Rat anti lamp 1 monoclonal antibody 1d4b

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.

The following immunoreagents were used: rabbit anti-Irga6 antiserum 165/3 [17 (link)], mouse anti-Irga6 monoclonal antibodies (mAb) 10E7 and 10D7 [33 (link)], rabbit anti-Irgb6 antiserum 141/3 [65 (link)], antibody A20 (Santa Cruz Biotechnology, TX, USA), rabbit anti-Irgd antiserum 81/3 [65 (link)], anti-Irgb10 antiserum 940/6 [19 (link)], rat anti-LAMP1 monoclonal antibody 1D4B (Abcam, Cambridge, United Kingdom), anti-GM130 antibody (BD Biosciences, NJ, USA), rabbit anti-LC3B antiserum L7543 (Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-Tom20 antiserum (Santa Cruz Biotechnology), mouse anti-β-Actin monoclonal antibody (Sigma-Aldrich), and mouse anti-cathepsin B antibody (R&D systems, MN, USA). The following secondary antibodies were used: Alexa Fluor 488/555/647-labeled donkey anti-mouse/donkey anti-rabbit/donkey anti-rat antibody (all Life Technologies), HRP-conjugated goat anti-mouse antibody and HRP-conjugated donkey anti-rabbit antibody (both Sigma-Aldrich). Nuclear staining was performed with 0.5 mg/mL DAPI (Life Technologies), 1 μg/mL PI (Sigma-Aldrich), or 1 μg/mL Hoechst 33342 (Sigma-Aldrich); 50 nM Lysotracker (Life Technologies) was used for lysosome staining; 0.1 μg/mL LD540 neutral lipid dye (kindly provided by Christoph Thiele, LIMES, Bonn) was used for the staining of lipid droplets as previously described [66 (link)].