Cyanine3 (cy3)

For the assessment of novel miRNA-2 and CPR1 colocalization, antisense RNA detection probes targeting novel miRNA-2 and the CPR1 gene were designed and labeled with the dual fluorophores Cy3 and FAM, respectively, at Servicebio Biotechnology Company (Wuhan, China). A scrambled sequence and the sense probe for the target gene were used as NCs. Two days after microinjection, the tick nymphs were dissected in cold PBS buffer, fixed overnight in 4% paraformaldehyde, and incubated overnight at 37°C for probe hybridization (Nuovo et al., 2009 (link); Yang et al., 2016 (link)). The tick samples were washed in PBS containing 5% Triton X-100 (v/v) for 10 min and stained with DAPI G1012 (Servicebio, Wuhan, China) at room temperature for 8 min. The signals of CPR1 and novel miRNA-2 were detected using an Eclipse CI upright fluorescence microscope (Nikon, Japan). The probes for the miRNA and target genes are listed in Table 1.

The paraffin sections were dewaxed and placed in a repair box of EDTA antigen retrieval buffer (PH8.0) for antigen retrieval in a microwave oven, and 3% BSA was used for blocking for 30 min, after which the blocking solution was discarded. Slides were placed in a wet box and incubated with the corresponding primary antibodies, including anti-CCL3 (1:40, AF-450-SP, R&D Systems), anti-CCR4 (1:200, sc-101375, Santa Cruz Biotechnology), and anti-E-cadherin (1:200, 14472S, CST) at 4°C overnight. The slides were then incubated with the secondary antibodies CY3(G1223, Servicebio, Wuhan, China), FITC (G1222, Servicebio), and CY5(GB27303, Servicebio), and incubated at room temperature for 50 min in the dark. The slides were then incubated with DAPI solution at room temperature for 10 min to stain the nuclei. The fluorescent images were observed and preserved using a fluorescence microscope.

After behavioral tests, mice were perfused. They were sequentially perfused with 40 ml of normal saline and 40 ml of a 4% paraformaldehyde solution (0.1 mol/L PBS, pH 7.4) through the ascending aorta after anesthesia with chloral hydrate. The mouse brain was fixed in 4% paraformaldehyde solution for more than 24 h, then dehydrated in gradient alcohol. Brain slices (thickness: 4 µm) were cut using a Frozen platform (JB-L5, Wuhan Junjie Electronics Co., Ltd., China) and collected in an EDTA antigen retrieval solution (pH 8.0). Next, the brain slices were rinsed with PBS (pH 7.4) three times for 5 min, then placed in 3% BSA and incubated at room temperature for 30 min. The brain slices were transferred into PBST with a primary antibody (Anti-P2X7R, 1:200, Bioss; Anti-Iba1, 1:500, Servicebio; Anti-Brdu, 1:200; Servicebio) and incubated at 4°C overnight. After rinsing with PBS (pH 7.4) for 5 min three times, the brain slices were transferred into PBS (pH 7.4) with a secondary antibody (FITC, 1:400, Servicebio; Cy3, 1:400, Servicebio) and incubated in the dark at room temperature for 50 min. For immunofluorescence staining, the patches were rinsed three times with PBS for 5 min three times, then dried with a 4’, 6-diamidino-2-phenylindole staining solution. Images were obtained using an Ortho-Fluorescent Microscopy (Nikon Eclipse C1, Nikon, Japan).