Immunohistochemistry Techniques to Analyze Cellular Proliferation and Neurogenesis in Rats using the Thymidine Analog BrdU

Abstract

One of the most important things in the field of adult hippocampal neurogenesis (AHN) is the identification of the newly generated cells. The immunodetection of thymidine analogs (such as 5-Bromo-2'-deoxyuridine (BrdU)) is a standard technique used for visualizing these newly generated cells. Therefore, BrdU is usually injected in small animals intraperitoneally, so the thymidine analog gets incorporated into dividing cells during DNA synthesis. Detection is performed by immunohistochemical analysis of brain slices. Every research group that has been using this technique can appreciate that it requires special attention to minute details to achieve a successful stain. For instance, an important step is DNA denaturation with HCl, which allows it to reach the cell nucleus to stain it. However, the existing scientific reports describe very few of such steps in detail. Therefore, standardizing the technique is challenging for new laboratories as it can take several months to yield positive and successful outcomes. The purpose of this work is to describe and elaborate the steps to obtain positive and successful outcomes of the immunostaining technique in detail when working with the thymidine analog BrdU. The protocol includes the reagent preparation and setup, administration of thymidine analog in a rodent, transcardial perfusion, tissue preparation, peroxidase immunohistochemical reaction, use of avidin-biotin complex, immunofluorescence, counterstaining, microscopy imaging, and cell analysis.

Adult Neurogenesis: A Story Ranging from Controversial New Neurogenic Areas and Human Adult Neurogenesis to Molecular Regulation

“…The controversial results and lack of clear and compelling evidence were likely obtained because the generation of new neurons may be very low under basal conditions and/or due to technical problems of the time of sampling/analysis. The first technique developed to detect newly generated neurons in the adult brain consisted of intracranially injecting tritiated thymidine (thymidine-3 H) in rats and detecting cells in the cell cycle by reviewing autoradiograms [52,262]. Then, in approximately 1988, the BrdU immunohistochemistry technique was developed by Miller and Nowakowski.…”

“…In studies with human tissue, this issue becomes even more important and critical, and might explain many of the discrepancies or differences between studies. Protocol optimization for the detection of neurogenesis is not a straightforward task (see [52]). A few months later, a new result that undoubtedly confirmed persistent neurogenesis in humans was published [4].…”

“…A study performed by Lee et al sought to determine whether corticosterone suppressed cell proliferation in the SVZ and whether this effect was reversed by voluntary exercise [45]. The authors found that rats treated with chronic corticosterone (4 weeks) exhibited decreased cell proliferation (indicated by immunodetection of the thymidine analog 5-bromo-2 -deoxyuridine (BrdU), which is a standard technique used to visualize these newly generated cells) [52] in the SVZ, whereas rats that were only subjected to voluntary exercise exhibited increased cell proliferation in this neurogenic niche. Furthermore, rats receiving both treatments (chronic corticosterone and simultaneous wheel running) had approximately the same number of BrdU-labeled cells as those in the control group; additionally, these BrdU-labeled cells expressed doublecortin (DCX), a migrating neuroblast marker.…”

A novel NMDA receptor modulator: the antidepressant effect and mechanism of GW043

“…The experimental procedure is as described previously. 34 BrdU powder was dissolved in 0.9% saline and injected intraperitoneally with 50 mg/kg at 12 h intervals 3 days before execution. 1 d after the last BrdU injection, animals were anesthetized with an overdose of sodium pentobarbital and then perfused intracardially with 0.9% saline and 4% paraformaldehyde.…”

Early Consumption of Cannabinoids: From Adult Neurogenesis to Behavior

“…In a more refined approach, chronic oral treatment with VCE-003.2 (a cannabigerol-derived cannabinoid acting through PPARγ) improved NSC mobilization and subventricular neurogenesis in mice analyzed by double-labeled BrdU+ and NeuN+ cells in response to mutant huntingtin-induced striatal neurodegeneration [ 81 ]. In this respect, 5-bromo-2′-deoxyuridine (BrdU) is a thymidine analog used as a standard technique for cellular proliferation visualization [ 82 ], and NeuN is a neuronal nuclei protein widely accepted as a specific cell marker for mature neurons ( Figure 2 ). Similarly, the use of the phytocannabinoid CBD has been useful for studying the effect of the ECS on the production of new neurons.…”