T1299

After permeabilisation with saponin (Sigma), cells were blocked for 2 hours with PBS containing 10% goat serum, then incubated with mouse monoclonal primary antibody against Microtubule-Associated Protein 2 (MAP2, M4403, Sigma) for oxidative stress studies or against Tyrosine Hydroxylase (T1299, Sigma) for 6-OHDA cultures. These antibodies were revealed with Alexa Fluor 488 goat anti-mouse IgG (A-11001, Molecular probe). Hoechst (Invitrogen) counter-staining was used to visualise nuclei. For each condition, 10 pictures per well were taken using the InCell Analyzer^TM 1000 (GE Healthcare) with a 20× magnification.

Immunohistochemical staining to determine levels of DAT and D₂R proteins and TH activity was conducted with striatal slices. To prepare for staining, the slices were prewashed in PBS (0.01 M, pH 7.4) three times for 5 min each, then sequentially treated with 0.2% Triton X-100 in PBS for 5 min and with 0.3% H₂O₂ in PBS for 10 min, and washed in PBS three times for 5 min each, all at room temperature. Slices were initially incubated with 10% normal goat serum for 10 min (or normal donkey serum for TH measurement), then incubated for 20 h at 4°C with the primary antibodies (D₂R antibody AB5084P, Millipore, CA, USA, 1:200 dilution; DAT antibody MAB369, Millipore, CA, USA, 1:100 dilution; TH antibody T1299, Sigma, CA, USA, 1:10,000 dilution), washed three times in PBS, and incubated for 60 min at 37°C with the secondary antibodies (anti-rabbit antibody PV-6001, ZSGB-BIO, CA, USA; anti-rat antibody ZB-2307, ZSGB-BIO, CA, USA; anti-mouse antibody PK4002, Vector, CA, USA). Slices were then washed five times for 3 min each in PBS, and incubated in 100 μL of 3,3′-diaminobenzidine tertrahydrochloride (DAB) for 3 min. The slices immunostained for TH were incubated in the ABC (VECTASTAIN ABC kit, ZSGB-BIO, Beijing, China) reagent for 30 min at 37°C and washed five times for 3 min each in PBS prior to the DAB treatment. Final wash of the slices was done in distilled water.

Midbrain was cut into 30-μm slices to detect tyrosine hydroxylase (TH) using a freezing microtome (Leica M1950, Nussloch, Germany) as described previously [26 (link)], Brain slices were incubated with anti-TH antibody (T1299, 1:1000, Sigma, St Louis, MO, USA) overnight. After that, the brain slices were incubated with secondary antibodies for 1 h and then visualized by incubation in substrate-chromogen solution. The total number of TH-positive neurons in the SNpc were counted stereologically using the Optical Fractionator (Stereo Investigator 7, MBF bioscience, Williston, VT, USA).
For immunofluorescence staining, the slices or the astrocytes were incubated with anti-GFAP (MAB360, 1:1000 dilution, Millipore, Billerica, MA, USA), anti-TH (AB152, 1:2000 dilution, Sigma-Aldrich, USA), anti-IBA-1 (ab5076, 1:1000 dilution, Abcam), anti-lamin B1 (Abcam, ab16048, 1:400 dilution), anti-p16 (Santa Cruz Biotechnology, sc-56330, 1:200 dilution) or anti-cGAS (31659S, 1:400 dilution, Cell Signaling Technology, USA) overnight at 4 °C, followed by incubation in Alexa Fluor 555-conjugated antibody (Invitrogen, A21432; 1:1000) or Alexa Fluor 488-conjugated antibody (Invitrogen, A21202; 1:1000) for 1 h at 20 °C. The nuclei was stained with DAPI (P36931, Life Technologies). Images were obtained by a confocal microscope (Axiovert LSM510, Carl Zeiss Co., Germany) and then processed by Image J.