4 6 diamidino 2 phenylindole dihydrochloride dapi

Hemolymph from normal or challenged shrimp was fixed with a 1 ml mixture of anticoagulant (pH 7.4) and 4% paraformaldehyde (1:1) and then centrifuged at 600 × g for 3 min at 4 °C. Collected hemocytes were washed three times with PBS and then deposited on a glass slide, washed with PBS and then incubated in 0.2% triton at 37 °C for 5 min. After washing with PBS, the hemocytes on the glass slide were blocked with 3% BSA (30 min, 37 °C), and incubated with an antibody recognizing phosphorylated Stat (anti-p-Stat) (1:400 in 3% BSA) overnight at 4 °C. On the second day, the glass slide with the hemocytes was washed with PBS, and incubated with 3% BSA for 10 min, and then with Alexa fluor 488-conjugated anti-rabbit secondary antibody (1:800 ratio, diluted in 3% BSA) for 1 h at 37 °C in the dark. After washing six times, the hemocytes were incubated with 4′-6-diamidino-2-phenylindole dihydrochloride (DAPI, AnaSpec Inc., San Jose, CA, USA; 1 μg/ml in PBS) for 10 min at room temperature and then washed six times. Hemocytes on the slide were observed under an Olympus BX51 fluorescence microscope (Shinjuku-ku, Tokyo, Japan).

Hemolymph was collected from three shrimps (10 g per shrimp) using a 5 ml syringe preloaded with 1 ml of anticoagulant (0.45 M NaCl, 10 mM KCl, 10 mM EDTA, and 10 mM HEPES, pH 7.45) and then fixed by adding 1 ml 4% paraformaldehyde. Hemocytes were collected by centrifugation at 700 ×g for 5 min at 4°C, and then washed with PBS for three times, incubated in 0.2% Triton X-100 at 37°C for 5 min, washed with PBS five times, and then blocked by 2% bovine serum albumin (BSA, dissolved in PBS) at 37°C for 30 min. Then, anti-Dorsal (1:200 in blocking regent) was added and samples were incubated overnight at 4°C. After washing with PBS, the hemocytes were incubated with 2% BSA for 10 min, then the second antibody (goat anti-rabbit-Alexa Fluor 488, 1:1,000 diluted in 3% BSA) was added, and samples were incubated for 1 h at 37°C and washed with PBS. Hemocyte nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI, AnaSpec Inc., San Jose, CA) for 10 min at room temperature and washed again. The results were observed under a fluorescence microscope (Olympus BX51, Tokyo, Japan) and ImageJ was used to calculate the colocalization percentage of Dorsal with nuclei stained with DAPI.