A protocol for immunofluorescence staining of floating neurospheres

Abstract This protocol describes the immunofluorescence staining of floating neurospheres in culture plates. Although this protocol is similar to conventional immunofluorescence staining, the staining procedure of floating neurospheres in multiwell culture plates and the washing procedure are different. Neurospheres in culture plates are transferred to a 12-well plate using a 200–1000 μL pipette. The spheres are precipitated by gravity for 3 min. Then, the 12-well plate is tilted slightly, and the culture medium is aspirated by the pipette. After aspiration, the spheres are visually verified to be at the bottom of the wells. PBS (400 μL) is added to the well for washing the spheres. This procedure is repeated three times. This protocol is easier than a conventional procedure using cryostat sections and can give clear sphere structures.