Fitc conjugated goat anti mouse ig

The FG and HINAE cells were seeded on the cover slips according to the method described by Morel et al. [20 (link)]. Briefly, acid etched circular cover slips were kept in 24-well plates, and 10⁴ cells per well were seeded and incubated to allow cells to attach to the cover slips. After 24 h incubation, the media was removed and the wells were carefully washed with PBS. Then cells were inoculated with 4 TCID₅₀/ml LCDV at 22°C for 2 h. After three washes with PBS, cells were fixed with 4% paraformaldehyde (Sangon Biotech, China) at 22°C for 30 min, followed by incubation overnight with anti-27.8R MAbs (1:5000, 3D9: 2G11 = 1:1, v/v) and rabbit anti-LCDV serum (1:500) at 4°C in a moisture chamber. After washing three times with PBS, the cells were incubated for 1 h at 37°C in the dark with fluorescein isothiocyanate (FITC)-conjugated goat-anti-mouse Ig (Sigma) and Cy3-labeled goat-anti-rabbit (Sigma) at a dilution ratio of 1: 256 in PBS in a moisture chamber. 4, 6-diamidino-2-phenylindole (DAPI, Roche) staining (blue) was used to visualize cell nuclei. Slides were rinsed again, and then mounted with 90% glycerin and observed under a fluorescence microscope.

The leucocytes (1.0 × 10⁷ cells/mL) were incubated with IgM MAb 2D8, rabbit polyclonal antibodies against CD4-1, CD4-2, and CD8β for 1.5 h at 37 °C. After washing three times with PBS containing 5% (v/v) newborn calf serum, the cells were incubated with FITC-conjugated goat-anti-mouse Ig (1:256 diluted in PBS, Sigma) for 45 min at 37 °C and washed again. The percentages of the CD4-1⁺, CD4-2⁺, CD8β⁺ T lymphocytes and sIgM⁺ lymphocytes in PBLs, SPLs and HKLs were analyzed with Accuri C6 cytometer (BD Accuri, Piscataway, NJ, USA). The myeloma culture supernatant instead of IgM MAb 2D8 and rabbit negative serum instead of polyclonal antibodies were used as negative controls.

The 27.8R localization and LCDV antigens were detected in peripheral blood cells of turbot at 3 h p.i. Blood samples were averagely divided into two groups, group one was directly diluted in PBS to 10⁶ cells/mL, and group two was centrifuged at 100× g for 20 min at 4 °C and the red blood cell pellet was re-suspended and diluted in PBS to 10⁶ cells/mL. The whole blood cells and red blood cells were then settled on glass slides for 2 h, and fixed for 20 min at 22 °C with 4% paraformaldehyde. For immune detection of 27.8R localization and LCDV antigens, both blood cell smears were incubated with MAbs against 27.8R (1:1000) and MAb 1A8 against LCDV (1:1000), respectively. Incubating with MAb 1D5 against WSSV, instead of anti-27.8R MAbs and anti-LCDV MAb 1A8, acted as the negative control. After washing three times with PBS, the slides were incubated with FITC-conjugated goat-anti-mouse Ig (1:256, Sigma, St. Louis, MO, USA) for 1 h at 37 °C in the dark. DAPI nuclear staining is shown in blue. After three washes with PBS, slides were mounted with 90% glycerin before observation under a fluorescence microscope.