Halt protease inhibitor cocktail mix

Mice were killed by an overdose of euthasol (150 mg/kg) i.p. and the brains excised and bisected. The hemisphere ipsilateral to the cannula was submerged in 4% paraformaldehyde in phosphate buffered saline overnight, followed by immersion in 30% sucrose prepared in phosphate buffered saline. This tissue was then sectioned as 40 µm sections and processed for tyrosine hydroxylase (TH) staining. The remaining brain hemisphere was homogenized in ice cold lysis buffer (25 mM Tris–HCl, 0.25 M sucrose, 2 mM EDTA, 10 mM EGTA, 1% Triton X-100, and 10 µL/mL Halt protease inhibitor cocktail mix (Thermo Scientific, Pittsburgh, PA)) at 100 mg tissue/mL, centrifuged at 12, 000×g for 20 min at 4 °C, and resulting supernatants analyzed for protein and cytokine content. Protein concentrations were determined with the Bradford assay [64 (link)].

The ELISA kit for Tumor Necrosis Factor-α (TNFα) was obtained from BD Biosciences (San Jose, CA). Pre-cast 15% gels were bought from Bio-Rad (Hercules, CA) and Immbilon PDVF membranes from Millipore (Millipore, Billerica, MA). The Halt protease inhibitor cocktail mix was obtained from Thermo Scientific (Pittsburgh, PA). Thermo Scientific supplied the 1.5 and 6.2 µm size markers (Duke Standards). The chemiluminescence kit was obtained from GE Healthcare (Pittsburgh, PA) and film from Denville Scientific (South Plainfield, NJ). Reagent grade sodium hypochlorite and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO). Ultrapurified water was used in the preparations of stock solutions and the concentrations of HOCl were determined using 5-thio-2-nitrobenzoic acid [63 (link)]. The latter reagent was generated by reduction of 5,5’-dithiobis-(2-nitrobenzoic acid) by sodium borohydride in sodium phosphate buffer (pH 7.9) as described by Jeitner et al. [63 (link)].

The Diff-Quik staining kit was obtained from Siemens Healthcare Diagnostics Inc. (Newark, DE). Primary antibodies were bought from Abcam (Cambridge, MA) whereas the secondary antibodies were supplied in the kits purchased from ImmunoCruz (Santa Cruz, CA) for immunohistochemical staining and the Amersham ECL Western Blotting Analysis System (GE Healthcare). Ladd industries (Burlington, VT) supplied the Epon epoxy resin. Annexin V Alexa Fluor® 488 and propidium iodide were bought from Invitrogen (Carlsbad, CA). The ELISA kit for Tumor Necrosis Factor-α (TNFα was obtained from (BD Biosciences, San Jose, CA). Pre-cast 15% gels were bought from Bio-Rad (Hercules, CA) and Immbilon PDVF membranes from Millipore (Millipore, Billerica, MA). The Halt protease inhibitor cocktail mix was obtained from Thermo Scientific (Pittsburgh, PA). PlasticsOne (Roanoke, VA) supplied the cannulae for microinjection. Duke Standards (0.5, 1.0 and 1.5 µm) were obtained from Thermo Scientific. All other reagents were purchased from Sigma-Aldrich (St. Louis, MO).