Carlzeiss lsm710 system

The hemocytes of mud crab were seeded onto the polysine-coated cover slips, fixed with 4% polyformaldehyde for 15 min at room temperature. After that, the . CC-BY 4.0 International license perpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for this this version posted February 5, 2020. ; https://doi.org/10.1101/2020.02.03.932970 doi: bioRxiv preprint cover slips were dehydrated in 70% ethanol overnight at 4°C，followed by incubation with hybridization buffer [1× SSC (15 mM sodium citrate, 150 mM sodium chloride, pH 7.5), 10% (w/v) dextran sulfate, 25% (w/v) formamide, 1× Denhardt's solution] containing 100 nM probe for 5 h at 37°C. The miR-137 probe (5'-FAM-ACGTGTATTCTCAAGCAATAA-3'), miR-7847 probe (5'-FAM-AATCCTCCTACT CCTCCAG-3') and AIF probe (5'-Cy3-TCCATCTTCTGTACTCTTGACT-3') were used. Then the slips were washed with PBS for three times, and after that the hemocytes were stained with DAPI (4', 6-diamidino-2-phenylindole) (50 ng/ml) (Sigma, USA) for 5 min [55] . The images were captured using a CarlZeiss LSM710 system (Carl Zeiss, Germany).

The hemocytes were seeded onto polysine-coated coverslips and then fixed with 4% polyformaldehyde for 15 min at room temperature. Followed by dehydrated in 70% ethanol at 4 °C overnight. After that, the coverslips were incubated with hybridization buffer [1× SSC (15 mM sodium citrate, 150 mM sodium chloride, pH 7.5), 10% (w/v) dextran sulfate, 25% (w/v) formamide, 1× Denhardt's solution] containing 100 nM of probe at 37 °C for 5 h. The probe used is listed below, miR-2 probe (5'-FAM-ATACAACAGCCACTTTGTGAG-3'), Caspase 2 mRNA probe (5'-Cy3-TCCAGCA AGAGACTTGCACTGA-3'). Subsequently, the slips were washed with PBS three times and stained with DAPI (4ʹ, 6-diamidino-2-phenylindole) (50 ng/mL) (Sigma, USA) for 5 min, and observed by CarlZeiss LSM710 system (Carl Zeiss, Germany).