Alexa fluor 488 labeled goat anti mouse igg

Tongue sole head kidney was surgically removed and passed through a nylon mesh with L15 medium (Gibco, Grand Island, NY, USA). The cell suspension was then placed on the top of a 61% Percoll (Pharmacia, Uppsala, Sweden) gradient and centrifuged at 4000 rpm for 10 min at 4 °C. Leukocytes were collected from the interphase, and the remaining red blood cells were removed as previously reported [47 (link)]. The cell viability of head kidney leukocytes was measured using the trypan blue (Sigma) exclusion assay, and the percentage of live cells was more than 95% of the total leukocytes. The cells (1 × 10⁶ leukocytes) were incubated with 5% bovine serum albumin (BSA) at 22 °C for 1 h followed by PBS washing for three times. Antibody against rCsCD209 (1:1000 dilution) was added to the cells, followed by incubation at 22 °C for 1 h. After washing three times with PBS, the cells were incubated with Alexa Fluor 488-labeled Goat anti-mouse IgG (Abcam, 1:1000 dilution) at 22 °C for 1 h. The cells were washed as above and determined for fluorescence intensity with a FACSAria II flow cytometer (BD Biosciences, Heidelberg, Germany). The experiment was performed in triplicate.

Cells were fixed, penetrated with 0.01% Triton X-100, and blocked with 5% goat serum for 30 min. After that, the cells were reacted with anti-PTEN (MA5-12278, 1:2,000, Thermo Fisher Scientific) and anti-SP1 (1:100, sc-420, Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA) overnight at 4°C, and then with Alexa Fluor® 488-labeled goat anti-mouse IgG (1:200, ab150113, Abcam) at 22–25°C for 1.5 h. The nuclei were counter stained by 4’, 6-diamidino-2-phenylindole. The cell slides were then sealed for observation under the fluorescence microscope [32 (link)].