Ab85015

The L4-L6 dorsal root ganglions (DRGs) of rats from different groups were sampled at specific timepoints, 0, 3, 7, and 14 d following CCI surgery. After 3 washes with icy PBS, the dissected tissues were rapidly transferred into the RIPA lysis buffer (#20-188, 0.5 M Tris-HCl, pH 7.4, 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, and 10 mM EDTA) containing protease inhibitor (#I3786), all from [17]. Total protein was determined by a BCA kit (#P0012, Beyotime, Shanghai, China). For each sample 30 μg protein was loaded, then separated on 10% SDS-PAGE gel, and transferred to a PVDF membrane (#IPVH00010, Merk Millipore, Billerica, MA). Then the membrane was blocked with 5% bovine serum albumin for 2 h at room temperature and incubated with primary antibodies, rabbit anti-Sirt1 (#ab220807, Abcam, Cambridge, UK), mouse anti-Nav1.7 (#ab85015, 1:500, Abcam), and rabbit anti-GAPDH (#ab9485, 1:2000, Abcam, Cambridge, MA, USA), at 4°C overnights. After 2-h incubation at room temperature with corresponding horseradish peroxidase-conjugated secondary antibodies (#AB6721 and #ab6728, 1:3000, Abcam), the targeted proteins were detected using Pierce ECL Western Blotting reagents (#32,106, ThermoFisher, Rockford, IL, USA) and imaged using FluorChem E (Alphalmager Proteinsimple, San Jose, California). GAPDH served as the inner control.

Na_V1.7 was immunostained using an anti-Na_V1.7 monoclonal antibody (ab85015, Abcam, Cambridge, UK). Positive and negative immunohistochemistry (IHC) controls were generated from cell lines that represent high or no expression of Na_V1.7. MZ-CRC-1, which showed high expression of Na_V1.7, was used as a positive control, and Nthy-ori3-1 (normal thyroid), which did not have an expression of Na_V1.7, was used as a negative control. Na_V1.7 expression was quantified within each core using an automated digital quantification (custom MATLAB code). IHC samples were automatically segmented to extract tissue boundaries and transition from RGB images to HSV, followed by a saturation mask to distinguish tissue. The distribution of saturation was plotted, and Otsu’s automated threshold for separating positive vs. negative staining was employed to extract out percentage of positive tissue expression.