Hnpp fast red

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).

Plasmids containing the coding regions of OBP and CSP genes (see Supplementary Table S2) were linearized with Sac I or Sal I (TaKaRa, Dalian, China). Digoxigenin (DIG)-labeled and biotin-labeled RNA probes were synthesized using DIG RNA Labeling Kit (SP6/T7) and Biotin RNA Labeling Mix (Roche, Mannheim, Germany) following the protocols.
RNA in situ hybridization was performed as previously described^{43 (link)}. In brief, antennae were dissected and embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, USA). Antennal cryosections (12 μm) were collected on Superfrost Plus microscope slides (Fisher Scientific, USA). Sections were fixed at 4 °C and pre-treated at room temperature. After pre-hybridized, the sections were incubated with hybridization solution containing RNA probes at 60 °C overnight. Detection of DIG-labeled probes was performed by using an anti-Dig AP-conjugated antibody (Roche) in combination with HNPP/Fast Red (Roche); a streptavidin-HRP (Perkin Elmer, USA) and Fluorescein Tyramide (TSA, Perkin Elmer, USA) were used for simultaneous detection of biotin-labeled probes. The sections were analyzed using a Zeiss LSM 880 laser scanning microscope (Zeiss, Oberkochen, Germany) and images were processed with ZEN 2012 software.

Ccl2 and Cxcl1 were cloned from PCR products (550-bp and 504-bp amplicons respectively) using cDNA synthesised from retinal RNA (as described above). Digoxigenin (DIG)-labelled riboprobes were then prepared as described in our previous publication [13 (link)]. In situ hybridisation on retinal cryosections was carried out according to our established methodology [55 (link)]; briefly, each riboprobe was hybridised to sections overnight at 55 °C, then was washed in decreasing concentrations of saline sodium citrate (pH 7.4) at 60 °C. The bound probe was visualised with either NBT/BCIP or HNPP/Fast-Red (Roche Applied Science).