Fitc goat anti mouse igm

Bacteria were cultured at 37 °C for 18 h in MRS medium (Hi-Media, Mumbai). The bacterial isolates were centrifuged 2 × in PBS (4000 g, 5 min) and re-washed using PBS. Afterwards they were fixed in 4% paraformaldehyde at 25 °C for 30 min before being washed one more time in PBS. The harvested cells after washing were incubated with 3% Human Serum Albumin (I, Sigma-Aldrich) in Phosphate-buffered saline at 25 °C for 1 h. Then, further incubation was done with the α-Gal Epitope (Galα1-3Galβ1-4GlcNAc-R) monoclonal antibody (M86, Enzo Life Sciences, Farmingdale, NY) that have been diluted 1:50 in 3% I/PBS for 14 h at 4 °C. For secondary antibody, FITC-goat anti-mouse IgM (Abcam, Cambridge, UK) onfuse antibody (diluted 1/200 in 3% I/PBS; 1 h at 25 °C) was used. Samples were analyzed on a FAC-Scalibur flow cytometer equipped with CellQuest Pro software (BD Bio-Sciences, Madrid, Spain). The viable cell population was gated according to forward-scatter and side-scatter parameters while bacteria incubated with the secondary antibody only and E. coli O86:B7 were used as negative and positive controls respectively according to the method of Cabezas-Cruz et al.^{61 (link)} with some modification. Results were presented in geo mean and median of the Fluorescence Intensity (FI) measured.

α-Gal detection was performed as previously described [12 (link)]. Briefly, human, tick, and bacterial cells were washed in PBS, then fixed, and permeabilized. The cells were then incubated with 3% HSA (Sigma-Aldrich, USA) in PBS for 1 h at RT. Then, the cells were incubated for 14 h at 4 °C with the mAb M86 diluted 1:50. The viable cell population was gated according to forward-scatter and side-scatter parameters. FITC-goat anti-mouse IgM (Abcam, Cambridge, UK) labelled Ab (diluted 1:200 in 3% HSA/PBS, 1 h at RT) was used as a secondary Ab. The mean fluorescence intensity (MFI) was determined by flow cytometry and compared between the test and control cells by Student’s t-test with unequal variance (p < 0.05, n = four biological replicates). Aliquots of fixed and stained samples were used for immunofluorescence assays, mounted in ProLong Antifade with a DAPI reagent (Molecular Probes, Eugene, OR, USA) and examined using a Zeiss LSM 800 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany) with oil immersion objectives.