Goat anti mouse igg alexa 568 conjugate

BHK-21 cells were infected with the reporter virus, JEV (RP-9) expressed enhanced green fluorescent protein (JEV-eGFP) (34 (link)), at an MOI of 2 for 15 h. Following fixation and permeabilization, the cells were incubated overnight at 4°C with a serial dilution of different sera from mice vaccinated with B5G, rEDIII, or BJLPET5G followed by detection with goat anti-mouse IgG-Alexa 568 conjugate (Invitrogen, Waltham, MA, USA), and examined under a fluorescence microscope. Fluorescent signal images were captured at 200× magnification with an Olympus IX71 inverted fluorescence microscope (Olympus, Tokyo, Japan).

Polyclonal immune sera from CD-1 mice were pre-absorbed with the Bucl8-lacking mutant cells of Bp82 [19 (link)] to diminish a cross binding to whole bacterial cells. For the ELISA, wells were coated with ~10⁴ cells, either Bp82 or Bp82Δbucl8-fusE, and incubated overnight at 4 °C. Wells were washed with 0.05% Tween-20/PBS and blocked with 1% BSA in 0.05% Tween-20/PBS at 37 °C for 2 h. Assay was completed as above.
To assess whole-cell binding by flow cytometry, Bp82 or Bp82Δbucl8-fusE bacteria were grown from an overnight liquid culture to OD 0.4 and ~10⁷ cells were pelleted. Cells were washed with FACs buffer (PBS + 5% LBM) and then resuspended in 500 μL of cold FACs buffer. Absorbed immune sera were added to cell suspension at a dilution of 1:500 and incubated on ice for 30 min. Cells were washed and resuspended in solution with goat anti-mouse IgG Alexa 568 conjugate (Invitrogen) diluted 1:300, then incubated on ice for 30 min. Cells were washed and resuspended in 0.4% paraformaldehyde overnight at 4 °C. For analysis, cells were washed, resuspended in FACs buffer, and analyzed on a BD LSRFortessa flow cytometer.