Elisa reader

The ability of bacteria to develop biofilms was assessed by staining attached cells with crystal violet as described previously^{25 (link)–27 (link)}. A bacterial suspension (1 × 10⁸ CFU/mL, 500 μL) was seeded onto titanium specimens and incubated at 37 °C for 24 h to allow biofilm formation. The nonadherent bacteria were removed with a pipette, and the specimen was washed three times using 5 mL PBS. A 0.1% crystal violet solution was used to determine biofilm formation on the surface of the titanium discs. The PBS-washed titanium discs were placed into a 12-well plate, submerged in 1 mL of crystal violet solution, and incubated at room temperature for 10 min. The discs were then transferred into a new 12-well plate and rinsed three times with 5 mL PBS to remove excess crystal violet solution. To elute the crystal violet, 500 μL of 30% acetic acid was added, and the plate was incubated at room temperature on an orbital shaker (Biofree, Buchen-si, Gyeonggi-do, Korea) at 250 rpm for 15 min. One hundred microliters of acetic acid solution containing the crystal violet stain retained by the biofilms was added to each well of a 96-well plate (SPL, Pochein-Si, Gyeonggi-Do, Korea), and the amount of biofilm that developed was measured at a wavelength of 595 nm using an ELISA reader (Epoch, Biotek, Winooski, VT, USA).

Rats were anesthetized with isoflurane and decapitated, and then L4‐L6 DRG tissues in both sides were exposed, removed, and dissected. Both ipsilateral and contralateral sides of the L4‐L6 DRGs from the same rat were removed 24 h after the end of femoral artery ligation and 18 h after the end of I/R procedures (I/R group). β‐NGF levels (Bax et al., 1997 (link)) in the rat L4‐L6 DRGs were assayed with a modified ELISA (Li et al., 2021b (link)). This assay was performed at room temperature as instructed by the manufacturer (RayBiotech Co. Cat# ELR‐bNGF‐CL). Briefly, the diluted samples (2 mg/mL) and rat β‐NGF standard solution were distributed in polystyrene 96‐well immunoplates (100 μL per well; Thermo‐Fisher Co. Cat#9205) and incubated for 2.5 h with gentle shaking. After being thoroughly washed for four times, 100 μL of diluted biotin‐labeled antibody mixture was added to each well and incubated for 1 h. After thoroughly washing, 100 μL of diluted streptavidin‐HRP conjugate (Invitrogen Co. Cat#43423) per well was added and incubated for 45 min. Then 100 μL of substrate per well was incubated for 30 min in the dark, then 50 μL of stop solution was added. The optical density at 450 nm was immediately examined using an ELISA reader (BioTek).