Click it cell reaction kit

Cultured cells and tissues were prepared as previously described using a combination of glutaraldehyde fixation, ferrocyanide reduced osmium tetroxide post-fixation, thiocarbohydrazide and osmium tetroxide liganding, followed by en bloc uranyl acetate and lead aspartate staining (Deerinck et al., 2010 , Ngo et al., 2016 (link), Williams et al., 2011 (link)). Cultured cells were also stained for DNA using click-chemistry as previously described (Ngo et al., 2016 (link)). Briefly, HeLa cells were incubated overnight in media containing 10 micromolar 5-ethynyl-2′-deoxyuridine (EdU, Life Technologies) and the following day, copper-mediated click-chemistry (Click-iT Cell Reaction Kit, Invitrogen) was used to attach dibromofluorescein-azide (1 micromolar) to the EdU incorporated into cellular DNA during replication, which was then used to photooxidize diaminobenzidine into a reaction product prior to treatment with osmium tetroxide (Ngo et al., 2016 (link)). Cultured cells were also prepared using a genetically targeted ascorbate peroxidase in order to stain the endomembrane system (Martell et al., 2012 (link)). Epoxy embedded samples were mounted to aluminum pins (Gatan) using either silver epoxy (Ted Pella, Redding CA) or cyanoacrylic adhesive, or mounted on a custom designed tip-tilt holder and sputter coated with a thin layer of Au/Pd prior to block-face imaging.

S. Typhi wild type, ΔttsA and ΔycbB mutant strains were grown in TTIM for 24 hs, and subsequently incubated in TTIM containing alkyne-D-alanine (2 mM) (Boao Pharma, Boston) for 4 hs. To analyze peptidoglycan remodeling in bacteria that had been allowed to re-enter the cell cycle after growth to stationary phase, S. Typhi strains were grown to stationary phase in TTIM and labeled with alkyne-D-alanine as described above. Bacterial cultures were then split, casamino acids or eluent were added, and cultures were further grown for the indicated times (always in the presence of alkyne-D-alanine). In all cases, after the various growth conditions bacteria were washed with PBS and fixed for 15 min in 4% paraformaldehyde (PFA) at room temperature. Fixed bacteria were resuspended in Click-iT Cell Reaction Buffer (Invitrogen) and copper-catalyzed click-chemistry was performed in the dark at room temperature for 30 min using the Click-iT Cell Reaction Kit (Invitrogen) with 10 μM azido-Alexa-Fluoro488 fluorophore (Invitrogen). Bacteria were then washed three times and attached to poly-(D) lysine coated coverslips. If applicable bacterial cells were counterstained for LPS (red) with primary anti-Salmonella O poly A-1 & Vi rabbit antiserum (Becton, Dickinson & Co.) (1:10,000) and secondary anti-rabbit Alexa-Fluor594 (Invitrogen) (1:2,000) antibody.