Sc 991

Coverslips were placed on the bottom of six-well plates and cells were plated as described earlier. After the exposures, the media were removed and the plates were washed 3× with PBS. Cells were fixed with 4% paraformaldehyde in PBS (MP Bio 199983) for 10 min. Cell plates were kept in PBS at 4°C until staining. Cells were permeabilized with ice-cold methanol for 2 min and blocked for 1 h with 10% rabbit serum and subsequently incubated in rabbit anti-Nurr1 (1 : 100) (SC 991; Santa Cruz Biotechnology) at 4°C overnight. After washing, the cells were incubated in AlexaFluor 488 donkey anti-goat for 1 h (A21206; Life Technologies, Carlsbad, California, USA). Coverslips were mounted onto slides with Vectashield containing DAPI (H-1500; Vector Laboratories Inc., Burlingame, California, USA).

All brain tissue processed for immunofluorescence (IF) was frozen with optimal cutting temperature compound on a microtome stage and sectioned/collected for ST (25 μm in thickness) and SN (40 μm in thickness) regions. Tissue sections were stored in cryoprotectant (30% sucrose, 30% ethylene glycol, and 0.5 M phosphate buffer, pH 7.2) at 20°C until selected for immunostaining. IF staining was conducted as previously described by Miller et al. (2011) (link), and all antibody dilutions were 1:500, unless stated otherwise (Miller et al., 2011 (link)). For stereology, SN sections were immunostained with anti-TH (cat. no. AB152; Merck Millipore, Burlington, MA) and anti-MAP2 (cat. no. AB5392; Abcam). For gliosis, SN and ST sections were immunostained with either anti–ionized calcium-binding adapter molecule 1 (IBA-1) (1:250; cat. no. 016-20001; Wako Chemicals USA, Inc., Richmond, VA) or anti–glial fibrillary acidic protein (GFAP) (cat. no. Z0334; Agilent Technologies, Santa Clara, CA) and anti-TH (cat. no. AB76442, Abcam). SN tissue was also immunostained with anti-TH and anti-Nurr1 (1:200, SC991; Santa Cruz Biotechnology, Dallas, TX) for mean intensity measurements of Nurr1. All secondary antibodies used for IF were Alexa Fluor 488, 555, and 647 (Thermo Fisher Scientific, Carlsbad, CA).