A method for pulse and chase BrdU-labeling of early chick embryos.
Pulse and chase BrdU labeling of early chick embryos represents a serious technical problem due to the hindrance of removing unincorporated BrdU after the pulse. We have developed a simple method that allows BrdU washout and control of pulse/chase duration. In this method, BrdU pulses are carried out in ovo. Afterwards, embryos are removed from the yolk, BrdU is washed out, and the embryos are maintained in a wholemount culture. Under these conditions, HH8-12 embryos continue with their normal development for at least 30 h. Morphological development of the nervous system and cell cycle kinetics of precursor cells seem to be normally maintained in cultured embryos. To prove the feasibility of the method, it has been applied to determine the onset of TUJ1 expression. TUJ1 is frequently considered an early neuronal marker, yet some reports have shown its expression in dividing progenitor cells and differentiating neurons. The application of this new method demonstrates that TUJ1 is expressed in newborn neurons as early as 1 h after cell cycle exit.
Hämmerle
B
B
Unidad de Neurobiologia del Desarrollo, Universidad Miguel Hernandez y CSIC, Campus de San Juan, 03550, Alicante, Spain.
Tejedor
F J
FJ
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D001973:Bromodeoxyuridine / Q000008:administration & dosage / Q000493:pharmacokinetics*; D002453:Cell Cycle; D002642:Chick Embryo; D004848:Epithelium / Q000196:embryology / Q000378:metabolism; D005240:Feasibility Studies; D007150:Immunohistochemistry / Q000379:methods*; D066298:In Vitro Techniques; D009474:Neurons / Q000378:metabolism; D020033:Protein Isoforms; D013116:Spinal Cord / Q000166:cytology* / Q000196:embryology* / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods*; D014404:Tubulin / Q000032:analysis / Q000378:metabolism*
D015415:Biomarkers; D020033:Protein Isoforms; D014404:Tubulin; D001973:Bromodeoxyuridine