institutes of Health, Bethesda, MD, USA) for each slice by two blinded investigators.

Male C57BL/6 mice, 25–30 g, were administrated with P7C3 or LPS described in the animal experiments above. After treatment, the mice were perfused with 0.9% saline followed by 4% paraformaldehyde in 0.1 M PBS (pH 7.4). The mice brains were then removed and post-fixed in the same fixation agent overnight at 4°C, followed by the treatment with the 30% sucrose at 4°C with another night. Serial 20 μM-thick mouse midbrain slices were cut with a freezing microtome. Immunohistochemical staining was conducted with anti-Iba1 antibody (Wako Chemicals, Tokyo, Japan), anti-GFAP and anti-TH antibodies (Millipore, Billerica, MA, USA) against six slices per mouse (120 μm interval). After incubation with primary antibodies at room temperature for 6 h, the slices were incubated with rhodamine (red)-or FITC (green)-conjugated secondary antibody (Invitrogen, Carlsbad, CA, USA) for 2 h. TH⁺ neurons were counted as described previously (Gu et al., 2017 (link)). Finally, the slices were stained with DAPI for 5 min and observed using an inverted IX71 microscope system (Olympus, Tokyo, Japan). Fluorescence intensity of Iba-1 and GFAP were anylized using ImageJ software (National Institutes of Health, Bethesda, MD, USA) as described previously (Guo et al., 2018 (link)).