*2.1. Expression of the ICAM-1 and PECAM-1 Proteins in the Cremaster Muscle*

The microcirculation of the cremaster muscle in animals injected with the toxins Jar, Jar-C, and BnP1 presented positive labeling for both adhesion molecules compared to the microcirculation of the control group. When the levels of these proteins were quantified using ELISA, these three toxins induced a similar pattern of protein expression based on the level of fluorescence and time course of appearance (Figure 1A–E).

Toxins induced an increase ICAM-1 expression in the early stages (2 h and 4 h) compared with the control group (Figure 1A,B); however, at 24 h postinjection, the group that received BnP1 did not exhibit a morphological difference from the control group (Figure 1C). Quantification using the immunoassay confirmed the increase in the expression of ICAM-1 in groups injected with the toxins 2 and 4 h after the injection compared to the control group. A statistically significant difference was observed in animals injected with Jar between 2 and 4 h, and this pattern was exclusively induced by Jar (Figure 1D). Twenty-four hours after the injection of those three toxins, the concentration of ICAM-1 returned to a value similar to its initial level and did not differ from the control group (Figure 1D).

PECAM-1 expression in the microvasculature of cremaster muscle was significantly increased 4 h and 24 h after the injection of the three toxins when compared to the control group, as evidenced by the results of immunofluorescence assays (Figure 1B,C). The quantification of PECAM-1 levels using ELISA revealed constitutive expression in the control group, and an evident increase in the expression of this molecule was induced by the three toxins over time. This difference was greater at 24 h after toxin injection (Figure 1E). Notably, no nonspecific immunofluorescence labeling for the primary antibody was observed, since staining was also conducted using samples from in naive animals (data not shown).

**Figure 1.** Expression of ICAM-1 and PECAM-1 in the cremaster muscle at different time points. The panels present photomicrographs of immunofluorescence labeling analyzed using confocal microscopy at2h(**A**), 4 h (**B**) and 24 h (**C**) after the subcutaneous injection of PBS, Jar, Jar-C or BnP1 (0.5 μg/100 μL) in the mouse scrotum. Bar, 50 μm. Quantification of the protein levels of ICAM-1 (**D**) or PECAM-1 (**E**) in homogenates of cremaster muscle determined using ELISA. Absorbance was measured at 492 nm. The results are presented as the means ± S.E.M. (*n* = 5). \* *p* < 0.05 compared to the PBS group at any time point. Different letters (a, b, c) in the same group represent significant differences (\* *p* < 0.05).
