Evans blue dye assay

The Evans blue assay was performed as previously described.[16] Briefly, animals were anesthetized and maintained on 1–1.2% isoflurane in oxygen. Evans blue dye, 100 μl of a 20 mg/ml solution in saline, was injected intra-jugularly 5 min before the mice were injected with NS, histamine and/or neuraminidase, and tissues were harvested 2h later. Injected and contralateral muscles were harvested, weighed, sectioned, and freeze-dried overnight. Eight volumes of formamide (ml/g tissue) were added, and samples were incubated at 60 °C for 2 h to extract Evans blue dye. After an additional 10h at room temperature, the Evans blue dye was collected and absorption at 595 nm was measured on a microplate reader (Model 3550, Bio-Rad, Hercules, CA). Sample concentrations were determined using a calibration curve generated in parallel from dilutions of Evans blue dye in formamide.