Dig oligonucleotide labeling kit

The fragments of Spot/vp (310bp) and Spot/vpr-like (288bp) sequences were cloned into pGEM-T easy vector (Promega, Madison, WI, USA). The linear DNAs, amplified by PCR, were used as templates for riboprobes construction using DIG-Oligonucleotide Labeling Kit (Roche Molecular Biochemicals, Mannheim, Germany). Cerebral ganglia, hepatopancreas and ovaries at early vitellogenic stage were dissected and prepared for the paraffin-cut section. The serial seven-micron sections were used for Hematoxylin-eosin (H&E) staining and in situ hybridisation [64 (link)], visualised by the BCIP/NBT Chromogen Kit (Solarbio, Beijing, China) and mounted in Clear-Mount. All sections were observed and photographed by fluorescence confocal microscope (version, Axio Imager A2) equipped with digital camera (version, AxioCam MRc) (Carl Zeiss, Jena, Germany). Three technical replicates were performed.

The spatial distribution of PGESs expression in the ovarian tissue sections was detected by ISH. Briefly, the ovaries were dissected out and fixed in fresh 4% paraformadehyde fixative in 0.1M PBS, pH 7.4 at 4 °C overnight. Then the tissues were processed by routine paraffin method. Paraffin embedded blocks were sectioned at 6 μm thickness. MroPGES2 gene was PCR-amplified with M13 primers using the plasmid containing the MroPGES2 gene as a template (forward; 5′ GTAAAACGACGGCCAGT 3′ and reverse primer; 5′ AACAGCTATGACCATG 3′). The PCR products were processed through separation and extraction using QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany) and used as a template for riboprobe synthesis using a DIG-oligonucleotide labeling kit (Roche, Germany). The in situ hybridization was performed following the previous described protocol (Thongbuakaew et al., 2019 ). The stained sections were observed and photographed under Nikon E600 microscope equipped with a DXM1200F digital microscope (Nikon, Tokyo, Japan).