similis-venom serum or pre-immune serum in a shaved region of the back. PBS was used as a negative control. Animals were then euthanized at 2, 4, and 8 h post-injection, and skin samples were taken for histological studies. Rabbit skin samples
were fixed in 10% buffered formalin solution, pH 7.4, and then embedded in paraffin. Sections of 4 μm thickness were stained with hematoxylin-eosin (H&E), Masson Trichrome (with aniline blue, Easypath Special Stain Kit, Brazil) and Reticulin (Easypath Special Stain Kit, Brazil) according to the manufacturer’s instructions. Fixed tissue samples were evaluated by light microscopy. Qualitative microscopic analysis evaluated the presence of necrosis, edema, hyperemia, hemorrhage, and thrombosis. Moreover, inflammatory cell infiltrate was quantitatively assessed using the following approach: a cell count was performed in 50 random fields and the standard deviation BIBW2992 cell line was calculated using 10 samples according to Moro et al. (2003). We determined that the coefficient of variation was stabilized after counting cells in 30 fields (data not shown). Therefore, the total number of inflammatory cells was determined in 30 fields
per slide using a digital image analyser. Morphometry was performed using the specific software Image-Pro Plus 5.0 (Media Cybernetics, MD, USA). The two-way analysis of variance (two-way ANOVA) with Bonferroni post hoc test was used to compare the titration curves of rabbit find more sera. For the in vitro neutralization assay, the one-way ANOVA with Dunnett’s Multiple Comparison Cediranib (AZD2171) post hoc test was used to compare the sphingomyelinase activity of the venom with antivenom group versus the venom only group. For morphometry, the two-way ANOVA with Bonferroni post hoc test was used to test how the inflammatory cell infiltrate count was affected by the groups (venom or serum) or time (2, 4, or 8 h). The level of significance was set at p < 0.05 and statistical analysis was performed using GraphPad
Prism version 5.0 for Windows (GraphPad Software, CA, USA). The immunization protocol was performed in rabbits by injecting several boosters of L. similis venom. We observed that after the third booster, no statistically significant difference was found between the boosters given to the same animal (data not shown). The titration of rabbit sera generated against L. similis venom is shown in Fig. 1. Antivenom antibodies strongly reacted against L. similis venom extracted from male and female spiders ( Fig. 1A and B, respectively). No significant differences were observed between reactions of anti-L. similis and anti-L. intermedia venom sera. The absorbance values of anti-L. similis-venom serum against L. similis male venom ( Fig. 1A) were not statistically different from those obtained for anti-L. similis-venom serum against L. similis female venom ( Fig. 1B). The neutralization assay was evaluated in rabbits immunized with L. similis venom extracted from female spiders ( Fig.