Cell cycle analysis in the rat external granular layer evaluated by several bromodeoxyuridine immunoperoxidase staining protocols

Abstract

An important step in bromodeoxyuridine (BrdU) immunohistochemistry is the production of single-stranded DNA to make the incorporated BrdU accessible to the antibodies. This paper examines the effect of distinct DNA denaturation pretreatments (DNase I, sodium citrate buffer, endonuclease Eco RI and exonuclease III, and HCl hydrolysis) on detection of BrdU. We found that all the methods used in the partial denaturation of DNA combined good nuclear immunostaining with acceptable tissue integrity. We also observed that these immunohistochemical protocols revealed a spatial pattern in the distribution of DNA-synthesizing cells within the cerebellar external granular layer (EGL) of 10-day-old rats, allowing us to estimate the fraction of S-phase cells. Our results indicate that detection of BrdU-stained cells is affected by the distinct histological procedures used in such detection. Additionally, as the duration and phases of the cell cycle in EGL neuroblasts are estimated in accordance with BrdU detection, an effect on this detection can render the measurement of cell cycle inaccurate. The present work shows that DNase I and citrate buffer, at appropriate conditions, may be good alternatives for acid denaturation. However, they are less sensitive than autoradiographic techniques that use H-thymidine administration. Finally, current data reveal that short survival times after a single BrdU exposure do not seem to affect the cell cycle progression of the EGL neuroblasts.

Administration of 5‐bromo‐2′‐deoxyuridine interferes with neuroblast proliferation and promotes apoptotic cell death in the rat cerebellar neuroepithelium

“…No prominent cytotoxic effects on the cerebellar neuroepithelium were found, which suggests that these doses are appropriate, at least in the acute situation, for tagging proliferative neuroblasts during the development of the central nervous system. These results are in line with previous reports, in which the cell cycle kinetics of neural progenitors in the embryonic cerebral wall (Takahashi, Nowakowski, & Caviness, 1995) and cerebellar external granular layer (Molina et al, 2017) of rodents were not altered by the administration of 50 μg/g of BrdU. Despite this, we cannot exclude a more protracted effect of BrdU on neuroepithelial cell biology, that is, cell differentiation and final fate.…”

“…The halopyrimidine 5‐bromo‐2′‐deoxyuridine (BrdU) is a chemically synthesized bromine‐tagged base analogue that is incorporated instead of thymidine into newly synthesized DNA during the S‐phase of the cell cycle (Lehner et al, 2011). Since the introduction of monoclonal antibodies against BrdU (Gratzner, 1982), an increasing number of methodologies have been used for the immunodetection of this nucleoside in the replicating DNA (Molina, Rodríguez‐Vázquez, Owen, Valero, & Martí, 2017). One of the advantages of the use of BrdU is that it has allowed to microscopically analyze the developmental timetables, fate and survival of different neuron types in the central nervous system (Lanctot et al, 2017; Martí, Santa‐Cruz, Serra, & Hervás, 2015; Martí, Santa‐Cruz, Serra, & Hervás, 2016; Taupin, 2007).…”

Drp1 is widely, yet heterogeneously, distributed in the mouse central nervous system

“…The neurons in this layer have important inhibitory effects [39]. In addition, Drp1 is also expressed in the second layer of the cerebral cortex, which is the external granular layer [40]. The difference in Drp1 protein expression in the cerebral cortex may be related to the diverse cell types in each layer.…”

Drp1 widely, yet heterogeneously distributes in mice central nervous system

“…The neuron in this layer is the important inhibitory neuron [21]. In addition, Drp1 is also expressed in the second layer of the cerebral cortex, which is the external granular layer [22]. Drp1 expression difference in cerebral cortex may be related to the diverse cell types of each layer.…”