Alexa fluor 488 goat anti rat igg antibody

Mice were administered 150 mg/kg (ip) 5‐Bromo‐2´‐deoxyuridine (BrdU, from SIGMA) on day 1 and 50 mg/kg on day 2. Mice were transcardially perfused with ice‐cold saline followed by 4% paraformaldehyde 2 hours after the last BrdU injection. Brains were dissected by vibratome (Leica, CM1860) coronally on a cryostat into 20‐μm‐thick sections containing the HP. Sections were incubated for 2 hours in 50% formamide/standard saline citrate (SSC) at 65°C; washed with SSC and incubated for 30 minutes in 2 N HCL/PBS, then for 25 minutes in 0.1 M boric acid. The sections were removed and placed in 3% normal goat serum in PBST (10 mM PBS with 0.3% Triton X‐100) for 1 hour, and then incubated in a rat anti‐BrdU monoclonal antibody (1:800; Abcam; ab6326) overnight at 4°C. The sections were then washed in PBST and incubated with an Alexa Fluor^® 488 goat anti–rat IgG antibody (1:500; molecular probes; A11006) for 2 hours at room temperature. The sections were then washed in PBS and a fluorescence microscope (Olympus, Tokyo) used to visualize the sections at a magnification of × 400.

To assess the extent of Burkholderia escape from phagosomes of activated (50 U/mL IFN-γ) Nramp1⁺ or Nramp1⁻ macrophages, we examined co-localization of bacteria with lysosome-associated membrane protein 1 (LAMP1) at 4 h p.i. The bacteria were stained red with mouse anti-Burkholderia monoclonal antibody (9D5; 1:20; from the laboratory of Dr. Narisara Chantratita) and Alexa Fluor 568-goat anti-mouse IgG (1:500; Invitrogen, USA). LAMP1 was stained green with rat monoclonal antibody (1D4B; 1:200; Abcam, USA) and Alexa Fluor 488 goat anti-rat IgG antibody (1:500; Molecular Probes, USA), and nuclei were stained blue with 4,6-diamidino-2-phenylindole (DAPI; 1:500). All of the staining procedures were performed for 1 h at 37°C. Cells were examined by a laser-scanning confocal microscope equipped with LSM5 Image Browser (LSM 510 META, Carl Zeiss, Germany). The percentage of intracellular Burkholderia associated with LAMP1 was determined as the number of bacteria co-localized with LAMP1/total number of bacteria within macrophages × 100. The association of Burkholderia with LAMP1 was considered when the red fluorescent bacteria co-localized with the green fluorescence of LAMP1-positive vacuoles, represented as an area of yellow staining.