Immunofluorescence Assay for Brucella in RAW264.7 Cells

RAW264.7 cells cultured on glass coverslips (Thermo Fisher Scientific, Waltham, MA, USA) were infected with Brucella at a multiplicity of infection of 100. The infected cells were fixed with 3.7% (w/v) paraformaldehyde at 4 °C for 24 h post-infection. The immunofluorescence assay was performed as previously described [15 (link)] using rabbit anti-Brucella serum (diluted 1:1000) and Rat anti-LAMP-1 monoclonal antibody [1D4B] (diluted 1:500, Abcam, Cambridge, MA, USA) as the primary antibody, and Alexa Fluor 488-conjugated goat anti-rabbit IgG (diluted 1:1000) and Alexa Fluor 555-conjugated goat anti-rat lgG (diluted 1:1000) (Invitrogen) as the secondary antibody. The coverslips were mounted onto glass slides using Eukitt quick-hardening mounting medium for microscopy (Sigma-Aldrich) and the cells were observed under a Nikon Eclipse 80i microscope (Nikon Corporation, Tokyo, Japan) with 100× oil immersion objective. Images were saved in TIFF format and imported to Adobe Photoshop CS4 (Adobe Systems Incorporated, San Jose, CA, USA), where they were merged using RGB format. To determine the percentage of bacteria positive for the lysosome marker LAMP-1, 100 intracellular bacteria were counted randomly. The assays were performed in triplicate.