For infection 3 × 106L. major promastigotes were injected intradermally in 10 μl of PBS into the ear of 8- to 12-week-old female C57BL/6 mice. The course of infection was determined by measuring the ear swelling using photo analysis with Fiji software (ImageJ) three times a week. When ear swelling started to develop, the animals were treated three times a week with the compound Eh-1 at concentrations of 5, 10, and 25 μg in 10 μl of DPBS topically applied to the ear.
After reduction of the ear swelling, the mice were sacrificed, and ear tissue, as well as lymph node tissue, was used for histological staining and gDNA isolation. Blood serum was taken every 2 weeks p.i. The cytokine profile of the serum, as well as that of the tissue lysates, was analyzed using a LEGENDplex assay kit (BioLegend). Lysates of the infected ears were obtained by mincing with zirconia beads (2 mm; Carl Roth GmbH) and incubated on ice with 200-μl protease inhibitor tablets (Sigma-Aldrich). Samples were then mixed in a TissueLyser for 10 min at 50 ms and then centrifuged at 4°C at maximum speed. The supernatant was used for cytokine analysis.
After reduction of the ear swelling, the mice were sacrificed, and ear tissue, as well as lymph node tissue, was used for histological staining and gDNA isolation. Blood serum was taken every 2 weeks p.i. The cytokine profile of the serum, as well as that of the tissue lysates, was analyzed using a LEGENDplex assay kit (BioLegend). Lysates of the infected ears were obtained by mincing with zirconia beads (2 mm; Carl Roth GmbH) and incubated on ice with 200-μl protease inhibitor tablets (Sigma-Aldrich). Samples were then mixed in a TissueLyser for 10 min at 50 ms and then centrifuged at 4°C at maximum speed. The supernatant was used for cytokine analysis.