Z1 axioobserver

The staining was performed as described previously (Hellert et al., 2013 (link)) with following changes: cells were washed and resuspended in PBS to a concentration of 0.5 × 10⁶ cells/ml to 1 × 10⁶ cells/ml and 10–15 μl of the cell suspension was dropped and allowed to dry on Superfrost/Plus slides (Thermo Fisher Scientific). A circle surrounding the dried cells was drawn with a hydrophobic pen (PAP PEN). The fixation with 4% PFA, quenching, permeabilization and blocking was performed as in Hellert et al. (2013) (link). Incubation with the primary antibodies (α-ORF59, Advanced Biotechnologies Inc. #13-211-100, dilution 1:100; α-LANA, NovocastraLiquid^TM Mouse Monoclonal α-LANA NCL-L-HHV8-LNA, dilution 1:100), secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., Cy^TM 3-labeled Donkey Anti-Mouse IgG #715-165-150, dilution 1:200; and Cy^TM 5-conjugated AffiniPure Goat Anti-Mouse IgG #115-175-146, dilution 1:200) and washes were performed in 10% FCS in PBS + 0.2% Triton X-100. Slides were then washed with PBS, stained with DAPI (1 mg/ml, 1:1000) for 30 min at 37°C and mounted with MOVIOL containing DABCO (2.5%). Slides were dried overnight at room temperature in the dark and imaged the next day using the Zeiss Z1 AxioObserver using the Plan-Neofluar 20×/0.50 objective (ORF59) or the Plan-Apochromat 63×/1.40 Oil DIC M27 objective (kLANA).

To determine the production of infectious progeny, titer samples were thawed, vortexed and sonicated and then serially diluted for infection of a new monolayer of HeLa cells. After 40 h, coverslips were fixed with methanol and stained as in immunofluorescence assays to visualize chlamydial inclusions [57 (link)]. Inclusions were counted on three coverslips per sample using 200X magnification and a Zeiss Z1 Axio Observer inverted light and epifluorescence microscope. Data are expressed as the average inclusion forming units/mL +/- SEM.

HEK293T cells were transfected with pLVX vectors containing MYC-tagged BirA, BirA-SMN, or BirA-SMNY109C using Lipofectamine 3000 (Invitrogen) following the manufacturer’s protocol. 24 h after transfection, BirA expression was induced using 1 µg/ml doxycycline. 24 h after induction, cells were fixed for 10 min at room temperature in 2% PFA. Cells were then permeabilized with 0.5% Triton, diluted in 1× PBS and 3% NGS (normal goat serum), then incubated for 1 h with primary antibody and 1 h with secondary antibody. MYC-tagged BirA, BirA-SMN, and BirA-SMNY_109C were detected using an α-MYC primary antibody diluted 1:500 (ab9106; Abcam). COILIN was detected using an α-COILIN diluted 1:1,000 (ab11822; Abcam). Endogenous SMN was detected using an α-SMN diluted 1:200 (610647; BD Biosciences). Highly cross-adsorbed secondary antibody AF488 α-rabbit was diluted 1:1,000 (A32731; Thermo Fisher Scientific), and highly cross-adsorbed secondary antibody AF555 α-mouse was diluted 1:1,000 (A32727; Thermo Fisher Scientific). DNA was stained using DAPI. Coverslips were mounted using Fluoromount-G (Invitrogen), and samples were observed using a Z1 Axio Observer (Zeiss) at 1,000X magnification.