Anti tnf α antibody

Paraffin-embedded sections from mouse livers were stained with hematoxylin and eosin (HE). Histological evaluations were performed employing the histological scoring system for NAFLD37 (link). Oil Red-O staining was performed on frozen liver sections. Immunohistochemical staining with anti-Collagen α 1 antibody (Abcam, Cambridge, MA, USA), anti-αSMA antibody (Abcam), anti-F4/80 antibody (Abcam), or anti-TNF-α antibody (Abcam) was performed by SRL Co. Ltd (Tokyo, Japan). Immunohistochemical staining with mouse RELMβ of the liver sections was performed using anti-mRELMβ antibody (Abcam), and simple stain mouse MAX-PO (R) (Nichirei, Tokyo, Japan) was used as the secondary antibody. Immunofluorescence staining was performed using rat anti-mouse F4/80 antibody (Serotec, Oxford, UK) and anti-mRELMβ antibody. Alexa-Fluor 546 and 488 (Invitrogen, CA, USA) were used as the secondary antibodies. Digital images of lesions were randomly selected and positive cells were counted using a multifunctional microscope (BZ-9000; KEYENCE Co, Osaka, Japan), Image-J (National Institute of Health, MD, USA) or FSX 100 Olympus Microscope (Olympus America Inc., Center Valley, PA).

The D-galactose (DG) was purchased from Sigma-Aldrich (USA). The LR anthocyanin (LRA) sample was prepared as we described previously [15 (link)]. Briefly, the anthocyanins were obtained from dried LR fruit by ultrasound-assisted extraction using 40-fold 80% ethanol (extracted for 1 h and repeated three times). The extract was concentrated, purified with AB-8 macroporous resin, and dried by lyophilization. The LRA sample was stored at −20 °C. The purity of the anthocyanin was 87.54% of the sample, which was calculated by the pH-differential method. The anti-TNF-α antibody was obtained from Abcam Plc (Cambridge, UK) and the fluorescent anti-rabbit secondary antibody was purchased from Invitrogen Corporation (Carlsbad, USA).

The normal tissues and adjacent paracancerous and cancerous tissues were lysed with ice-cold RIPA buffer containing protease inhibitor, in accordance with the manufacturer's protocol applying the bicinchoninic acid assay (BCA) (Thermo Scientific, USA) methods for total protein, using BSA as standards. Meanwhile, the proteins were denatured at 100°C for 5 min. A total of 30 ng of proteins was separated in 12.5% SDS-PAGE and transferred to a PVDF membrane. Then, it was blocked with 5% skim milk for 2 h. The following antibodies were used in the study: rabbit anti-TIM-3 antibody (ABCAm, USA), anti-TNF-α antibody (ABCAm, USA), anti-IFN-γ antibody (ABCAm, USA), and mouse anti-β-actin antibody (Proteintech, China). The PVDF membranes were incubated with TIM-3, TNF-α, IFN-γ, and β-actin primary antibodies at 4°C overnight. The secondary antibodies (CST, USA) were used to incubate with the membranes for 1 h at room temperature. After washing with TBST, the membranes were visualized in ECL developing solution. Band intensity was scanned by ImageJ software. Quantification was determined by estimating the intensity value ratios of the reference β-actin and the target gene.

Twenty-six days after LPS induction, IL-1β, IL-6, TNF-α, cyclooxygenase-2 (COX-2), and NF-κB concentrations in the hippocampus were assayed according to a previously described procedure (Lee et al. 2014 (link)). Three rats from each group were deeply anesthetized via isoflurane inhalation (1.2%), and were sacrificed one day after behavioral testing. The IL-1β, IL-6, TNF-α, COX-2, and NF-κB concentrations were evaluated with competitive enzyme-linked immunoassays (ELISAs) using anti-IL-1β antibody (Abcam, Cambridge, MA, USA), anti-IL-6 antibody (Abcam), anti-TNF-α antibody (Abcam), anti-COX-2 antibody (Abcam), and anti-NF-κB antibody (Abcam) according to the manufacturer's protocols.

Curcuma (TCM Pharmacy of Longhua Hospital of Shanghai University of Traditional Chinese Medicine), DSS (MP Biomedicals, USA), absolute ethyl alcohol, Tween-20, xylene substitute (Sinopharm Group Chemical Reagent Co. Ltd.), RIPA Lysis buffer, PMSF, BSA, BCA Protein Quantitation Kit (Beyotime), PAGE gel rapid preparation kit, Multicolor Restrained Protein Ladder (Shanghai EpiZyme Biotechnology Co., Ltd.), β-actin, anti-STAT3 antibody, anti-TNF-α antibody (Abcam Company, England), HE dyeing (Shanghai Yixin Biotechnology Co., Ltd.), and neutral gum (Shanghai Yiyang Instrument Co., Ltd.) were used.

The following materials were used: anti-Bcl-2 antibody (Abcam, 182,858), anti-Bax antibody (Abcam, 32,503), anti-GAPDH antibody (Abcam, 8245), anti-cleaved caspase-3 antibody (Cell Signaling Technology, 9664), anti-IL-1β antibody (Cell Signaling Technology,12,242), anti-IL-6 antibody (Cell Signaling Technology, 12,912), anti-IL-10 antibody (Abcam, 189,392), anti-TNF-α antibody (Abcam, 183,218), anti-TLR4 antibody (Abcam, 22,048), anti-MyD88 antibody (Abcam, 133,739), anti-NF-κB antibody (Abcam, 16,502), anti-p-NF-κB antibody (Abcam, 76,302), goat anti-rabbit HRP-conjugated antibody (Abcam 6721), and goat anti-mouse HRP-conjugated antibody (Abcam 6789).