Superblock blocking buffer

Ten milligrams of spores were resuspended in 750 μl SuperBlock Blocking buffer (Thermo Scientific) and incubated for at least 20 min at room temperature. The spores were then harvested by centrifugation and the spore pellet was resuspended in 250 μl SuperBlock Blocking buffer with 1 μl primary antibody and incubated at room temperature for 20 min (with mixing every 5 min). Rabbit polyclonal anti‐rBclA antibodies were used (Thompson et al., 2007 (link)). Following incubation with the primary antibody, the spores were harvested by centrifugation and washed with 750 μl of SuperBlock Blocking buffer. The pellet was then resuspended in 250 μl of SuperBlock Blocking buffer with secondary antibody conjugate (1:250 goat anti‐rabbit IgG‐Alexa Fluor 568; Invitrogen). The spores were incubated at room temperature for 20 min, pelleted, and washed with 750 μl SuperBlock Blocking buffer, followed by three washes with 750 μl PBS, and finally resuspended in 250 μl PBS. The spores were examined by epi‐fluorescence microscopy using a Nikon E600 epifluorescence microscope using the mCherry filter set.

Cells were prepared for immunostaining and confocal microscopy by fixation in 2% paraformaldehyde for 15 minutes, permeabilization in 0.2% Triton X-100 for 1 hour, and blocking in SuperBlock Blocking Buffer (Thermo Fisher Scientific, Waltham, MA). Cleaved caspase-3 was immunostained by incubating HAE with a primary human cleaved caspase-3 (Asp175) antibody (catalog no. MAB835; R&D Systems, Minneapolis, MN, 1:100 in SuperBlock Blocking Buffer) for 1 hour. This was followed up with a 1-hour incubation of an Alexa 568 labeled anti-rabbit secondary antibody (catalog no. A-11036; Invitrogen, Waltham, MA, 1:1000 in SuperBlock Blocking Buffer). To stain for F-actin, HAE were incubated with Phalloidin-Alexa 647 (1:50 in PBS, catalog no. A22287; Thermo Fisher Scientific) for 30 minutes. The filters with the HAE were then cut from the rest of the transwell insert and mounted on glass microscope slides using VECTASHIELD Mounting Medium with DAPI (catalog no. H-1200-10; Vector Laboratories, Inc., Burlingame, CA). Confocal images were acquired using a Leica TCS SP3 confocal microscope (Leica Microsystems, Inc.) with 20x, 40x, and 63x objectives. Images were processed and z-stacks were compiled using ImageJ version 2.1.0. Live-image microscopy was performed using a Leica DMI6000-B inverted microscope (Leica Microsystems, Inc., Buffalo Grove, IL) using a 10x objective.

An ELISA method was used to compare binding efficiency of mAbs according to a published method [47] (link). LPS from E. coli 055:B5 (Sigma) and Leptospira licerasiae[48] (link) were used as specificity controls. B. melitensis LPS was dissolved (2 µg/ml) in 0.06 M sodium carbonate buffer (pH 9.6); 100 µl was used to coat wells by incubation overnight at 4°C. For the assay, Superblock blocking buffer (Thermo Fisher Scientific, IL, and USA) was used for blocking, TBST was used for washing the plate and 1% Superblock blocking buffer in TBST was used to dilute antibodies and conjugates. LPS-coated plates were blocked (for 1 hr at 22°C followed by three washes. mAbs were diluted (2 µg–0.02/ml) and added 200 µl per well in duplicate and incubated overnight at 4°C. The wells were washed three times with TBST and 200 µl of diluted (1∶5,000) goat anti-mouse immunoglobulin G (IgG) antibody conjugated to horseradish peroxidase was added to each well and incubated for 1 hr at room temperature. The plate was washed four times and 100 µl of chromogenic substrate (TMB Microwell Peroxidase Substrate system, KPL, Gaithersburg, MD) was added to each well and the reaction was stopped by the addition of 100 µl of 2N H₂SO₄. The plate was read using a microplate reader (Spectramax Plus, Molecular Devices, Sunnyvale, CA) at wavelength 450 nm.