Enhanced chemiluminescence ecl detection reagent

For protein isolation, cells were lysed using RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) supplemented with 1 mM PMSF (Beyotime Biotechnology, Shanghai, China). The extracted total proteins were then separated using 12.5% SDS-PAGE and subsequently transferred to a polyvinylidene difluoride membrane (0.2 μm; Millipore, Burlington, MA, USA) utilizing a semi-dry transfer system (Bio-Rad; Hercules, CA, USA). The membrane was blocked with a rapid blocking solution (YaMei, Hangzhou, China) for 12 min. After blocking, the membranes were incubated overnight with primary antibodies at 4 °C. Subsequently, the membranes were washed four times with TBST, each wash lasting 9 min. The membranes were then incubated with appropriate secondary antibodies for 1 h at room temperature, followed by four additional 9 min washes with TBST. For detection, the membrane was incubated with Enhanced Chemiluminescence (ECL) detection reagent (Thermo Scientific, Rockford, IL, USA) and imaged using a chemiluminescence detector (Tanon, Shanghai, China). Protein bands were scanned in grayscale using a Tanon Gel Imaging System (Tanon, Shanghai, China). Protein levels were quantified using ImageJ software (1.8.0), with each protein normalized against vinculin levels. The specific antibodies utilized are detailed in Table 2.

All the animals were sacrificed under deep sevoflurane (3%) anesthesia. The spinal dorsal horn segments (L₃–L₅), amygdala and cortex were removed quickly and snap-frozen in liquid nitrogen. The tissues underwent mechanical homogenization in chilled RIPA buffer with PMSF (Abcam, Cambridge, UK). The lysates were cleared by centrifugation, and supernatants were obtained for total protein extraction. Protein amounts were assessed by the bicinchoninic acid assay. Protein separation was carried out by 10% SDS-PAGE. After transfer onto nitrocellulose membranes, the specimens underwent successive incubations with rabbit monoclonal antibodies targeting pentraxin-3 and rabbit polyclonal antibodies nectin-1 (1:1000; Santa Cruz, CA, USA), respectively, and horseradish peroxidase-linked secondary antibodies (1:2000, Jackson ImmunoResearch, PA, USA). Enhanced chemiluminescence (ECL) detection reagents (Thermo Scientific, IL, USA) were utilized for development, followed by quantification with Gene Tools Match software (Syngene, Cambridge, UK). Antibodies targeting β-actin (1:5000; Sigma, MO, USA) were utilized for normalization.