Dig probe

NHDFs were infected with CHIKV_181/25, and total RNA was collected at 12 hpi by the TRIzol method. RNA was electrophoretically separated on a formaldehyde agarose gel and transferred onto a Hybond-N⁺ positively charged nylon membrane (Amersham). CHIKV RNA was detected using an E2-6K-E1-specific digoxigenin (DIG) probe (Roche) constructed by PCR using forward primer 5′-CGCAGTTATCTACAAACGGTA-3′ and reverse primer 5′-TTTACTCTCAGGTGTGCGA-3′. Human β-actin RNA was detected using a DIG probe constructed using forward primer 5′-ACCCTGAAGTACCCCATCGA-3′ and reverse primer 5′-CGGACTCGTCATACTCCTGC-3′. Detection was performed using the DIG-High Prime DNA labeling and detection starter kit II (Roche). DIG-labeled membranes were incubated with a CSPD [disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2′-(5″-chloro)tricyclo (3.3.1.1)decan}-4-yl)phenyl phosphate] alkaline phosphatase chemiluminescent substrate and visualized on CL-XPosure film (Thermo).

Plasmid replicons were typed using PCR-based typing approach as described by Carattoli et al. [20] (link). In order to define the presence of KPC-IncI2 plasmids, not detected using the PCR-based replicon typing but increasingly disseminated in New York and New Jersey, US states neighboring to Ontario, primers recently described by Chen et al were used [21] (link). The genetic surrounding of bla_KPC was studied by PCR mapping and sequencing [22] (link). Plasmid content and their estimated sizes were determined by S1 endonuclease-digested genomic DNA and PFGE (S1-PFGE) [23] (link). Plasmids carrying bla_KPC genes and their replicons were identified by S1-PFGE followed by Southern blot analysis using specific probes for bla_KPC and positive replicons (DIG Probe, Roche Diagnostics) [24] (link).

Whole-mount in situ hybridization was performed according to previously described procedures (Pizard et al., 2004 (link)). Briefly, E10.5 embryos were dissected in cold 1× PBS and immediately fixed with 4% paraformaldehyde (PFA) overnight at 4°C. Embryos were then rinsed in PBT (PBS with 0.1% Triton X-100) to remove fixative, dehydrated by an ascending methanol series and stored in 100% methanol at -20°C. Embryos were rehydrated through a graded methanol series into PBT, digested with 10 μg/ml protease K for 10 min and post-fixed in 4% PFA plus 0.2% glutaraldehyde for 20 min. After prehybridization at 65°C for 2 h’ embryos were incubated overnight in hybridization buffer containing antisense probe at 65°C. Embryos were then washed with a series of washing buffer and blocked in 1× TBST containing 20% sheep serum for 3 h at room temperature. Embryos were incubated overnight with anti-digoxygenin-labeled (DIG)-probes (1:5000; Roche). Following probe incubation, embryos were washed eight times with 1× TBST and rinsed twice for 15 min in fresh NTMT solution. When hybridization signal is optimal, embryos were refixed in 4% PFA.