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.

1. Pre-immunohistochemistry: Perfusion and Histology
<ol>
	<li>Perfuse mice as per a general transcardial protocol adapted for mice and extract the brains into 4% paraformaldehyde (PFA)9.</li>
	<li>Leave the brains in PFA overnight at 4 &#176;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 &#176;C until brains sink to the bottom of tube.</li>
	<li>Cut 40 &#956;m brain sections on a microtome. Consult a mouse brain atlas to start collecting sections from the beginning of the dentate gyrus till its end. Preserve sections in anti-freeze solution (a simple recipe is 300 g sucrose in 500 ml 0.1 M PBS and 300 ml ethylene glycol) until ready for immunohistochemistry.</li>
	<li>Mount the cut tissue on microscope slides specifically designed to adhere strongly to tissue. These slides provide a strong hold to tissue during the boiling stage. After mounting the tissue, please let the slide dry for 10 min or until it is fully dry. Drying can be expedited if the slide is rested against a vertical surface with the slide&#39;s bottom edge on a paper towel. If more than one slide is being mounted, the other slide(s) may be left dry during that time (1-2 hr is okay) until ready to proceed to the next step.</li>
	<li>Once slide is dry, wash with PBS three times for 5 min each and dry again (PBS buffer = 0.137 M NaCl, 0.0027 M KCl, 0.0119 M Na2PO4). This ensures any previous chemical (such as glycerol from anti-freeze storage solution) is sufficiently removed and cannot impair the tissue&#39;s adherence to the slide.</li>
</ol>
2. Preparation of Reagents and Minor Equipment
<ol>
	<li>Prepare Solution A (0.1 M or 19.21 g/l citric acid) and Solution B (0.1 M or 24.9 g/l tris-sodium citrate) in which tissue sections will be boiled.</li>
	<li>In a graduated cylinder, combine 9 ml of Solution A and 41 ml of Solution B. Add 450 ml of ddH2O to this mixture.</li>
	<li>Pour this mixture into an empty container (e.g. an empty pipette tips box) that is microwave safe.</li>
</ol>
3. Heat-induced Antigen Retrieval
<ol>
	<li>Boil solution made in step 2 for 5 min at standard setting in a standard microwave. The solution will start boiling at or above 100 &#176;C.</li>
	<li>Once boiling is complete, carefully remove container from microwave and place dried slides into it, ensuring the slide-face with hippocampal sections is facing down and fully exposed to the liquid. If possible, place slides at an angle against the walls of the box and stack other slides around them. Ensure their &#34;fit&#34; inside the box is tight so the slides have no room to move on top of each other.</li>
	<li>Boil this mixture with slides for 7 min at standard settings in the microwave. The solution will boil during this time at or above 100 &#176;C.</li>
	<li>During this boiling stage, fill two ice buckets halfway with ice.</li>
	<li>Once boiling in step 3.3 is complete, remove the container from the microwave and place it inside one of the ice buckets. Pour the rest of the ice from the other bucket all around the container and completely cover it. Let it sit for 1 hr.</li>
</ol>
4. Primary and Secondary Antibody Staining
<ol>
	<li>At the end of the 1 hr waiting period, remove the slides from the container and wash with TBS-T buffer 3x for 5 min each. Any apparatus may be used to perform these basic washes. (Buffer TBS = 50 mM Tris HCl, 150 mM NaCl, TBS-T = 0.05% Triton X-100 TBS buffer, TBS-TT = 4% donkey serum TBS-T buffer).</li>
	<li>After washing, let the slides dry in a dark place (e.g. inside a bench drawer, place a paper towel inside the drawer and tilt the slide against it) for 5-10 min or until slide is fully dry. During this period, prepare a chamber for overnight primary antibody staining. A simple staining chamber may include a container with large surface area filled with ddH2O and a stage inside the container, on top of which the slide may be rested above the ddH2O.</li>
	<li>Once the slide has dried, draw an outline around the tissue sections using a water-repellant pen. During step 4.5, this outline acts as a barrier preventing the antibody mixture from flowing over.</li>
	<li>Prepare the primary antibody mixture in TBS-TT. At least 500 &#956;l is required to cover one entire slide for instance, if antibody stock is at 1:500 concentration, add 1 &#956;l of particular antibody to 500 &#956;l of TBS-TT). Utilize primary antibodies as mentioned in the reagents table.</li>
	<li>Place the dried slide on top of the stage in the staining chamber. Add 500 &#956;l of the primary antibodies slowly and cover entire surface. Cover entire apparatus and wrap in aluminum foil to block external light. Let it sit overnight at room temperature.</li>
	<li>The next day, wash the slide three times for 5 min each with TBS-T buffer. Let it dry in a dark area as described in step 4.2.</li>
	<li>Prepare secondary antibody mixture similar to step 4.4. Refer to reagents table for secondary antibodies.</li>
	<li>Place the dried slide in the staining chamber as described in steps 4.2 and 4.5. Add 500 &#956;l of the secondary antibodies slowly and cover entire surface. Wrap the staining chamber in aluminum foil to protect it from external light. Let it sit covered and protected from external light for at least 2 hr at room temperature.</li>
	<li>After completion of secondary staining, wash the slide three times for 5 min each with TBS-T buffer. Let the slide dry in a dark area. After drying, put mounting solution and cover the slide with a cover slip carefully preventing any introduction of bubbles. Let the slide sit in a dark area for at least 2 hr before subsequent analysis.</li>
	<li>Perform time-lapse or confocal analysis on the stained tissue.</li>
</ol>

<ol>
	<li>Ming, G. L., Song, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adult+neurogenesis+in+the+mammalian+central+nervous+system.">Adult neurogenesis in the mammalian central nervous system.</a> Annual Reviews of Neuroscience. 28, 223-250 (2005).</li><li>Santarelli, L., Saxe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+hippocampal+neurogenesis+for+the+behavioral+effects+of+antidepressants.">Requirement of hippocampal neurogenesis for the behavioral effects of antidepressants.</a> Science. 301 (5634), 805-809 (2003).</li><li>Sahay, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increasing+adult+hippocampal+neurogenesis+is+sufficient+to+improve+pattern+separation.">Increasing adult hippocampal neurogenesis is sufficient to improve pattern separation.</a> Nature. 472 (7344), 466-4670 (2011).</li><li>Clelland, C. D., Choi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+role+for+adult+hippocampal+neurogenesis+in+spatial+pattern+separation.">A functional role for adult hippocampal neurogenesis in spatial pattern separation.</a> Science. 325 (5937), 210-213 (2009).</li><li>Wang, J. W., David, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+fluoxetine+stimulates+maturation+and+synaptic+plasticity+of+adult-born+hippocampal+granule+cells.">Chronic fluoxetine stimulates maturation and synaptic plasticity of adult-born hippocampal granule cells.</a> Journal of Neuroscience. 28 (6), 1374-1384 (2008).</li><li>D'Amico, F., Skarmoutsou, E., Stivala, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State+of+the+art+in+antigen+retrieval+for+immunohistochemistry.">State of the art in antigen retrieval for immunohistochemistry.</a> Journal of Immunological Methods. 341 (1-2), 1-18 (2009).</li><li>Daneshtalab, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Troubleshooting+tissue+specificity+and+antibody+selection:+Procedures+in+immunohistochemical+studies.">Troubleshooting tissue specificity and antibody selection: Procedures in immunohistochemical studies.</a> Journal of Pharmacological and Toxicological Methods. 61 (2), 127-135 (2010).</li><li>Jang, M. H., Bonaguidi, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+frizzled-related+protein+3+regulates+activity-dependent+adult+hippocampal+neurogenesis.">Secreted frizzled-related protein 3 regulates activity-dependent adult hippocampal neurogenesis.</a> Cell Stem Cell. 12 (2), 215-223 (2013).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Animal+Perfusion+Fixation+for+Rodents.">Whole Animal Perfusion Fixation for Rodents.</a> J. Vis. Exp. (65), e3564 (2012).</li><li>Porcelli, M., Cacciapuoti, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-thermal+effects+of+microwaves+on+proteins:+Thermophilic+enzymes+as+model+system.">Non-thermal effects of microwaves on proteins: Thermophilic enzymes as model system.</a> FEBS Letters. 402 (2-3), 102-106 (1997).</li><li>Stone, J. R., Walker, S. A., Povlishock, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+visualization+of+a+new+class+of+traumatically+injured+axons+through+the+use+of+a+modified+method+of+microwave+antigen+retrieval.">The visualization of a new class of traumatically injured axons through the use of a modified method of microwave antigen retrieval.</a> Acta Neuropathologica. 97 (4), 335-346 (1999).</li></ol>

The authors declare that they have no competing financial interests.

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...

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, and learning and memory. Thus, there has been a concerted effort to understand the development and regulation of neurons in this rich neurogenic niche.
An unavoidable and important aspect of studying adult hippocampal neurogenesis is the identification of separate stages that an activated neural stem cell passes through on its way to becoming a fully functional neuron. In this process, the quiescent neural stem cell is activated and proceeds through a series of early active and late active progenitor cell types (Figure 1). These distinct populations of neural progenitors can be identified by morphology and their expression of molecular markers such as MCM2, nestin, Tbr2 and Doublecortin (DCX) and NeuN that guide the conversion of RGLs into their respective progenitor cell types. Nestin is an intermediate filament protein that is expressed across the radial glia-like cells and some early progenitor cell types. Another marker is Tbr2 that is specifically expressed in the amplifying progenitor cells. The Tbr2 transgene expression is initially switched off in the Type-1 cells (radial-glia like), turned on across the Type-2a, Type-2ab and Type-2b progenitors and switched off among the more differentiated Type-3 and immature neurons (Figure 1). DCX is a neuronal migration marker which is expressed in the Type-2ab, Type-2b and Type-3 neural progenitors. Using this combination of three markers, we can label distinct subtypes of neural progenitors. These include the Type-1 (MCM2+nestin+Tbr2-), Type-2a (MCM2+nestin+Tbr2+), Type-2ab (partial MCM2+nestin-Tbr2+ and partial MCM2+Tbr2+DCX-), Type-2b (MCM2+Tbr2+DCX+) and Type-3 (MCM2+Tbr2-DCX+). Post-mitotic cells such as the immature and functionally integrated neurons can be identified by utilizing DCX and the mature neuronal marker, NeuN.
Traditional methods of IHC utilize antibodies to identify antigens for specific cell types based on their gene expression. Subsequent visualization through high resolution imaging techniques such as confocal microscopy can be utilized for their identification. However, currently there are problems that exist with these methods that don't allow efficient identification of the radial glia-like cells and early progenitor cell types. It is difficult to identify these particular cell types because the applied antibody is not able to penetrate and efficiently bind the nestin protein. Nestin is the intermediate filament protein that comprises the radial processes produced by the radial glia-like cell. The inefficient binding of an antibody to the antigen can be a result of many factors including fixation time, temperature and technique utilized6-7. To help solve such issues, we have developed an antigen-retrieval or "boiling" method. Besides nestin, this method of antigen-retrieval also improves staining for other markers such as Ki67, BrdU, glial fibrillary acidic protein (GFAP), Tbr2 and MCM2. Our method combines together chemical and physical approaches. It involves chemical fixation of the tissue in paraformaldehyde and subsequent high temperature boiling of thin coronal hippocampal sections in a buffer of a denaturant and chaotropic treatment. This enhances accessibility of the antigen to the antibody, improves antibody specificity and allows improved identification of progenitor cell types. Our approach is simple to use requiring mostly already available tools and reagents in the laboratory. We have utilized it extensively to study development of neural stem cells and their regulation via intrinsic genetic or extrinsic environmental factors8.

<table><tbody><tr><td>SuperFrost Plus Slide</td><td>Fisherbrand</td><td>12-550-15</td><td>Provides strong adherence to hippocampal tissue during boiling</td></tr><tr><td>PVA/DABCO Mounting Media</td><td>Sigma Aldrich</td><td>10981-100 ml</td><td> </td></tr><tr><td>Nestin (chicken) </td><td>Aves Lab</td><td>NES</td><td>Neural stem cell marker</td></tr><tr><td>GFAP (rabbit)</td><td>Dako North America</td><td>Z033401-2</td><td>Astrocyte and neural stem cell marker</td></tr><tr><td>MCM2 (mouse)</td><td>BD Transduction Laboratory</td><td>610701</td><td>Proliferation marker</td></tr><tr><td>DCX (goat)</td><td>Santa Cruz Biotechnology</td><td>SC8066</td><td>Immature neuron marker</td></tr><tr><td>Tbr2 (rabbit)</td><td>Abcam</td><td>Ab23345</td><td>Amplifying progenitor marker</td></tr><tr><td>NeuN (mouse)</td><td>Millipore</td><td>MAB377</td><td>Mature neuron marker</td></tr><tr><td>Cy2 (anti-rabbit)</td><td>Jackson Immunoresearch</td><td>111-226-047</td><td> </td></tr><tr><td>Cy2 (anti-chicken)</td><td>Jackson Immunoresearch</td><td>303-165-006</td><td> </td></tr><tr><td>Cy5 (anti-mouse)</td><td>Jackson Immunoresearch</td><td>315-175-047</td><td> </td></tr><tr><td>Cy5 (anti-goat)</td><td>Jackson Immunoresearch</td><td>305-165-047</td><td> </td></tr><tr><td>DAPI</td><td>Life Technologies Corporation</td><td>D1306</td><td> </td></tr><tr><td>Dako Pen</td><td>S2002</td><td>Dako</td><td>Water-repellant pen</td></tr></tbody></table>

heat-induced antigen retrieval: an effective method to detect and identify progenitor cell types during adult hippocampal neurogenesis

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.

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...

Watch this Scientific Journal Video about Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis at JoVE.com

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

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.

Traditional  methods of immunohistochemistry (IHC) following tissue fixation allow visualization  of various cell types. These typically proceed with the ...

heat-induced-antigen-retrieval-an-effective-method-to-detect-identify

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

12.5K 次观看.  Mayo Clinic College of Medicine. 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.

视频: Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Progenitor-derived Oligodendrocyte Culture System from Human Fetal Brain

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell lineage development. In vitro expansion of neural progenitors from fetal CNS tissue has been well characterized. Despite the identification and isolation of glial progenitors from adult human sub-cortical white matter and development of various culture conditions to direct differentiation of fetal neural progenitors into myelin producing oligodendrocytes, acquiring sufficient human oligodendrocytes for in vitro experimentation remains difficult. Differentiation of galactocerebroside+ (GalC) and O4+ oligodendrocyte precursor or progenitor cells (OPC) from neural precursor cells has been reported using second trimester fetal brain. However, these cells do not proliferate in the absence of support cells including astrocytes and neurons, and are lost quickly over time in culture. The need remains for a culture system to produce cells of the oligodendrocyte lineage suitable for in vitro experimentation.
Culture of primary human oligodendrocytes could, for example, be a useful model to study the pathogenesis of neurotropic infectious agents like the human polyomavirus, JCV, that in vivo infects those cells. These cultured cells could also provide models of other demyelinating diseases of the central nervous system (CNS). Primary, human fetal brain-derived, multipotential neural progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons (progenitor-derived neurons, PDN) and astrocytes (progenitor-derived astrocytes, PDA) This study shows that neural progenitors can be induced to differentiate through many of the stages of oligodendrocytic lineage development (progenitor-derived oligodendrocytes, PDO). We culture neural progenitor cells in DMEM-F12 serum-free media supplemented with basic fibroblast growth factor (bFGF), platelet derived growth factor (PDGF-AA), Sonic hedgehog (Shh), neurotrophic factor 3 (NT-3), N-2 and triiodothyronine (T3). The cultured cells are passaged at 2.5e6 cells per 75cm flasks approximately every seven days. Using these conditions, the majority of the cells in culture maintain a morphology characterized by few processes and express markers of pre-oligodendrocyte cells, such as A2B5 and O-4. When we remove the four growth factors (GF) (bFGF, PDGF-AA, Shh, NT-3) and add conditioned media from PDN, the cells start to acquire more processes and express markers specific of oligodendrocyte differentiation, such as GalC and myelin basic protein (MBP). We performed phenotypic characterization using multicolor flow cytometry to identify unique markers of oligodendrocyte.

Primary, human fetal brain-derived, multipotential progenitor cells proliferate in vitro while maintaining the capacity to differentiate into neurons and astrocytes. This work shows that neural progenitors can be induced to differentiate through stages of the oligodendrocytic lineage by conditioning with select growth factors.

来自祖，人脑少突胶质细胞培养系统

Differentiation of human neural progenitors into neuronal and glial cell types offers a model to study and compare molecular regulation of neural cell ...

科研

神经科学

Functional Interrogation of Adult Hypothalamic Neurogenesis with Focal Radiological Inhibition

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral delivery or transgenic animals have potential confounds that make interpretation of results from these studies difficult. New radiological tools are emerging, however, that allow one to noninvasively investigate the function of select groups of adult-born neurons through accurate and precise anatomical targeting in small animals. Focal ionizing radiation inhibits the birth and differentiation of new neurons, and allows targeting of specific neural progenitor regions. In order to illuminate the potential functional role that adult hypothalamic neurogenesis plays in the regulation of physiological processes, we developed a noninvasive focal irradiation technique to selectively inhibit the birth of adult-born neurons in the hypothalamic median eminence. We describe a method for Computer tomography-guided focal irradiation (CFIR) delivery to enable precise and accurate anatomical targeting in small animals. CFIR uses three-dimensional volumetric image guidance for localization and targeting of the radiation dose, minimizes radiation exposure to nontargeted brain regions, and allows for conformal dose distribution with sharp beam boundaries. This protocol allows one to ask questions regarding the function of adult-born neurons, but also opens areas to questions in areas of radiobiology, tumor biology, and immunology. These radiological tools will facilitate the translation of discoveries at the bench to the bedside.

The function of adult-born mammalian neurons remains an active area of investigation. Ionizing radiation inhibits the birth of new neurons. Using computer tomography-guided focal irradiation (CFIR), three-dimensional anatomical targeting of specific neural progenitor populations can now be used to assess the functional role of adult neurogenesis.

成人下丘脑神经发生局灶性放射性抑制功能审讯

The functional characterization of adult-born neurons remains a significant challenge. Approaches to inhibit adult neurogenesis via invasive viral ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

树突分支中的海马区对小鼠的齿状回评

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly committed intermediate progenitor subtypes. Each of these subtypes expresses a set of specific molecular markers that, together with specific morphological criteria, can be used for their identification. Typically, immunofluorescent techniques are applied involving subtype-specific antibodies in combination with exo- or endogenous proliferation markers. We herein describe immunolabeling methods for the detection and quantification of all stages of adult hippocampal neurogenesis. These comprise the application of thymidine analogs, transcardial perfusion, tissue processing, heat-induced epitope retrieval, ABC immunohistochemistry, multiple indirect immunofluorescence, confocal microscopy and cell quantification. Furthermore we present&#160;a sequential multiple immunofluorescence protocol which circumvents problems usually arising from the need of using primary antibodies raised in the same host species. It allows an accurate identification of all hippocampal progenitor subtypes together with a proliferation marker within a single section. These techniques are a powerful tool to study the regulation of different progenitor subtypes in parallel, their involvement in brain pathologies and their role in specific brain functions.

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

免疫组化和多标签与同一宿主物种抗体研究成人神经发生

Adult neurogenesis is a highly regulated, multi-stage process in which new neurons are generated from an activated neural stem cell via increasingly ...

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

组蛋白3赖氨酸 79 Dimethylation 标记沉淀后神经祖细胞的分离培养

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate new neurons, which integrate into an existing neural circuit and contribute to specific hippocampal functions. Importantly, adult neurogenesis is highly susceptible to environmental stimuli, which allows for activity-dependent regulation of various cognitive functions. A vast range of neural circuits from various brain regions orchestrates these complex cognitive functions. It is therefore important to understand how specific neural circuits regulate adult neurogenesis. Here, we describe a protocol to manipulate neural circuit activity using designer receptor exclusively activated by designer drugs (DREADDs) technology that regulates NSCs and newborn progeny in rodents. This comprehensive protocol includes stereotaxic injection of viral particles, chemogenetic stimulation of specific neural circuits, thymidine analog administration, tissue processing, immunofluorescence labeling, confocal imaging, and imaging analysis of various stages of neural precursor cells. This protocol provides detailed instructions on antigen retrieval techniques used to visualize NSCs and their progeny and describes a simple, yet effective way to modulate brain circuits using clozapine N-oxide (CNO) or CNO-containing drinking water and DREADDs-expressing viruses. The strength of this protocol lies in its adaptability to study a diverse range of neural circuits that influence adult neurogenesis derived from NSCs.

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

成人希马神经前体细胞的乳化电路特定调节

Adult neurogenesis is a dynamic process by which newly activated neural stem cells (NSCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate ...

Fluorescence-Activated Nuclei Negative Sorting of Neurons Combined with Single Nuclei RNA Sequencing to Study the Hippocampal Neurogenic Niche

Adult Hippocampal Neurogenesis (AHN), which consists of a lifelong maintenance of proliferative and quiescent neural stem cells (NSCs) within the sub-granular zone (SGZ) of the dentate gyrus (DG) and their differentiation from newly born neurons into granule cells in the granule cell layer, is well validated across numerous studies. Using genetically modified animals, particularly rodents, is a valuable tool to investigate signaling pathways regulating AHN and to study the role of each cell type that compose the hippocampal neurogenic niche. To address the latter, methods combining single nuclei isolation with next generation sequencing have had a significant impact in the field of AHN to identify gene signatures for each cell population. Further refinement of these techniques is however needed to phenotypically profile rarer cell populations within the DG. Here, we present a method that utilizes Fluorescence Activated Nuclei Sorting (FANS) to exclude most neuronal populations from a single nuclei suspension isolated from freshly dissected DG, by selecting unstained nuclei for the NeuN antigen, in order to perform single nuclei RNA sequencing (snRNA-seq). This method is a potential steppingstone to further investigate intercellular regulation of the AHN and to uncover novel cellular markers and mechanisms across species.

Presented here is a method to sequence single nuclei isolated from the mouse dentate gyrus that excludes most neurons through fluorescence-activated nuclei (FAN)-sorting. This approach generates high-quality expression profiles and facilitates the study of most other cell types represented in the niche, including scarce populations such as neural stem cells.

荧光激活神经元核负分选结合单核RNA测序研究海马神经源性生态位

Adult Hippocampal Neurogenesis (AHN), which consists of a lifelong maintenance of proliferative and quiescent neural stem cells (NSCs) within the ...

Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using interneurons to replace or mitigate the effects of altered neuronal circuits is an attractive therapeutic avenue. To this end, more knowledge is needed about how human stem cell-derived GABAergic interneuron-like cells (hdINs) mature, integrate, and function over time in the host circuitry. Of particular importance in neurodevelopmental disorders is a better understanding of whether these processes in transplanted cells are affected by an evolving and maturing host brain. The present protocol describes a fast and highly efficient generation of hdINs from human embryonic stem cells based on the transgenic expression of the transcription factors Ascl1 and Dlx2. These neuronal precursors are transplanted unilaterally, after 7 days in vitro, to the hippocampus of neonatal 2-day-old mice. The transplanted neurons disperse in the ipsi- and contralateral hippocampus of a mouse model of cortical dysplasia-focal epilepsy syndrome and survive for up to 9 months after transplantation. This approach allows for investigating the cellular identity, integration, functionality, and therapeutic potential of transplanted interneurons over an extended time in developing healthy and diseased brains.

Transplantation of human pluripotent stem cell-derived GABAergic neurons generated by neuronal programming could be a potential treatment approach for neurodevelopmental disorders. This protocol describes the generation and transplantation of human stem cell-derived GABAergic neuronal precursors into the brains of neonatal mice, allowing long-term investigation of grafted neurons and evaluation of their therapeutic potential.

将人干细胞衍生的GABA能神经元移植到出生后早期小鼠海马体中以减轻神经发育障碍

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using ...

Immunofluorescence Staining for Visualizing Proliferated Neural Stem Cells in Mouse Brain Sections

Source: Quintanilla, L. J., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/59237">Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells.</a> J. Vis. Exp. (2019)
This video demonstrates the procedure for staining newly proliferated neural stem cells in brain tissue sections using EdU incorporation, followed by immunofluorescence to visualize specific proteins, enhancing the understanding of cell dynamics and proliferation.

免疫荧光染色用于观察小鼠脑切片中增殖的神经干细胞

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Immunohistochemistry

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JoVE Encyclopedia of Experiments

Neuropathology

Developmental and Pediatric Disorders

An Immunofluorescence Staining Technique to Detect Adult Hippocampal Neurogenesis

Source: Ansorg, A., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/52551">Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis.</a> J. Vis. Exp. (2015)
The video illustrates an immunofluorescence staining method used to examine neurogenesis in the adult hippocampus. Initially, a mouse brain section undergoes treatment with primary antibodies directed at marker proteins associated with neurogenesis in neuronal progenitor cells. Next, it is labeled with secondary antibodies tagged with fluorophores. The brain section is then observed under a confocal microscope to distinguish various subtypes of progenitor cells, based on the expression patterns of distinct combinations of marker proteins.

一种检测成人海马神经发生的免疫荧光染色技术

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Tissue ...