Fluorescein tyramide

Double fluorescence in situ hybridization (dFISH) was performed in the gut of viruliferous planthopper. Prehybridization solution contained biotin-labeled miR-263a probe (1 μmol mL^-1; RiboBio) and digoxigenin (DIG)-labeled RNA1 3’-EUTR probe (50 ng μL^-1) for hybridization. Approximately 200 bp of the RNA probe for 3’-EUTR of RNA1 was synthesized by a T7/SP6 RNA transcription kit (Roche). The gut was dissected from viruliferous fourth-instar nymphs and fixed in 4% (wt vol^-1) paraformaldehyde at 4°C overnight. After washing with PBST, the gut was digested with proteinase K (20 μg mL^-1; Tiangen) at 37°C for 15 min and hybridized with the miRNA probe and viral RNA probe at 37°C overnight. The midgut was washed with 0.2× SSC at 37°C to remove nonspecific binding. An anti-DIG alkaline phosphatase-conjugated antibody (1:500) and an anti-biotin antibody (1:100) were used for signal detection. Probes for cel-miR-67-3p and rice PsbP gene (KF460579) were used in the control group. The fluorescent signal of DIG or biotin was generated by HNPP/Fast Red or fluorescein-tyramide (Perkin-Elmer, Waltham, MA, USA) and recorded using a Leica TCS SP5 confocal microscope (Leica Microsystems). Primers used for probe synthesis are listed in S1 Table.

miRNA and circRNA probes were designed and synthetized at Shanghai GenePharma Co., Ltd. mRNA probes were prepared using an NTP labeling mixture and an SP6/T7 Transcription Kit (Roche, Basil, Switzerland). They were then broken into small fragments of about 250 bp by alkaline lysis. RNA fluorescence in situ hybridization (FISH) was carried out as per the manufacturer’s instructions and previous methods [74 (link)], with a FISH kit (Guangzhou RiboBio Co., Ltd.). Briefly, the adherent cells were prepared with 4% PFA and immersed in PBS containing 0.3% Triton X-100. The samples were hybridized with miR-novel-53 probe (10 pM) and ModSP probe (20 ng/μL) or circRNA probes (20 pM) at 37 °C approximately 12 h after treatment with proteinase K (25 g/mL) at 37 °C for 20 min and pre-hybridization for 5 h. Then, they were rinsed, in order, in 2 × SSC, 1 × SSC, and 0.2 × SSC at 37 °C. Mouse anti-digoxin antibody (1:150) and rabbit anti-biotin antibody (1:150) were used at 4 °C approximately 12 h for probe detection. The cells were then incubated with FITC-conjugated goat anti-rabbit and Alexa Fluor 594-conjugated goat anti-mouse (Bioss Antibodies, China) for 60 min in darkness. HNPP/Fast Red (Roche, Basil, Switzerland) or fluorescein-tyramide (Perkin Elmer, USA) was used to acquire the fluorescent signal. All images were captured with a LSM 710 (Carl Zeiss AG, Germany).

Tissue sections were subjected to antigen retrieval in citrate buffer pH6 and processed according to standard laboratory protocols. Sections were first incubated with mouse monoclonal ERβ5 (clone 5/25. BioRad, cat no. MCA4676T) diluted 1:200 in normal goat serum (NGS) overnight at 4°C, followed by goat anti-mouse peroxidase fab (Abcam) 1:500 in serum for 30 min at room temperature and finally incubated with Tyramide Fluorescein (PerkinElmer) at 1:50 in kit diluent for 10 min. Antibody elution was carried out by boiling sections in citrate buffer for 2.5 min followed by 30 min rest, incubated in NGS for 30 min at RT, blocked by streptavidin/biotin following manufacturer’s instructions (Vector, Peterborough, UK). Sections were washed and incubated with ERα mouse monoclonal (Vector, cat no. VP-E614) at 1:80 in NGS overnight at 4°C. Slides were incubated with goat anti-mouse biotinylated (Abcam) at 1:500 in serum for 30 min at RT, followed by Streptavidin Alexa fluor 546 (Molecular Probes) 1:200 in PBS for 1 h. Sections were washed, counterstained with DAPI (Sigma) at 1:1000 in PBS for 10 min before finally mounting in Permafluor (PerkinElmer). All washes between antibodies were carried out three times in TBS. Full details of antibodies used in the study are provided in Supplementary Table 2.