CVF was obtained from CalBiochem (San Diego, CA) and 25 μg injected IP into mice 24 hours before P. aeruginosa infection. In some experiments, 0.5 μg in 5 μL of CVF was also applied topically to the eye at 0, 8, 24, and 32 hours post-infection (along with MAb F429 at 8, 24, and 32 hours post-infection) to ascertain the effect of depletion of C3 in the local environment of the eye. Controls received 5 μL of PBS. CVF applied topically by itself showed no obvious effect on the appearance or clarity of the cornea. Measurement of the level of C3 in the serum and in the corneas after CVF treatment and in comparison to controls was done as follows: For measuring C3 in corneas, tissues were obtained from CVF-treated or untreated control mice 48 hours after bacterial application to the scratch-injured eye. Corneas were excised from enucleated eyes, washed in PBS, and homogenized in PBS. The corneal homogenates were centrifuged and the supernatant used to coat an ELISA plate (Immulon IV; Thermo Fisher Scientific, Waltham, MA), starting with a 1:10 dilution in 0.05 M carbonate buffer (pH 9.6). To measure C3 levels in the sera of control and CVF-treated mice, dilutions of sera in the same carbonate buffer were used to coat an ELISA plate (Immulon IV). The sensitization step proceeded overnight at 4°C, after which the plate was washed in PBS with 0.02% Tween-20, then wells blocked by addition of 200 μL of 1% bovine serum albumin (BSA) in PBS plus 0.002% sodium azide for 2 hours at room temperature. After washing, a goat antibody to mouse C3 (GeneTex, Inc., San Antonio, TX) diluted 1:500 was added to the wells of the plates and incubated for 1 hour at room temperature; a rabbit antibody to goat IgG conjugated to alkaline phosphatase (1:1000 dilution in 1% BSA–0.02% tween-20) was then added and left for 1 hour at room temperature. After washing, the alkaline phosphatase substrate, para-nitrophenylphospate in 0.05 M carbonate buffer (pH 9.6) with 20 mg/L MgCl2, was added, and incubation proceeded for 20 minutes at room temperature. Plates were read at 405 nm and the percentage reduction in C3 determined by comparing serum or corneal samples from CVF-injected mice to the mean values from serum or corneal samples from mice not given CVF.