Texas red labeled goat anti rabbit igg

Phagocytic cells (10⁵) were seeded in 2-chamber slides, and duplicate wells were infected with fluorescein-labeled live or heat-killed (100°C) M. avium 104 with multiplicity of infection [27 (link)] of 10 for 1 h at 37°C. Some monolayers were inactivated for AP3B1 with a gene specific siRNA or transfected with MAV_2941 recombinant protein before infection. The MAV_2941 recombinant protein was made as follow: the MAV_2941 gene was fused in frame with the TAT peptide/Protein Transducing Domain (PTD) and HIS-tag by inserting the PCR-generated fragment into the EcoRI and HindIII sites of pET28b TAT v1 vector kindly provided by Dr. Steven F. Dowdy at University of California, San Diego [35 (link)]. The overexpressed protein was purified with HIS-columns (Clontech). Forty eight hours after infection, cells were fixed in 4% formaldehyde for 30 min, permeabilized with 0.1% Triton X-100 for 10 min and blocked in 2% bovine serum albumin. Slides were then incubated with either rabbit anti-LAMP-1 or Rab5 antibody (1:200) (Santa Cruz) for 2h, washed three times and incubated with Texas-Red labeled goat anti-rabbit IgG (1:1000, Molecular Probe, Eugene OR) for 1h. The slides were mounted and observed under a Leica DM4000B fluorescent microscope (Leica).