Wga hrp

PIA production in biofilms formed in TSBg or milk was quantified by ELISA. Briefly, the final culture density from biofilms during 24 h at 37 °C was measured by reading the OD₅₉₅ to normalize PIA quantification. Then, the fixed biofilms were washed and 100 μl of blocking solution (1%, w/v, BSA (Sigma-Aldrich, St. Louis, MO, USA) in PBST (PBS + 0.05 % Tween 20) (Honeywell Fluka, Thermo Fisher Scientific, UK) was added to each well. After incubation for 1 h at 37 °C, the blocking solution was removed and 100 μl per well of 75 ng/ml wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) (Sigma-Aldrich, St. Louis, MO, USA) was added. The level of PIA was measured at OD₄₉₂ and normalized to the OD₅₉₅ of growth as OD₄₉₂/OD₅₉₅. The OD₅₉₅ of growth of TSB was used to normalize the OD₄₉₂ measuring of biofilms formed in milk.

The sciatic nerve injury model was modified from our previous study (White and Kocsis, 2002 (link)). 8–10-week old mice were anesthetized with an intraperitoneal injection of ketamine (90 mg/kg) and xylazine (4 mg/kg), and the right sciatic nerves were exposed at the level of the piriform tendon. The nerves were ligated with a suture with 6.0-nylon and cut immediately distal to the ligature site. A 10 to 15 mm section of the distal nerve was removed and the distal stump was retracted. The sciatic nerve stump was sutured into a sterilized polyethylene tubing cuff (15 mm long, 3.27 mm outer diameter, 1.65 mm inner diameter, Baxter Scientific) and on the end of the cut nerve containing a small piece of sterile gelfoam permeated with the 100 μl of anterograde neurotracer wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) (Sigma, 4% in sterile saline). Animals with unilateral sciatic nerve exposure served as sham controls. After surgery, the overlying skin and muscles were sutured, and the wound was treated with Betadine to prevent infection. Animals were administered with buprenorphine (0.05 mg/kg/day, subcutaneous) for 48 h and ibuprofen (5 mg/kg/day, per os) for 72 h. Recovery was uneventful in all cases (White and Kocsis, 2002 (link)).