Horseradish peroxidase

Coronal 5‐µm thick brain sections harvested 20 days after surgery were processed for immunohistochemical staining to count dopaminergic neurons as previously described.^[72] Briefly, sections were deparaffinized, antigen retrieved, and endogenous peroxidase activity inactivated. Dopaminergic neurons were identified using an anti‐TH polyclonal antibody (1:1000, Invitrogen, catalogue number: PA1‐4679) and goat anti‐sheep IgG antibody conjugated with horseradish peroxidase (1:500, Santa Cruz Biotechnology, catalogue number: C0513). Immunoreactivity was detected with a bright‐field microscope (Olympus DP70) and analyzed by Image J software. Three sections per mouse were analyzed.

Western blot analysis was performed as described previously [48 (link)] using the following antibodies: cIAP1 (R&D Systems, Inc., Wiesbaden, Germany), RIP1 (BD Biosciences, Heidelberg, Germany), RIP3 (Novus Biologicals, Littleton, CO, USA), MLKL (GeneTex, Irvine, CA, USA), phospho-MLKL (Cell Signaling Technologies, Danvers, MA, USA), β-Actin (Sigma-Aldrich) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (HyTest, Turku, Finland) as loading controls and secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Enhanced chemiluminescence was used for detection (Amersham Bioscience, Freiburg, Germany). Alternatively, secondary antibodies labeled with IRDye infrared dyes were used for fluorescence detection (Odyssey Imaging System, LI-COR Bioscience, Bad Homburg, Germany). All Western blots shown are representative of at least two independent experiments.

Whole-cell lysates were generated by incubating cells in RIPA buffer for 30 min on ice, followed by sonication using a Covaris S-220 Ultrasonic Processor for 5 min. Lysates were separated in an 8% polyacrylamide gel and transferred to polyvinylidene fluoride membranes (Millipore) using an OWL semi-dry transfer apparatus. Membranes were blocked using 1% Blotting Grade Blocker Non-Fat Dry Milk (Bio-Rad) and incubated overnight at 4°C with the following primary antibodies: a rabbit polyclonal RUNX1 (Cell Signaling #4334, 1:1,000); a goat polyclonal to POU5F1 (Santa Cruz Biotechnology #sc-8628, 1:1,000); a rabbit polyclonal to CDK2 (M2) (Santa Cruz #sc-163, 1:2,000); a mouse monoclonal to GAPDH (0411) (Santa Cruz #sc-47724); a rabbit monoclonal to SMAD2 (D43B4) (Cell Signaling #5339); and a rabbit monoclonal to pSMAD2 (Ser465/467) (138D4) (Cell Signaling #3108). Secondary antibodies conjugated to horseradish peroxidase (Santa Cruz) were used for immunodetection, along with the Clarity Western ECL Substrate (Bio-Rad) on a Chemidoc XRS+ imaging system (Bio-Rad).

(-)-epigallocatechin-3-gallate (EGCG), N-acetylcysteine (NAC), and N-vanillylnonanamide (NVN) were obtained from Sigma Aldrich Co. Ltd. (St. Louis, MO). Recombinant human IL-1β was purchased from GIBCO BRL (Grand Island, NY). The inhibitors, BAY 11–7085 for NF-κB, SB 203580 for p38, and PD 98059 for MEK 1, were purchased from Calbiochem (La Jolla, CA). EGCG and NAC were dissolved in H₂O. NVN, BAY 11–7085, SB 203580, and PD 98059 were dissolved in dimethyl sulfoxide, methyl alcohol, or H₂O. The final vehicle concentration was adjusted to 0.1% (v/v), and the control medium contained the same quantity of vehicle. Antibodies against phospho-p38, p38, phospho-ERK, and ERK were obtained from Cell Signaling Technology (Beverly, MA). Antibodies against IκBα, β-tubulin, horseradish peroxidase, and Cy3-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

The clinical glioma specimens were obtained from the Neurosurgery Department, the Second Affiliated Hospital of Kunming Medical University, China. Immunohistochemical staining was conducted according to standard procedures. Briefly, paraffin‐embedded sections were incubated with antibodies of rabbit anti‐human TNIP1 (Abcam, USA) overnight at 4°C. The sections were then further stained with goat anti‐human secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology, USA). The immunohistochemical signals were developed with DAB reagent (Boster Biological Technology Ltd., China) and further examined under a light microscope (Olympus, Japan).

Six randomly chosen injury muscle samples from each group were homogenized in lysis buffer (150 mM NaCl, 10 mM HEPES, pH 7.9, 1 mM EDTA, 0.6% NP-40, 0.5 mM PMSF, 1 μg/mL leupeptin, 1 μg/mL aprotonin, and 10 μg/mL trypsin inhibitor). Samples were then sonicated and incubated on ice for 15 minutes. Protein was extracted by Cytosol and Nucleus Protein Extraction Kit (KeyGEN Bio TECH). Protein concentration was determined by Bradford's method. Samples were separated by SDS-PAGE gel and electrophoretically transferred to nitrocellulose membrane. Nonspecific binding sites were blocked with Trisbuffered saline (TBS; 40 mM Tris, pH 7.6, 300 mM NaCl) containing 5% BSA for 12 h at 4°C. And the membranes were incubated with anti-NF-κB (p65), anti-COX-2, anti-iNOS, anti-IκBα, anti-phosphor-AKT (Ser⁴⁷³), anti-AKT, anti-phosphor-p38 (Thr¹⁸⁰/Tyr¹⁸²), anti-p38, anti-phosphor-JNK (Thr¹⁸³/Tyr¹⁸⁵), anti-JNK, anti-β-actin, and anti-Lamin B1 antibodies (all from Signalway Antibody Co., Ltd., USA) at 1 : 1000 concentration overnight at 4°C. After being washed with TBST (TBS with 0.1% Tween-20), membranes were incubated with secondary antibody for 2 h at room temperature, which was conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunopositive bands were visualized by a chemiluminescent method (ECL, Tanon-5200).