Axioskop 2 upright epifluorescence microscope

HeLa cells were seeded on coverslips in 24-well plates (50,000 cells/well) and incubated overnight. For primary infections, cells were infected with purified EBs from either C. trachomatis L2 434/Bu or its mutant Rif^R derivatives, as indicated at an MOI of 1. At 29 hpi, secondary infections were performed using a C. trachomatis L2 434/Bu strain transformed with the GFP-expressing plasmid pGFP-SW2 [35 (link)]. After 1h, cells were fixed (3% formaldehyde-0.025% glutaraldehyde, 20 min, RT) and immunostained with mouse anti-phospho tyrosine monoclonal antibodies without permeabilization and counter-stained with anti-mouse Alexafluor 555-conjugated secondary antibodies (Invitrogen). Coverslips were mounted in Slow Fade Gold Antifade media and images were acquired using a Zeiss Axioskop 2 upright epifluorescence microscope with Axiovision v3.0 software. Phosphotyrosine-containing foci were counted in 35 cells for each of two biological replicates, in two independent experiments performed by two independent observers. One-way ANOVA with Bonferroni’s multiple comparison test was performed using GraphPad Prism.