A protocol for immunofluorescence staining of floating neurospheres.
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 200-1000microL pipette. The spheres are precipitated by gravity for 3min. 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 (400microL) 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.
Sasaki
Ryo
R
Department of Oral and Maxillofacial Surgery, and Global Center of Excellence (COE) Program, Tokyo Women's Medical University, School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. sasaki@oms.twmu.ac.jp
Aoki
Shunsuke
S
Yamato
Masayuki
M
Uchiyama
Hiroto
H
Wada
Keiji
K
Ogiuchi
Hideki
H
Okano
Teruo
T
Ando
Tomohiro
T
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D001562:Benzimidazoles; D002478:Cells, Cultured; D003782:Dental Pulp / Q000166:cytology* / Q000294:innervation*; D005455:Fluorescent Antibody Technique / Q000379:methods*; D005456:Fluorescent Dyes; D007381:Intermediate Filament Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D064231:Nestin; D051381:Rats; D013194:Staining and Labeling / Q000379:methods; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D060225:Tetraspanin 28
D015703:Antigens, CD; D001562:Benzimidazoles; D005456:Fluorescent Dyes; D007381:Intermediate Filament Proteins; D009419:Nerve Tissue Proteins; C577486:Nes protein, rat; D064231:Nestin; D060225:Tetraspanin 28; C017807:bisbenzimide ethoxide trihydrochloride