Anti mouse immunoglobulin g igg coupled to alkaline phosphatase

For western blotting analysis, the Paracoccidioides protein samples were probed using polyclonal antibodies produced to the rFBA. Protein samples were loaded onto a 12% SDS-PAGE gel and separated by electrophoresis. The gels were run at 150 V for approximately 2 h and the proteins were transferred to nitrocellulose membranes at 30 V for 16 h in a buffer containing 25 mM Tris–HCl (pH 8.8), 190 mM glycine and 20% (v/v) methanol. The gels were stained with Ponceau red to verify complete protein transfer. Next, each membrane was incubated in blocking buffer [1X PBS, 1.4 mM KH₂PO₄, 8 mM Na₂HPO₄, 140 mM NaCl, 2.7 mM KCl (pH 7.3), 5% (w/v) nonfat dried milk and 0.1% (v/v) Tween 20] for 2 h. The membranes were washed with PBS-T, and incubated with anti-rFBA polyclonal antibodies (1:1000), followed by washing in blocking buffer three times, during 15 min each wash. The membranes were incubated with the secondary antibody anti-mouse immunoglobulin G (IgG) coupled to alkaline phosphatase (Sigma Aldrich) diluted 1:5000 in blocking buffer, for 1 h. After that, the membranes were washed and the reaction was developed using 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and Nitro Blue Tetrazolium (NBT).

J774 1.6 macrophages (Rio de Janeiro Cell Bank – BCRJ/ UFRJ, accession number 0273) were used for phagocytosis assays. The J774 1.6 cells were cultured in RPMI medium containing bovine fetal serum 10% (v/v) (Vitrocell Embriolife,) containing IFN-γ (1U per mL) and MEM non-essential amino acid solution (Sigma Aldrich, Missouri, USA) at 36°C and 5% CO₂, until complete confluence. The macrophages were incubated with 50 μg/mL of rFBA, at 36°C for 5 h, and washed. Next, the cells were lysed by incubating with distilled water for 1 h. The lysate was centrifuged at 1,400 × g for 5 min. The proteins contained in the supernatant were submitted to SDS-PAGE and transferred to nitrocellulose membrane. The membrane was incubated blocking buffer [PBS 1X with 5% (w/v) nonfat dried milk and 0.1% (v/v) Tween 20] for 2 h, and then successively with anti-rFBA polyclonal antibodies (1:1000) and with the anti-mouse immunoglobulin G (IgG) coupled to alkaline phosphatase (Sigma Aldrich). The reactions were developed with BCIP-NBT.