Alexa flour 488 labeled goat anti mouse igg

NP cells were rinsed with PBS for three times, fixed with 4% formaldehyde for 15 min, and permeabilized with 0.3% Triton-X100 (Sigma, CO, USA) for 10 min. Subsequently, the cells were blocked with 6% BSA for 1 h and incubated with primary antibody against Ki67 (dilution, 1:200; Rabbit polyclonal antibody, Abcam, Shanghai, China) overnight at 4 °C. Then, cells were incubated with Alexa Flour 488 labeled goat anti-mouse IgG (Invitrogen 1:200; Invitrogen, OR, USA) for 1 h at room temperature. Cells were analyzed using the Olympus DP72 fluorescence microscope (Olympus, Tokyo, Japan).

We crossed Gal4 or Gal4-Gal80 transgenic flies with UAS-mCD8-GFP flies to produce progeny expressing GFP in peptidergic neurons and used them for GFP immunohistochemical staining. CNS of prepupae were dissected in phosphate buffered saline (PBS) and fixed in 4% paraformaldehyde in PBS overnight in 4°C, washed with PBS containing 0.2% Triton X-100 (PBST), and incubated in 5% normal goat serum in PBST for 30 minutes at room temp. They were incubated in PBST with primary mouse antibody to GFP (Invitrogen) for 2 days at 4°C. Tissues were then washed with PBST and incubated with Alexa Flour 488-labeled goat anti-mouse IgG (Invitrogen). GFP expression was observed under a confocal microscope (Leica model SP2) with FITC filter.