Pna lectin

A Pierce™ Direct IP Kit (Thermo Fisher Scientific) was used according to the manufacturer’s instructions to selectively immunoprecipitate HOMER3 from membrane protein lysates using a rabbit polyclonal anti-HOMER3 antibody (PA5–59383, Thermo Fisher Scientific). The isolated glycoproteins were then run on 4–20% precast SDS-PAGE gels (Bio-Rad), transferred into nitrocellulose membranes and screened for HOMER3 and ST antigens expressions by western blot using the above-mentioned anti-HOMER3 antibody and a biotinylated PNA lectin (Vector laboratories), respectively. ST antigens detection by PNA lectin was preceded by overnight α-neuraminidase from Clostridium perfringens (Sigma-Aldrich) in membrane digestion 0.1 U/mL at 37 °C.

Leishmania amazonensis (WHOM/BR/Josefa) and Leishmania major (MHOM/IL/80/Friedlin) were grown in Schneider medium (Sigma-Aldrich) supplemented with 10% fetal calf serum and 1% penicillin–streptomycin at 26 °C The stationary phase promastigotes were washed three times with PBS (1045 g, 10 min, 4 °C) and, resuspended in medium RPMI 1640. L. major promastigotes were washed with PBS and incubated for 15 min with PNA lectin (Sigma) to separate the metacyclic form. 4% formaldehyde was used for parasite fixation, followed by two washes with PBS.

Frozen sections of mouse testes were prepared at 5 μM thickness, permeabilized with 0.1% Triton X-100 in PBS for 20 min, and boiled for antigen retrieval in 20 mM citrate buffer (pH 6.0) for 10 min [27 (link)]. After blocking with 3% goat serum in PBST overnight at 4°C, the sections were then incubated with a 1:2000 dilution of anti-EFCAB2 antiserum as the primary antibody for 1 h, followed by incubation for an additional hour with a 1:1000 dilution of the secondary antibody Alexa Fluor 594 (Life Technologies, Gaithersburg, MD, USA) together with PNA-lectin, a FITC-conjugated peanut agglutinin (Sigma; L7381). The sections were counterstained using DAPI and observed under a fluorescence microscope (BX50/BX-FLA; Olympus, Tokyo, Japan).