Anti p p65

Rat and human IVD tissues were obtained, fixed with 4% PFA for 48 h and then decalcified and embedded in paraffin. The tissues were cut into 5-μm-thick sections that were successively dewaxed and hydrated with xylene, anhydrous ethanol and an alcohol gradient (95%, 85% and 75%). The sections were then subjected to antigen retrieval with pepsin for 20 min at 37°C, incubated with neutralized endogenous peroxidase in a humidified box for 15‒20 min and sealed with goat serum for 30 min. The slides were then incubated overnight at 4°C with primary antibodies including anti-p-p65 (1:100; ABclonal), anti-HMGB1 (1:100; ABclonal), anti-NLRP3 (1:1000; Cell Signaling Technology), anti-caspase-1/p20 (1:100; Affinity), and anti-p21 (1:100; Santa Cruz Biotech, Santa Cruz, USA). The sections were subsequently rewarmed and incubated with biotin-labeled goat anti-mouse/rabbit secondary antibodies for 20‒30 min at room temperature and HRP-conjugated streptavidin (ZSGB-BIO, Beijing, China) for 25 min. DAB (ZSGB-BIO) staining and hematoxylin staining were performed for 1 min. Finally, images were captured using an Eclipse 80i fluorescence microscope (Nikon, Tyoko, Japan).

All the sections were blocked with 10% donkey serum with 0.3% Triton X-100 and 1% BSA for 2 h and incubated with anti-Cav1 (1:400, Cell Signaling Technology, MA, USA), anti-p-p65 (1:200, ABclonal, MA, USA), anti-COX-2 (1:200, HuaBio, Zhejiang, China), anti-Collagen I (1:200, Abcam, Cambridge, UK) and anti-IgG (1:400, Cell Signaling Technology, MA, USA) antibodies at 4 °C overnight, followed by incubation with secondary antibody (HRP conjugated goat anti-Rabbit IgG, 1:500, Beyotime, Shanghai, China) for 2 h. Sections were visualized with DAB (Sangon Biotech, Shanghai, China) for 30 min and counterstained with hematoxylin. Evaluation of the immunocytochemistry was done by light microscopy.

Total protein was extracted by lysing cells in lysis buffer (20 mM HEPES, pH 7.4, 20 mM NaCl, 10% glycerol, and 1% Triton X-100). Colonic membrane proteins were prepared using a Membrane Protein Extraction Kit (Biovision, Milpitas, CA, USA) and soluble proteins were isolated from colon homogenates using methanol and chloroform (28 (link)). All protein samples were subjected to 12% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Merck KGaA, Darmstadt, Germany). Immunoblotting was performed with the following primary antibodies: anti-tmTNF-α (home-made) (31 (link)), anti-TNF-α (Cat# 3707s), anti-PARP (Cat# 9532s), anti-cleaved caspase 3 (Asp175) (Cat# 9661s) from Cell Signaling Technology (Danvers, MA, USA), anti-IκB-α (Santa Cruz, CA, USA, Cat# sc-1643), anti-p65 (Cat# A19653), anti-p-p65 (Cat# AP0475), anti-caspase 3 (Cat# A17900), anti-Na⁺/K⁺ ATPase (Cat# A12405), and anti-β-actin (Cat# AC026) from Abclonal (Wuhan, China). HRP-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA, Cat# 7074) were subsequently applied to the membrane. Bands were visualized using an enhanced chemiluminescence system (ECL; TIANGEN, Beijing, China).