Anti tnf α sc 52746

Sections (5 µm) were cut for immune staining. The tissue sections were incubated with primary antibodies of IL-1 β, TNF- α, NF-κβ, and VEGF (anti-IL-1β (SC-12742), anti-TNF-α (SC-52746), anti-NF-κβ (SC-8008), and anti-VEGF (SC-57496), Santa Cruz, Biotechnology, Inc., at a dilution of 1:200) for 1 h at room temperature, followed by washing with TBS. Endogenous peroxidases were blocked using hydrogen peroxide. Afterward, an HRP-labeled detection kit (Bio SB, BSB0001, USA) was used as per the manufacturer’s instructions to develop the positive reaction. Negative controls were obtained by deleting incubation with primary antibodies. Positive immune staining was quantified as area percent using LAS-X software (Leica v4.13, Germany).

Brains fixed in 4% paraformaldehyde were processed for paraffin embedding. Hematoxylin and eosin (H&E) staining was conducted on 5 μm sections according to previously published procedures (Wang et al., 2013 (link)). Immunohistochemistry (IHC) analyses were performed on hippocampus sections with the following antibodies: Anti-IL-1β (sc-12742, 1:200, Santa Cruz), Anti-TNF-α (sc-52746, 1:200, Santa Cruz), Anti-Synaptophysin (MAB5258-I, 1:200, Millipore). Zeiss Axio Scope.A1 or Zeiss Discovery.V8 Stereo microscopes (Carl Zeiss MicroImaging GmbH, Göttingen, Germany) was used and integrated with an Axio-Cam ICc3 camera (Spectra Service, Ontario, NY, United States). Images were acquired by AxioVision Rel. 4.7 software from Zeiss. The images were digitized and quantified using ImageJ.

The protein expression levels of cAMP, phosphorylated Akt, phosphorylated CREB, TNF-α, COL1, and COL3 were measured via Western blotting. Proteins were extracted from the Achilles tendon tissues by homogenization using Pro-Prep Protein Extraction Solution (iNtRON Biotechnology, Seongnam, Korea). Homogenates were centrifuged at 13,000× g for 20 min at 4 °C. The protein concentration was measured using BCA Protein Assay Reagent (Pierce Biotechnology, Rockford, IL, USA). After gel electrophoresis and hybridization, the proteins were incubated with the following primary antibodies: 1:1000 anti-cAMP (500-9534; Abbomax, San Jose, CA, USA), anti-pAKT (sc-377556; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-pCREB (ab32096; Abcam, Waltham, MA, USA), anti-TNF-α (sc-52746; Santa Cruz Biotechnology), anti-COL1 (MA1-26771; Invitrogen, Carlsbad, CA, USA), anti-COL3 (PA5-27828; Invitrogen), 1:2000 goat anti-rabbit IgG (H + L), horseradish peroxidase (HRP, 31463; Invitrogen), 1:2000 goat anti-mouse IgG (H + L), and HRP (31430; Invitrogen). Signals were detected using an Immobilon Chemiluminescence Kit (Merck, San Jose, CA, USA) and then quantified using ImageJ software version 1.53 (NIH). The band intensity per protein was normalized using β-actin as a control.