*4.11. Evaluation of Paw Edema*

Mice (*n* = 6) were injected (30 μL) with Cryptoxin-1 (45 μM), GST (45 μM), or PBS (negative control) in the right hind paw. The edema-forming activity was studied after 1, 24, 48, and 72 h, by pachymeter. The results were expressed as the difference in paw thickness before (control) and after (experimental) injection (mean ± S.E.M).

#### *4.12. Histological Analysis*

Mice (*n* = 5) were injected in the right paw with Cryptoxin-1 45 μM/30 μL, GST 45 μM/30 μL or PBS (negative control) and 24 h after the injection, the animals were euthanized, and the right paws were collected for footpad skin removal. The samples were then fixed in 4% paraformaldehyde in PBS, pH 7.2, for 24 h. After dehydration in a crescent ethanol series up to 95%, the samples were embedded in glycol methacrylate (Leica Microsystems Nussloch GmbH, Heidelberg, Germany). Sections of 4 μm were obtained in a Microm HM340 microtome and stained with the hematoxylin-eosin solution for morphological studies of tissues.

### *4.13. Analysis of the Neutrophil Infiltrate in Footpad Tissue by Flow Cytometry*

The Neutrophil migration in the footpad after 24 h of Cryptoxin-1 injection was analyzed by flow cytometry. Groups of mice (*n* = 5) were euthanized, and the right paws were removed at the tibiotarsal joint and macerated. The debris was resuspended into 1 mL of PBS and then centrifuged (5 min/1200 rpm/4 ◦C). The cell pellets were recovered and counted by Trypan blue exclusion (Sigma–Aldrich, St. Louis, MO, USA) using a hemocytometer. The cells that were resuspended in an RPMI-1640 cell culture media (Gibco Thermo Fisher Scientific, Waltham, MA, USA), were then incubated with anti-FcγRII/III mAb for 30 min at 4 ◦C. Afterward, the cell suspensions were centrifuged (5 min/1200 rpm/4 ◦C) and resuspended in the culture medium (106 cells/well) and incubated with anti-leukocyte (CD45-APC) and anti-neutrophil (Ly6G-PE/CD11b-PeCy7) monoclonal antibodies (BD Biosciences, Franklin Lakes, NJ, USA) for 30 min at 4 ◦C. The cells were then washed and resuspended in PBS containing 0.1% paraformaldehyde (Merck, Darmstadt, Germany). All samples were acquired in the flow cytometer (FACS Canto II, BD Biosciences, Franklin Lakes, NJ, USA). Around 20,000 events were collected for each sample The data were analyzed using FlowJo software 7.5 (BD Biosciences, Franklin

Lakes, NJ, USA). The forward and side scatter density plots (FSC × SSC) were used to exclude the debris and select the cell population, followed by the selection of the single cells. After this, the CD45<sup>+</sup> cells were selected and then, using the fluorescent minus one (FMO) methodology, the CD45+CD11b+Ly6G+ cells were determined. The results were expressed as the mean of the percentage of CD45+CD11b+Ly6G+ cell population of individual mice/group ± standard error of the mean (S.E.M).

#### *4.14. Statistical Analysis*

All statistical analyses and graphical representations were analyzed using the Graph-Pad Prism 9.1.2 program. Statistical tests performed using ANOVA followed by the Bonferroni test and t Student's test. The *p* values followed the pattern recommended by the software: \* *p* < 0.05; \*\* *p* < 0.001; \*\*\* *p* < 0.0001; \*\*\*\* *p* < 0.00001.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/toxins13120858/s1, Figure S1: Capillary electrophoresis of the RNA sample extracted from *C. iheringi*; Figure S2: Size distribution of *C. iheringi* cDNA library evaluated in Agilent 2100 Bioanalyzer; Table S1: *Cryptops iheringi* transcriptome data.

**Author Contributions:** Conceptualization: G.S.M., L.H.D.L.C., I.d.L.M.J.-d.-A. and M.Y.N.-J. Methodology: G.S.M., M.Y.N.-J., E.L.F.-M., I.d.L.M.J.-d.-A. and L.H.D.L.C. Validation: E.L.F.-M., G.S.M., M.Y.N.-J., L.H.D.L.C. and B.d.C.L.F.T. Formal analysis: E.L.F.-M., G.S.M., M.Y.N.-J., L.H.D.L.C. and B.d.C.L.F.T. Investigation: E.L.F.-M., G.S.M., M.Y.N.-J., L.H.D.L.C., B.d.C.L.F.T. and U.C.d.O. Resources: G.S.M., L.H.D.L.C., I.d.L.M.J.-d.-A. Data curation: M.Y.N.-J., L.H.D.L.C. and G.S.M. Writing original draft preparation: G.S.M., L.H.D.L.C. Writing—review and editing: G.S.M., L.H.D.L.C., M.Y.N.-J. Visualization: G.S.M., L.H.D.L.C., E.L.F.-M., I.d.L.M.J.-d.-A., M.Y.N.-J., B.d.C.L.F.T. and U.C.d.O. Supervision: G.S.M. and M.Y.N.-J. Project administration: G.S.M. and L.H.D.L.C. Funding acquisition: G.S.M. and L.H.D.L.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by São Paulo Research Foundation (FAPESP) (2017/16999-8) granted to Geraldo Santana Magalhães. A fellowship, from the National Coordination of High Education Personnel Formation Programs (CAPES- Demanda Social) and FAPESP (2017/13812-4), was granted to Lhiri Hanna De Lucca Caetano. A fellowship from National Council for Scientific and Technological Development (CNPq) (312096/2018-6) was granted to Eliana L. Faquim-Mauro. Computational Infrastructure from the Bioinformatics and Computational Biology Core from Laboratório de Toxinologia Aplicada, Butantan Institute was also funded by FAPESP (2013/07467-1).

**Institutional Review Board Statement:** Ethics Committee Name: Committee on Animal Use of the Butantan Institute (CEUAIB); Approval code: CEUAIB n◦ 4300061120; Approval Date: 22 December 2020; Approval code: CEUAIB n◦ 886/12; Approval Date: 19 November 2014.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We would like to thank the staff of the Butantan Institute and Butantan Foundation for their technical support, as well as the availability of the institute's facilities, especially the Immunopathology Laboratory, the Laboratory of Applied Toxinology Laboratory (LETA), the computational infrastructure from Bioinformatics and Computational Biology Core (NBBC) and the graduate course of Butantan Institute (ESIB).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

