Alkaline phosphatase labeled goat anti rabbit igg

At 72 hours after spinal cord injury, five rats in each group were sacrificed under anesthesia and spinal cord tissue was harvested. Samples were centrifuged at 1,500 r/min for 30 minutes, and total protein concentration in the supernatant was determined. The samples were then electrophoresed in 5% stacking gel at 40 V for 1 hour and 10% separating gel at 60 V for 3.5 hours, and transferred onto a PVDF membrane at 14 V for 14 hours. The membrane was blocked at 37°C for 2 hours, rinsed three times in TBS for 10 minutes each time, and incubated with rabbit anti-AQP9/AQP4 polyclonal antibody (1:500; Sigma, St. Louis, MO, USA) and rabbit anti-β-actin polyclonal antibody (1:500; Sigma) at room temperature for 60 minutes. After three washes in TBST, samples were incubated with alkaline phosphatase-labeled goat anti-rabbit IgG (1:2,000; Sigma) at room temperature for 60 minutes, developed with DAB (Beijing CellChip Biotechnology Co., Ltd., Beijing, China), and analyzed using Quantity One imaging software (Bio-Rad, Hercules, CA, USA). The AQP4/9 protein expression level was calculated as the integrated optical density ratio of AQP4/9 to β-actin.

A functional lectin pathway activity assay was done using EDTA-plasma diluted 2.5% in barbital-buffered saline (BBS) incubated for 15′ at 37° C on 10 µg/mL mannan-coated plates.^{27 (link)} C3 deposition was revealed using a polyclonal anti-human-C3c antibody (2.4 µg/mL, Dako) and an alkaline-phosphatase labeled goat anti-rabbit IgG (1 µg/mL, Sigma). Absorption at optical density (OD) 405 nm measured using Infinite M200 spectrofluorimeter (Tecan, CH).