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

Indirect immunofluorescence assay of target protein in shrimp hemocytes. Hemolymph was collected from shrimp using a syringe that contained cold modified Alsever solution (Lin et al. 2002). Hemocytes were placed on glass coverslips, washed with PBS and fixed in 4% paraformaldehyde for 10 min at 4°C. After acetone treatment (3 min on ice), the hemocytes were incubated with 3% normal goat serum for 16 h at 4°C to prevent non-specific antibody adsorption. After this blocking, the hemocytes were incubated for 3 to 4 h at room temperature with a 1/500 dilution of target protein specific rabbit antiserum. Following 2 washes, each for 15 min in PBST (PBS containing 0.3% Tween-20), the cells were incubated for 1 h with a 1/200 dilution of fluorescein isothiocyanate (FITC)-conjugated polyclonal goat anti-rabbit IgG. Following extensive washing with PBST, the cells were mounted and viewed using an Olympus microscope. Then, 4’,6-diamidino-2-phenyl-indole (DAPI) was used to counter stain the nucleus.

References

1. Lin ST, Chang YS, Wang HC, Tzeng HF, Chang ZF, Lin JY, Wang CH, Lo CF, Kou GH. (2002) Ribonucleotide reductase of shrimp white spot syndrome virus (WSSV): expression and enzymatic activity in a baculovirus/insect cell system and WSSV-infected shrimp. Virology 304: 282-290.
2. Wang HC, Wang HC, Kou GH, Lo CF, Huang WP. (2007) Identification of icp11, the most highly expressed gene of shrimp white spot syndrome virus (WSSV). Dis Aquat Organ. 74: 179-189.

Some of our publications that have used this platform

1. Lin ST, Chang YS, Wang HC, Tzeng HF, Chang ZF, Lin JY, Wang CH, Lo CF, Kou GH. (2002) Ribonucleotide reductase of shrimp white spot syndrome virus (WSSV): expression and enzymatic activity in a baculovirus/insect cell system and WSSV-infected shrimp. Virology 304: 282-290.
2. Wang HC, Wang HC, Kou GH, Lo CF, Huang WP. (2007) Identification of icp11, the most highly expressed gene of shrimp white spot syndrome virus (WSSV). Dis Aquat Organ. 74: 179-189.