Horseradish peroxide conjugated secondary antibody

Protein samples were harvested in lysis buffer containing protease inhibitor cocktail (Roche, Germany). Equal amount or volume of protein was loaded and separated in 8% SDS-PAGE. Then, proteins were transferred to polyvinylidene difluoride membrane (Millipore Co., Billerica, MA, USA) at 200 mA for 2 hrs (100mA, 1 hr for ephrinA1) at 4°C. After blockage in TBS containing 5% BSA (w/v) and 0.1% Tween-20 for 1 hr, membranes were incubated with appropriate primary antibodies at 4°C overnight. The dilution ratios of primary antibodies were as in the following: ephrinA1 (1:300), HIF-1α (1:1000), MMP-2 (1:1000), MMP-9 (1:1000), β-actin (1:1000). After five 5 mins washes with TBST, the blocks were treated with horseradish peroxide-conjugated secondary antibody (dilution 1:10,000) (Jackson Immunoresearch Laboratories) for 1 hr at room temperature. Finally, bands were recorded by using ECL plus Western blotting detection reagents (Beyotime, People’s Republic of China).15 (link)

Animals were sacrificed with 10% chloral hydrate and, then, transcardially perfused with PBS, and the L5 lumbar SC segments were dissected. Then, the protein samples were quantified by a bicinchoninic acid protein assay kit (Pierce Biotechonology, Rockford, IL, USA). A total of 40 μg ipsilateral L5 spinal sample proteins were electrophoresed through a 10% sodium dodecyl sulfate-polyacrylamide gel and electrotransferred to 0.4 mm polyvinylidene fluoride membranes. The blots were first incubated overnight at 4°C with one of the following primary antibodies (CXCR4: 1:1000, rabbit; Abcam, Cambridge, UK; GlyRα3: 1:1000, rabbit; EMD Millipore, Billerica, MA, USA) and then with a horseradish peroxide-conjugated secondary antibody (1:4000; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA). An enhanced chemiluminescence detection system (Pierce Biotechonology) was applied for protein detection. An anti-GAPDH antibody (1:1000, rabbit; Cell Signaling Technology, Danvers, MA, USA) was used for normalization. The intensity of protein bands was analyzed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).