T7 transcription kit

DNA fragments encoding either the entire 5´ UTR of the virB operon or a shortened fragment lacking the first 73 nucleotides, were amplified from X. citri genome using forward primers which include the T7 promoter sequence (see S1 Table). RNA transcripts of the cloned 5´UTR_B7 fragments were produced in vitro from the resulting purified PCR products by using the T7 Transcription Kit (Roche) and labelled by using the RNA 3´ End Biotinylation Kit (Pierce). Recombinant CsrA protein was purified as previously described [27 (link)]. Approximately 70 nM of purified CsrA protein and 6.25 nM Biotin-labeled RNA were mixed with binding buffer (10 mM HEPES (pH 7.3), 20 mM KCl, 1mM MgCl₂, 1 mM DTT, 5% glycerol, 0.1 μg/μL yeast tRNA, 20 U RNasin (Promega)) in a total reaction volume of 20 μL. The binding reactions were incubated at 25°C for 20 min. A 5 μL aliquot of loading buffer (97% glycerol, 0.01% bromophenol blue, 0.01% xylene cyanol) was added to the binding reaction and immediately loaded and resolved by 5% native polyacrylamide gels. The binding assays and detection of RNA products were performed with the LightShift Chemiluminescent RNA EMSA Kit (Thermo Scientific). For the control reactions, 312.5 nM unlabelled (competitor) virB 5´UTR_B7 RNA was added to the binding reactions.

After euthanasia, ears were removed, fixed in 4% phosphate-buffered formalin for a minimum of 48hrs at 4°C, paraffin-embedded and sectioned (4μm thick). Sections taken from an area that bisected the circular wound were deparaffinized, rehydrated and stained with hematoxylin and eosin. For in situ hybridization, slides were probed with Angptl4, Cxcl12 and Cxcr4 RNA using the following primers Angptl4; F 5’-ccagactcctgagactctgc-3', R 5’-gcacagccaattggcttcc-3’, Cxcl12; F-5’-gtcctcttgctgtccagctc-3’, R-5’-taatttcgggtcaatgcaca-3’, and Cxcr4; F-5’-ttctcatcctggccttcatc-3’, R-5’-atggagttgagtgcatgctg-3’. A T7 sequence was included at the start of all reverse primers generated using T7 transcription kit (Roche, Indianapolis IN). Colorimetric visualization was performed using AP substrate-chromogen NBT/BCIP (Sigma). Negative controls were generated by eliminating incubation with the probe. For immunohistochemistry slides were blocked with Background Buster (Innovex Biosciences), and stained with anti-ANGPTL4 (ThermoFisher 40–9800, 1:20). Visualization for immunohistochemistry was achieved using Stat-Q AEC kit (Innovex Biosciences). Negative controls were generated by elimination of incubation with the primary antibody. All slides were evaluated by a board-certified veterinary pathologist, blinded to the groups.