Alexa fluor 488 labelled goat anti rabbit antibody

IF was performed as described previously (Tatavosian et al., 2015 (link); Zhen et al., 2014 (link)). Wild-type (PGK12.1), Eed^−/−, Ezh2^−/−, Y-Eed/Eed^−/−, and Y-Ezh2/Ezh2^−/− mES cells were cultured on coverslips and fixed using 2.0% paraformaldehyde. After permeabilizing with 0.2% Triton X-100, the cells were washed with basic blocking buffer (10 mM PBS pH 7.2, 0.1% Triton X-100, and 0.05% Tween 20) and then blocked with blocking buffer (the basic blocking buffer plus 3% goat serum and 3% bovine serum albumin). Anti-H3K27me3 antibody (07–449; Millipore, Billerica, MA) was incubated with the cells for 2 hr at room temperature. After washing with the basic blocking buffer, Alexa Fluor 488-labelled goat anti–rabbit antibody (A-11008; Life Technologies, Carlsbad, CA) was incubated with the cells for 1 hr. After incubating with 0.1 μg/ml hoechst, the cells were washed and then mounted on slides with ProLong Antifade reagents (P7481; Life Technologies, Carlsbad, CA). The images were taken and processed as described previously (Tatavosian et al., 2015 (link); Zhen et al., 2014 (link)).

C. trachomatis SvD (Trachoma type D strain UW-3/Cx, ATCC® VR-885™) was propagated in HeLa cells as previously described [27 (link)]. Briefly, the HeLa cells were cultured in six well plates and infected with 1.5 C. trachomatis SvD inclusion forming units (IFU) per HeLa cell. The infection was followed by centrifugation at 750 g for 1 h, and thereafter incubated at 35 °C for 2 h, after which the media was enriched with 0.5 % glucose and 1 μg/ml cyclohexamide. The plates were incubated at 37 °C for 48 h, and thereafter harvested and purified as described by Olsen et al. [27 (link)].
The concentration of infectious bacteria was determined by culturing bacterial suspensions on McCoy-cells [28 (link)]. The plates were incubated at 37 °C for 22 h. Visualization of inclusions was made by incubating the cells with polyclonal rabbit antibodies against chlamydial Major Outer Membrane Protein and chlamydial Heat Shock Protein-60 [29 (link)], and thereafter stained with 4 μg/ml Alexa Fluor 488 labelled goat-anti rabbit antibody (Life Technologies) and kept in the dark at 4 °C until microscopy was carried out.
The cell plates were evaluated using a fluorescent microscope (Olympus IX71). Inclusion bodies were enumerated by counting positively stained inclusions in at least 20 fields in each well using the 40x objective on microscope. Results were calculated as average of duplicate samples.