Human serum albumin (hsa)

Uninfected and Toxoplasma infected cells were fixed with 4% paraformaldehyde and incubated with 3% HSA (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 1 h at room temperature. After subsequent incubation with the monoclonal Ab M86 (Enzo LifeScience Inc., Farmingdale, NY, USA) diluted 1:50 in 3% HSA/PBS for 14 h at 4 °C, the secondary antibody Alexa Fluor^® 647 (red) goat anti-mouse IgM (Abcam, Cambridge, UK) was used at 2 µg/mL in 3% HSA/PBS for 1 h at room temperature. Stained samples were mounted in ProLong Antifade reagent containing DAPI (Molecular Probes, Eugene, OR, USA). The cell preparations were examined using a Zeiss LSM 800 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) with oil immersion objectives (×63).
Identification and relative expression of α-Gal epitopes in S. mansoni cercariae and A. fumigatus conidia was performed by immunofluorescence and flow cytometry following the previously described procedures [27 (link)]. The α-Gal-specific monoclonal antibody M86 (Enzo LifeScience Inc., Farmingdale, NY, USA) and FITC-labelled goat anti-mouse IgM (Abcam, Cambridge, UK) were used as primary and secondary antibodies, respectively.

S1B clones with unique (previously unreported) sequences were transformed into E. coli BL21 (DE3) BirA (Table 1) and expressed for 4 h in 2.5 mL cultures with the addition of 1 mM IPTG and 50 μM D-biotin. Bacteria were pelleted at 15000 × g for 5 min and resuspended in 0.01% PBST with 200 μg/mL lysozyme. Cells were lysed with three cycles of freezing at -80°C and thawing at 37°C for 30 min. Cell lysate was centrifuged for 15 min and 50 μL of supernatant was loaded to a PolySorp microtiter plate (Nunc), previously coated with 10 μg/mL of recombinant Stx1B and blocked with 1% BSA (Carl Roth GmbH, Karlsruhe, Germany). After 1 h incubation at RT, the plate was washed five times with PBST, and ABD-binders were detected with HRP-conjugated streptavidin (1:5000 in 1% BSA/PBST). The color was developed with the addition of 0.5 mg/mL o-phenylenediamine (OPD, Sigma-Aldrich, St. Louis, USA) and 0.01% H₂O₂ in 0.1 M citrate buffer (pH 5.0) for 5 min. The reaction was stopped with the addition of 2 M H₂SO₄ and absorbance read at 492 nm. Lysate containing the wild-type albumin binding domain (ABDwt) in fusion with TolA protein was loaded to wells coated with 10 μg/mL of human serum albumin (Abcam, Cambridge, UK) to serve as a positive control. Negative background was recorded in wells loaded with 1% BSA/ PBST without S1B-TolA lysate. All samples were measured in triplicates.

Primary antibodies against CD68, Connexin32, Cytokeratin, E-cadherin, human serum albumin, P450 reductase and Vimentin were purchased from Abcam (MA, USA). Donkey anti Rabbit:HRP and Goat anti mouse HRP were purchased from Jackson Immuno Research (PA, USA) while Goat anti Rabbit IgG: AF595 and Goat anti Mouse IgG were purchased from Life Technologies (CA, USA).