Rabbit anti rage

Cisplatin (#T1564) is from Topsicence (United States); FPS-ZM1 (#HY-19370) is from MedChemExpress (United States); rabbit anti-RAGE (#ab3611) is from Abcam (United Kingdom); rabbit anti-Bax (#CPA1092), and Bcl-2 (#CPA1095) antibodies are from Cohesion Bioscience (United Kingdom); rabbit anti-Phospho-NF-κB p65 (Ser536) (#3033) and NF-κB p65 (#3034) antibodies are from Cell Signaling Technology (United States); rat anti-F4/80 antibody (#14-4801-82) is from Invitrogen United States; mouse anti-GAPDH (#AC033), rabbit anti-β-actin (#AC026), Cpt1a (#A5307) and PGC-1α (#A12348) and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (#AS014) antibodies are from ABclonal (China). One Step Terminal transferase dUTP nick-end labelling (TUNEL) Apoptosis Assay Kit (#C1090) is from Beyotime (China). SuperKine™ West Femto Maximum Sensitivity Substrate (#BMU102-CN) is from Abbkine (China).

We used whole extracts from whole tDRGs collected from adult WT mice for Western blotting^{48 (link)}. Whole tDRG extracts were homogenized in ice-cold CelLytic MT Cell Lysis Reagent (Sigma-Aldrich) containing a protease and phosphatase inhibitor cocktail. We used 4 replicas per condition (PBS-control and LPS), and each replica was generated from 2 adult mice. Equal amounts of protein were loaded per group, separated on 12% SDS polyacrylamide gels and then electrotransferred onto a PVDF or nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). Membranes were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-RAGE (1:1000; Abcam) and mouse anti-β-actin (1:2000; Sigma); followed by horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (1:20,000; Bio-Rad Laboratories). Protein signals were visualized using enhanced chemiluminescence reagents (Bio-Rad) and quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA).

Sensory neuron-like cells were incubated with ECM-NC, ECM-GC, or ECM-NC matrix with IL-1β for 24 h to detect AGEs receptor expression. Then the cells were fixed, permeabilized, and blocked as described previously. Cells were incubated with primary antibodies diluted in PBS and 3% BSA: chicken anti-neuron specific β-III tubulin diluted 1:500 (Merck Millipore, Burlington, MA, USA) and rabbit anti-RAGE diluted 1:200 (Abcam, Waltham, MA, USA); or incubated with chicken anti-neuron specific β-III tubulin diluted 1:500 and rabbit anti-Galectin-3 (1:500, Abcam, Waltham, MA, USA) overnight at 4 °C.
Cells were then incubated with secondary PE-conjugated goat anti-chicken antibodies (1:500 dilution), and Alexa Fluor 647-conjugated goat anti-rabbit (1:300), for 1 h at room temperature. Negative controls were incubated with secondary antibodies. Nuclei were stained with Hoechst-33342 (1:3000), and the fluorescence was determined using a Confocal Microscope (Zeiss) and images were captured with a confocal laser-scanning microscope (Confocal TCS, SP8, Leica, Wetzlar, Germany).