DNAse I pre-treatment markedly enhances detection of nuclear cyclin-dependent kinase inhibitor p57Kip2 and BrdU double immunostaining in embryonic rat brain

Abstract

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.

A 2-Step Laemmli and Antigen Retrieval Method Improves Immunodetection

“…29 A similar selective additive effect has been shown for DNAse treatment to enhance the detection of nuclear antigens. 30,31 We tried TCEP as a substitute for 2-ME and we showed that it can be an alternative which works at a much lower molar ratio than 2-ME. At 1 mM the cost is, however, 5-fold.…”

Cytoarchitecture and ultrastructure of neural stem cell niches and neurogenic complexes maintaining adult neurogenesis in the olfactory midbrain of spiny lobsters, Panulirus argus

“…To overcome the common problem that the pre-treatment of sections required for intense BrdU labeling severely compromises general nuclear staining (Tang et al, 2007), we developed a protocol based on the mild digestion of double-stranded DNA with DNase I (Ye et al, 2007). In this optimized triple-labeling protocol, free-floating sections were first incubated in 2 N HCl for 20 minutes and rinsed for 4 × 10 minutes in SPB.…”

Selection of buffer pH by the isoelectric point of the antigen for the efficient heat-induced epitope retrieval: re-appraisal for nuclear protein pathobiology

“…Therefore, we suggest that if the antigen is adjacent to the DNA-binding domain, it may be naturally exposed so that it can easily bind to antibody without being aVected by the HIER buVer pH value. For antigens containing DNA-binding domains, it may be eVective to use DNAse as pre-treatment in combination with HIER for IHC (Ye et al 2007). …”