Immunofluorescent assays of proteins in L. vannamei hemocytes

Description

Indirect immunofluorescence is a useful technique for determining the cellular localization of proteins in L. vannamei hemocytes. Application include the study of 1.cellular adhesion 2. interactions between hemocyte receptors and their ligands. 3. protein translocation within the cell in response of specific stimulation.

Brief protocol

Preparation of the dsRNA, the PCR-generated partial DNA fragments of target genes were used as linearizing DNA templates for single-stranded RNA (ssRNA) synthesis. The T7 promoter sequence was incorporated into the DNA by using appropriate primer. After transcribing the ssRNAs in vitro by using the T7 RiboMAX™ Express Large Scale RNA System (Promega, USA), complementary ssRNA strands were mixed and annealed to dsRNAs by incubation at 70 °C for 20 min followed by gradual cooling to room temperature for over 30 min. The dsRNAs were purified by phenol/chloroform/isoamyl alcohol extraction according to the Promega instruction manual. After purification, dsRNAs were verified by agarose gel electrophoresis, quantified by UV spectrophotometry and stored at −80 °C for further in vivo experiments. For the gene knockdown experiments, shrimps in the experimental groups (mean body weight 10 g/shrimp) were injected with dsRNA (1 μg/g shrimp) by intramuscular injection. To determine the earliest time point of maximal silencing, samples from the hemolymph, lymphoid organ and heart were taken from 3 shrimps in each treatment protocol at 0.25. 1, 3, 7 and 10 day post dsRNA injection. Total RNA was extracted from these samples and reverse-transcribed to cDNA using Superscriptase II Reverse Transcriptase (Invitrogen) and anchor-dTv primer. The efficiency of the gene knockdown was checked using both the reverse transcription polymerase chain reaction (RT-PCR). The optimum time point of gene silencing was found to be at 3 days after dsRNA injection.

References

1. Maningas MB, Kondo H, Hirono I, Saito-Taki T, Aoki T. (2008) Essential function of transglutaminase and clotting protein in shrimp immunity. Mol Immunol 45: 1269-1275.
2. Wang KC, Kondo H, Hirono I, Aoki T. (2010) The Marsupenaeus japonicus voltage-dependent anion channel (MjVDAC) protein is involved in white spot syndrome virus (WSSV) pathogenesis. Fish & Shellfish Immunology 29: 94-103.

Some of our publications that have used this platform

1. Wang KC, Kondo H, Hirono I, Aoki T. (2010) The Marsupenaeus japonicus voltage-dependent anion channel (MjVDAC) protein is involved in white spot syndrome virus (WSSV) pathogenesis. Fish & Shellfish Immunology 29: 94-103.