Dapi dihydrochloride

The hemocytes collected from oysters were resuspended in L15 cell culture media (extra addition of 20.2 g/L NaCl, 0.54 g/L KCl, 0.6 g/L CaCl, 3.9 g/L MgCl₂, and 1 g/L MgSO₄) and deposited on dishes pre-coated with poly-L-lysine, and then incubated at 37°C for 1 h to adhere to the glass slides. The hemocytes on the object slides were fixed with 4% paraformaldehyde, washed with PBS, and permeabilized with 1% Triton X-100 for 5 min. After three times of PBS wash, the hemocytes were blocked with 3% (w/v) fetal bovine serum albumin (BSA) at 37°C for 30 min and incubated with the antibody [anti-CgIRF-8, anti-CgcGAS, 1:500 (v/v) in 3% BSA] at 37°C for 1 h. Then the samples were washed with PBS and incubated with Alexa Fluer 488-labeled goat-anti-mouse antibody (Solarbio life sciences, China) diluted at 1:1,000 (v/v) with 3% BSA at 37°C in the dark for 1 h. The DAPI dihydrochloride (1 mg/ml in PBS; Solarbio Life Sciences, China) was added to stain the nucleus for 5 min followed by three times wash with PBS. Fluorescence was observed using Carl Zeiss Axio Imager A2 microscope (Carl Zeiss, Germany).

Formalin-fixed, paraffin-embedded ovarian specimens were sectioned at five μm. After dewaxing, antigens were retrieved by pressure cooking in sodium citrate buffer at 90 °C for 40 min. Sections were blocked for 30 min in 3% diluted in phosphate-buffered saline (PBS; pH7.4). The immunoreactions were performed with the TRKB (Novus biologicals, Bio-Techne, USA) antibody (10 μg/ml) and DDX4 (Abcam, USA) antibody (1:100) simultaneously. Slides incubated in the absence of primary antibodies were used as a negative control. After an overnight incubation at 4 °C, the primary antibody was detected using fluorochrome-tagged secondary antibodies by incubating the sections with Alexa Fluor 488 Goat Anti-Mouse (Abcam, USA; 1:400) and Cy™3 AffiniPure Donkey Anti-Goat IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA; 1:400) for 1 h at room temperature. Cell nuclei were stained with DAPI dihydrochloride (Solarbio Life Sciences, China). Confocal images were acquired using a Leica Corp. TCS SP8 confocal system (Leica Corp. Microsystems, Heidelberg, Germany). In general, six to eight representative sections were acquired for each image. Images were further processed using Photoshop.