*2.16. Dermonecrosis In Vivo*

For assessment of dermonecrotic e ffects, 10 and 20 μg of LiRecTCTP, 1 μg of LiRecDT1 (wild type dermonecrotic toxin), and 20 μg of GFP (a recombinant protein without relevant biological activity) [2,27] were injected subcutaneously into a shaved area of the rabbit dorsal skin. Animals were observed over the course of dermonecrotic lesion progression. Animal skin was photographed immediately after injection and after 24 h of toxins application. The same rabbit received all the 7 samples (divided in the both dorsal sides of the animal) (PBS, GFP, LiRecDT1, LiRecTCTP 10 μg, LiRecTCTP 20 μg, LiRecDT1+LiRecTCTP 10 μg, and LiRecDT1+ LiRecTCTP 20 μg). Experiments were initially performed in two animals and then repeated using four animals. Images show a photograph from the skin of a representative rabbit. Animals were euthanized using intramuscular injection of ketamine (240 mg/kg) and xylazin (27 mg/kg). After euthanasia of animals, skin samples were harvested for histopathological analysis and myeloperoxidase (MPO) activity assay.

### *2.17. Histological Methods for Light Microscopy*

Rabbit skin pieces from animals, which were previously subcutaneously inoculated with recombinant toxins, were collected. The tissue samples were fixed in "ALFAC" (ethanol 85%, formaldehyde 10%, and glacial acetic acid 5%) for 16 h at room temperature. After fixation, samples were dehydrated in a graded series of ethanol before para ffin embedding (for 2 h at 58 ◦C). Then, thin tissue sections (4 μm) were processed and stained with hematoxylin and eosin (H & E). Histological sections were analyzed in Image J analysis software (v.1.x) to quantify edema formation by the measurement of the area between the epidermis and adipose tissue, histological section area observed after GFP protein inoculation was considered the control for comparisons.
