Alexa fluor 647 labeled goat anti mouse igg

The haemolymph was collected from nine oysters and mixed with an anticoagulant solution (pH 7.4). The haemocytes were harvested by centrifugation at 600g for 10 min and resuspended in M-L15 medium at a concentration of 10⁶ cell/ml. The bacteria V. splendidus were grown in 2216E media at 28 °C with shaking at 160 rpm for 12 h to the mid-log phase and collected via centrifugation at 6000g for 10 min. The bacteria were treated with absolute formaldehyde for 10 min, washed with NaHCO3 (0.1 M, pH 9.0) four times, and incubated with FITC (1 mg/ml; Sigma) with continuously gentle stirring at 4 °C overnight. After four times of washing with PBS, the FITC-labeled V. splendidus were resuspended in PBS at a concentration of 2 × 10⁸ CFU/ml. Twenty microliters of FITC labeled V. splendidus was incubated with 1 ml of haemocytes in dark at room temperature with slight rotation for 0.5 h. The immunocytochemical assay of haemocytes was conducted as the previous description (58 ) with anti-CgC3 as the primary antibody and Alexa Fluor 647-labeled Goat Anti-Mouse IgG (Beyotime Biotechnology) as the secondary antibody. Fluorescence was observed by using confocal laser scanning microscope (LSM 800, ZEISS).