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4 protocols using hrp labeled goat anti mouse igg antibody

1

Cell Viability and Protein Analysis

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Cell counting kit-8 (CCK-8) was purchased from Sigma-Aldrich. BCA protein quantitative kit was purchased from Boster (Wuhan, China). Mouse Nrf2, HO-1, SMAC, Cyt-C, Lamin-B, and β-actin monoclonal antibodies were purchased from Abcam. HRP-labeled goat anti-mouse IgG antibody and goat anti-rabbit IgG antibody were purchased from Santa Cruz. Commercial kit for the determination of lactate dehydrogenase (LDH) cytotoxicity was purchased from Thermo Fisher Scientific. Western blot electrophoresis and exposure system were purchased from Bio-Rad (CA, USA) and the automatic microplate reader was purchased from Yongchuang Medical Instruments (Shanghai, China). The other facilities used in this study included cell culture plate (Corning, USA), ultra-clean workbench (Yatai Kelong Instrument, Beijing, China), centrifuge (Shanghai Lu Xiangyi Centrifuge Instrument, China), cell counting plate (Germany), and carbon dioxide incubator (USA).
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2

Nrf2/HO-1 Pathway Modulation in PC12 Cells

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Rat adrenal pheochromocytoma cells (PC12 cells; highly differentiated type) were purchased from the Cell Center of Shanghai Academy of Life Sciences, Chinese Academy of Sciences. Aβ25-35 (purity ≥ 95%, no. Y0044) was from Beijing Biosynthesis Biotechnology Co., Ltd. (China). DMEM, streptomycin, trypsin, and fetal bovine serum were from HyClone Laboratories Inc. (Logan, Utah, USA). MTT was from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). A lactate dehydrogenase (LDH) kit was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). Superoxide dismutase (SOD), malondialdehyde (MDA), myeloperoxidase (MPO), and glutathione peroxidase (GSH-Px), as well as catalase (CAT), total antioxidant capacity (T-AOC), and acetylcholinesterase (AChE) kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The primary antibodies against sirtuin1 (Cat. No. 13161-1-AP), total Nrf2 (Cat. No. 16396-1-AP), nuclear Nrf2 (Cat. No. 66504-1-Ig), HO-1 (Cat. No. 66743-1-Ig), β-actin (Cat. No. 60008-1-Ig), and PCNA (Cat. No. 60097-1-Ig) were from Proteintech Co., Ltd. (Chicago, USA). HRP-labeled goat anti-mouse IgG antibody was purchased from Santa Cruz Biotechnology, Inc. (Cat. No. sc-2005; Santa Cruz, California, USA). HRP-labeled goat-rabbit IgG antibody was from Proteintech Co., Ltd. (Cat. No. 10285-1-AP; Chicago, USA).
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3

Quantification of MBL Isoforms in Plasma and Tissue

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MBL isoforms were analyzed after sample incubation on mannan-coated (10 µg/mL) plates prepared according to a procedure reported previously.18 (link) Plates were incubated for 1 h at 37 °C with EDTA-plasma diluted in barbital-buffered saline (BBS; 4 mm barbital, 145 mm NaCl, 2 mm CaCl2, 1 mm MgCl2 (pH 7.4)) to a final plasma concentration of 2.5% or with cortical homogenates (ipsilateral and contralateral to the lesion) diluted in BBS to a final concentration of 4 mg/mL total protein. Samples were then incubated for 1 h 30 min at room temperature (RT) with anti-MBL-A or anti-MBL-C antibodies (both 1 µg/mL, Hycult, # HM1035 and #HM1038, respectively). After washing, plates were incubated with an HRP-labeled goat anti-mouse IgG antibody (Santa Cruz, CA, USA), diluted to 0.4 µg/mL in wash buffer for 1 h 30 min at RT. After washing, the assay was developed by adding 100 µL of TMB substrate solution (TMB Substrate Kit, code 34021, Thermo Scientific, MA, USA; diluted 1:1 in H2O2 solution). The reaction was terminated by adding 100 μL of 2 m H2SO4, and the absorbance at 450 nm was measured using an Infinite M200 spectrofluorimeter managed by Magellan software (Tecan, CH).
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4

Antioxidant and Inflammatory Biomarker Assay

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Superoxide dismutase (SOD), malondialdehyde (MDA), myeloperoxidase (MPO), glutathione peroxidase (GSH-Px), catalase (CAT), and reactive oxygen species (ROS) kits were purchased from Nanjing Jiancheng Institute of Bioengineering (Nanjing, China). Lactate dehydrogenase (LDH) kits were purchased from Shanghai Beyotime Biotechnology Co. (Shanghai, China). Tumor necrosis factor-α (TNF-α), Interleukin-1 b (IL-1β), Interleukin-10 (IL-10), and Interferon-γ (IFN-γ) kits were purchased from Changzhou Dimenuo Biotechnology Co., (Changzhou, China). TUNEL cell apoptosis detection kits were purchased from Shanghai Beyotime Biotechnology Co. (Shanghai, China). In situ cell apoptosis detection kits were purchased from ROCHE (Switzerland). DAB Detection kits were purchased from Bohai Bioengineering Co. (Hebei, China). Antibodies against Bcl-2 (cat# 60178-1-Ig), Bax (cat# 60267-1-Ig), GSK-3β (cat# 67329-1-Ig), Fyn (cat# 66606-1-Ig), Nrf2 (cat# 66504-1-Ig), and β-actin (cat# 60008-1-Ig) were all purchased from Proteintech Co. (Chicago, USA). HRP-labeled goat anti-mouse IgG antibody was purchased from Santa Cruz Co. (cat# sc-2005; Santa Cruz, California, USA). The reverse transcription kits were purchased from TransGen Biotech Co., (Beijing, China). RT-PCR kits and Western blot kits were purchased from Semerfly Technology Co. (Shanghai, China).
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