DNAse I pre-treatment markedly enhances detection of nuclear cyclin-dependent kinase inhibitor p57Kip2 and BrdU double immunostaining in embryonic rat brain.
As a member of the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs), p57Kip2 binds tightly to G1 cyclin/cyclin-dependent kinase complexes to block cell cycle progression. CKIs play critical roles in regulating the transition from proliferation to differentiation in many tissues, including the nervous system. Conversely, CKI dys-regulation contributes to neoplasia and cancer progression. While the combined detection of CKI immunoreactivity and S phase entry using bromodeoxyuridine (BrdU) incorporation may be particularly informative, successful immunostaining may be limited due to "masked" antigen epitopes and acid-induced signal degradation. We now report an improved double immunofluorescent method for detecting p57Kip2 and BrdU in paraformaldehyde-fixed frozen sections of embryonic rat brain. We substituted deoxyribonuclease I (DNAse I) for HCl pre-treatment to expose antigenic sites in frozen sections, and employed a biotinylated tyramide-based system to enhance p57Kip2 visualization. We identified a time- and dose-dependent relationship between DNAse I treatment and double labeling of p57Kip2 and BrdU, increasing both the numbers and intensities of immunopositive nuclei. With excess DNAse I treatment, however, there was signal degradation for both BrdU and total DNA, as reflected by DAPI staining. The use of DNAse I pre-treatment significantly increases the reliability and sensitivity of immunodetection of CKI nuclear factors, and should be useful for both developmental neurobiology studies as well as cancer diagnostic applications.
Ye
Weizhen
W
Department of Neuroscience and Cell Biology, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, RWJ SPH 362, Piscataway, NJ, 08854, USA, diciccem@umdnj.edu.
Mairet-Coello
Georges
G
DiCicco-Bloom
Emanuel
E
D000818:Animals; D001921:Brain / Q000166:cytology / Q000196:embryology*; D001923:Brain Chemistry; D001973:Bromodeoxyuridine / Q000032:analysis* / Q000302:isolation & purification; D050761:Cyclin-Dependent Kinase Inhibitor p57 / Q000032:analysis* / Q000302:isolation & purification; D003850:Deoxyribonuclease I / Q000378:metabolism*; D004622:Embryo, Mammalian; D005260:Female; D007150:Immunohistochemistry / Q000379:methods*; D011247:Pregnancy; D051381:Rats; D017207:Rats, Sprague-Dawley
D050761:Cyclin-Dependent Kinase Inhibitor p57; D003850:Deoxyribonuclease I; D001973:Bromodeoxyuridine