During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness.
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Method Article
Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis
* These authors contributed equally
In This Article
Summary
Abstract
Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.
Introduction
Neurogenesis, the generation of new neurons from neural stem cells, is now known to constantly occur into adulthood in two specialized regions in the brain. These include the subventricular zone (SVZ) of the olfactory bulb and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus. Over the past decade, the field of adult hippocampal neurogenesis has seen significant work. The region has attracted much interest since newborn neurons in the SGZ contribute to enhanced neural plasticity that can sustain specific brain functions1-5. Adult hippocampal neurogenesis has been implicated to play important roles in mood regulation, regeneration, ....
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Protocol
1. Pre-immunohistochemistry: Perfusion and Histology
- Perfuse mice as per a general transcardial protocol adapted for mice and extract the brains into 4% paraformaldehyde (PFA)9.
- Leave the brains in PFA overnight at 4 °C (e.g. a 10 ml conical tube filled with PFA). Keep in PFA for at least 12-24 hr, no more than 36 hr for full fixation. After overnight fixation, empty out PFA from conical tube but leave brains inside. Pour 30% sucrose into tube and wash the brains for a brief 5 sec to remove any residual PFA. Finally, fill tube containing brains with 30% sucrose again. Keep brains in sucrose at 4 °C until brains sink ....
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Results
Adult hippocampal neurogenesis is a sequential process where the representative progenitor cell type at each stage may be identified with a combination of markers. Careful chemical fixation and antigen-retrieval improves the effectiveness and accessibility of markers that are otherwise difficult to detect. One of these is nestin which is expressed among the radial glia-like neural stem cells and some early active progenitor cell types. Nestin can be a key protein in identifying early progenitor cell types as its expressi.......
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Discussion
The most critical steps for successful antigen-retrieval and staining of progenitor cell types are: 1) utilizing well perfused and fixed tissue of the optimal coronal thickness; 2) allowing sufficient time for boiling and subsequent cooling during antigen retrieval; 3) manual dexterity in mounting fixed coronal sections and preventing their damage when doing so.
It is critical in IHC to use perfused tissue that has undergone sufficient fixation but not over-fixation. A primary difficulty can b.......
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Disclosures
The authors declare that they have no competing financial interests.
Acknowledgements
The methodology was originally developed in Dr. Hongjun Song's laboratory at the Institute for Cell Engineering, Department of Neurology, Johns Hopkins School of Medicine. This work was funded by NIMH (R00MH090115), NARSAD, the Fraternal Order of Eagles' Mayo Cancer Research Fund and a start-up package from Mayo foundation awarded to M.H.J. and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (A3014385) awarded to W.R.K.
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| SuperFrost Plus Slide | Fisherbrand | 12-550-15 | Provides strong adherence to hippocampal tissue during boiling |
| PVA/DABCO Mounting Media | Sigma Aldrich | 10981-100 ml | |
| Nestin (chicken) | Aves Lab | NES | Neural stem cell marker |
| GFAP (rabbit) | Dako North America | Z033401-2 | Astrocyte and neural stem cell marker |
| MCM2 (mouse) | BD Transduction Laboratory | 610701 | Proliferation marker |
| DCX (goat) | Santa Cruz Biotechnology | SC8066 | Immature neuron marker |
| Tbr2 (rabbit) | Abcam | Ab23345 | Amplifying progenitor marker |
| NeuN (mouse) | Millipore | MAB377 | Mature neuron marker |
| Cy2 (anti-rabbit) | Jackson Immunoresearch | 111-226-047 | |
| Cy2 (anti-chicken) | Jackson Immunoresearch | 303-165-006 | |
| Cy5 (anti-mouse) | Jackson Immunoresearch | 315-175-047 | |
| Cy5 (anti-goat) | Jackson Immunoresearch | 305-165-047 | |
| DAPI | Life Technologies Corporation | D1306 | |
| Dako Pen | S2002 | Dako | Water-repellant pen |
References
- Ming, G. L., Song, H. Adult neurogenesis in the mammalian central nervous system. Annual Reviews of Neuroscience. 28, 223-250 (2005).
- Santarelli, L., Saxe, M., et al. Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants....
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