*2.18. Myeloperoxidase (MPO) Activity Assay*

The activity of tissue myeloperoxidase (MPO) in rabbit skin was evaluated 24 h after subcutaneous injection of toxins in the animals as previously described [27]. Briefly, a 6 mm skin tissue punch (biopsy) was placed in 0.75 mL of 80 mM sodium-phosphate bu ffer, pH 5.4, containing 0.5% hexadecyltrimethylammonium bromide (HTAB) (Sigma Aldrich), and then homogenized (45 s at 0 ◦C) in a motor-driven homogenizer. The homogenates were decanted into a microfuge tubes, and the vessel was washed with 0.75 mL of HTAB-bu ffer. The wash was added to tube, and the 1.5 mL sample was centrifuged at 12,000 × *g* at 4 ◦C for 15 min. Samples in triplicate (30 μL) of the resulting supernatant were added into 96-well microtiter plates. For the assay, 200 μL of a mixture containing 100 μL of 80 mM PBS (pH 5.4), 85 μL of 0.22 M PBS (pH 5.4), and 30 μL of 0.017% hydrogen peroxide (w/w) were added to the wells. The reaction was started with the addition of 20 μL 18.4 mM TMB dihydrochloride (Sigma Aldrich) in dimethylformamide. Plates were incubated at 37 ◦C for 10 min, and then the reactions were stopped by the addition of 30 μL of 1.46 M sodium acetate, pH 3.0. Enzyme activity was determined colorimetrically using a plate reader set to measure absorbance at 630 nm and was expressed as mOD/biopsy. Results are shown as mean ± s.e.m of three independent biological replicates.
