Briefly, 50 pairs of salivary glands were dissected under sterile conditions in endotoxin-free PBS, placed in 50 μl of PBS and were kept at −70°C until use. Immediately before use, the glands were disrupted by sonication using a Sonifer 450 homogenizer (Branson, Danbury, Connecticut). AZD6094 molecular weight Endotoxin levels were evaluated by using the QCL-1000(r) Chromogenic LAL Endpoint Assay kit (Lonza, Switzerland), which revealed negligible

levels of endotoxin within the salivary gland supernatants. MEK inhibitor SGE intradermal inoculation The ear dermis of BALB/c mice was intradermally inoculated with different inoculums of SGE (SGE-1X and SGE-3X). Each inoculum consisted of 0.5 pair of SGE diluted in 10 μL of PBS /ear. SGE-1X group received one single inoculum of SGE and, other group, the mice received SGE-1X plus promastigote forms of L. braziliensis (1 × 105). The protocol of immunization with saliva consisted of three inoculums of SGE, with intervals of 10 days among each ones. Alternatively, the mice received three inoculums of SGE being that, in the third one, they also received the infection with parasites. The control group, the mice received one injection with 10 uL of PBS. Thus, the groups are: Group PBS = one injection of PBS; Group SGE-1X = one injection of SGE; Group SGE-3X = three injections of SGE; Group

PBS/parasite = PBS plus parasite; Group SGE-1X/parasite = SGE-1X plus parasite; Group SGE-3X/parasite = SGE 2X + SGE-1X plus parasite. Parasitic, intradermal infection and parasitic burden click here quantification L. braziliensis was cultured in Schneider (Sigma, Saint Louis, MO, USA) medium supplemented with 20% heat-inactivated fetal

calf serum see more (Cultilab, Campinas, SP, Brazil), 4 mM NaHCO3, 100 U/ml penicillin, 100 μg/ml streptomycin (all from Gibco, Grand Island, NY, USA), and 2% v/v male human urine at 25°C. Promastigotes of L. braziliensis were isolated from stationary phase cultures (5-6th day of culture), centrifuged at 1540 g at 4°C for 10 min and washed in PBS. Promastigotes of L. braziliensis were isolated from stationary phase cultures (5-6th day of culture), centrifuged at 1540 × g at 4°C for 10 min and washed in PBS. The L. braziliensis promastigotes (1 × 105) were inoculated intradermally into the ear of mice previously inoculated with SGE (−1X or -3X) or vehicle (PBS) using a 27.5-G needle in a total volume of 10 μl. The development of lesions was monitored by measuring the diameter of the ear lesion with a vernier caliper. To quantify the parasitic burden, the dermal sheets of the infected ears were separated, deposited dermal side down, and then homogenized using a Medimachine (Becton & Dickinson Biosciences, San Diego, CA, USA) tissue grinder in a microfuge tube containing 1000 μl of supplemented Schneider medium (Sigma, Saint Louis, MO, USA) for 4 min.