Fluorescein 12 utp

To test the filtration properties of the ventriculus-posterior midgut connection, polystyrene FMs of 50 nm (Polysciences) and a fluorescent tracer dye (Alexa Fluor 555, Thermo Fisher Scientific) were delivered using the method described above. In addition, fluorescein-labeled dextran of various sizes (500, 40, and 4 kDa, MilliporeSigma) were delivered to adult female mites as mixtures with 6% blue dye (erioglaucine; McCormick, Sparks Glencoe, MD) using the leaf coating method (Suzuki et al., 2017a (link)). To ensure the intactness of fluorescein-labeled dextran, the fluorescein-12-UTP (Roche) was delivered as a control. In both delivery systems, mites were allowed to feed for 24 h and fluorescence was visualized using an epifluorescence Zeiss Axioplan II microscope fitted with a FITC filter cube. Images were taken using an AxioCam Color HRc CCD Camera 412-312.

Fluorescence antisense and sense riboprobes were synthesized following a previously described protocol [29 (link)] with slight modifications. The mRNA probes were prepared from plasmid DNA of 626 bp fragments of the Hdh-FMRF2 cDNA that was subcloned into the pGEM-T easy vector (Promega, Madison, WI, USA). First, Hdh-FMRF2 plasmid DNA was linearized using 10 µg of plasmid DNA with the ApaI or SpeI restriction enzymes (Promega, USA) to obtain the antisense and sense probes, respectively. Then, the antisense and sense probes were separately labeled with fluorescein-12-UTP (Roche, Mannheim, Germany) using the T7 or SP6 RNA polymerases (Promega, Madison, WI, USA). The procedures were conducted using 20 μL of reaction mixture containing 1 μg of the linearized plasmid DNA, 4.0 µL of 5× optimized transcription buffer, 2.0 µL of 100 mM DTT, 2.0 µL of fluorescein-12-UTP labeling mix, 2.0 µL of RNase inhibitor, 2.0 µL T7 or SP6 RNA polymerase and RNase-free water (8.0 µL). The reaction mixture was incubated at 37 °C for 2 h. After incubation, the labeled linearized plasmid DNA template was digested at 37 °C for 15 min with DNase I (2.0 µL). The obtained riboprobes were then purified through ethanol precipitation with 1 μL of yeast tRNA (10 mg/mL). The purified riboprobes were stored at −80 °C until required for FISH.

For generation of RNA in vitro, the HIV 5′-UTR (nucleotides 1–497) was cloned into pSP72 vector (Promega) using BglII and EcoRI sites under the control of T7 promoter. All constructed plasmids were verified by DNA sequencing. Primers were obtained from IDT and are reported in Feng et al [73 (link)]. Fluorescently labeled RNA was produced by transcribing pSP72 DNA cut with EcoRI in vitro using T7 RNA polymerase with a nucleotide mixture containing fluorescein-12-UTP (Roche Applied Science). Steady-state rotational anisotropy reactions (60 μL) were conducted in buffer containing 50 mM Tris, pH 7.5, 40 mM KCl, 10 mM MgCl₂, and 1 mM DTT and contained 10 nM fluorescein-labeled 5’UTR RNA and increasing amounts of A3 (A3H_{hap II}, 0.1–61 nM; A3C-A3H_{hap II}, 0.0045–6.040 nM; and A3G, 0.36202 nM). A QuantaMaster QM-4 spectrofluorometer (Photon Technology International) with a dual emission channel was used to collect data and calculate anisotropy. Measurements were performed at 21°C. Samples were excited with vertically polarized light at 495 nm (6-nm band pass), and vertical and horizontal emissions were measured at 520 nm (6-nm band pass). The K_d was obtained by fitting to a hyperbolic decay curve equation using SigmaPlot version 11.2 software.

Riboprobes for ISH were generated from cDNA sequences as follows (NCBI GenBank accession numbers in parenthesis): mouse Pomc, bases 502–1008 (short probe) or bases 1–1008 (long probe; NM_001278584.1); Vglut2, bases 1762–2390 (NM_080853.3); and Gad67, bases 317–892 (NM_008077.4). The Vglut2 and Gad67 plasmid templates are a gift from Dr. Erik Hrabovszky (Institute of Experimental Medicine, Budapest), the long Pomc template was synthesized by Genscript. For dual-label ISH, the short Pomc probe was labeled with digoxigenin-11-UTP (Roche Applied Sciences), and the Vglut2 and Gad67 probes with [³⁵S]-uridine 5’-(α-thio) triphosphate (PerkinElmer). For triple-label ISH, the long Pomc probe was labeled with fluorescein-12-UTP (Roche), the Gad67 probe with digoxigenin-11-UTP, and the Vglut2 probe [³⁵S]-uridine 5’-(α-thio) triphosphate.