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...

Neurogenèse, la génération de nouveaux neurones à partir de cellules souches neurales, est maintenant connu pour se produire en permanence à l&#39;âge adulte dans deux régions spécialisées dans le cerveau. Il s&#39;agit notamment de la zone sous-ventriculaire (SVZ) du bulbe olfactif et la zone sous-granulaire (SGZ) du gyrus denté de l&#39;hippocampe. Au cours de la dernière décennie, le domaine de la neurogenèse hippocampique adulte a vu d&#39;importants travaux. La région a suscité beaucoup d&#39;intérêt depuis les nouveaux neurones dans le SGZ contribuent à une meilleure plasticité neuronale qui peut maintenir les fonctions spécifiques du cerveau 1-5. Adulte neurogenèse hippocampique a été impliqué à jouer un rôle important dans la régulation de l&#39;humeur, la régénération et l&#39;apprentissage et la mémoire. Ainsi, il ya eu un effort concerté pour comprendre le développement et la régulation des neurones dans cette riche niche neurogène. Un aspect inévitable et important d&#39;étudier la neurogenèse hippocampique adulte est l&#39;identification de station séparéeges qu&#39;une cellule souche neurale activé traverse sur son chemin pour devenir un neurone entièrement fonctionnel. Dans ce procédé, la cellule souche neurale de repos est activé et passe à travers une série de types de cellules progénitrices précoces actives actif et à la fin (Figure 1). Ces populations distinctes de cellules progénitrices neurales peuvent être identifiés par la morphologie et de leur expression de marqueurs moléculaires tels que MCM2, la nestine, TBR2 et Doublecortin (DCX) et NeuN qui guident la conversion de RGLs dans leurs types de cellules précurseurs respectifs. La nestine est une protéine de filament intermédiaire qui est exprimée dans les cellules gliales en forme radiales et certains types de cellules progénitrices précoces. Un autre marqueur est TBR2 qui est spécifiquement exprimé dans les cellules progénitrices amplification. L&#39;expression du transgène TBR2 est initialement désactivée dans les cellules de type 1 (radiale gliales comme), activé à travers les type 2a, progéniteurs de type-2ab et Type-2b et éteint parmi les plus différenciée de type 3 et immatureneurones (Figure 1). DCX est un marqueur de la migration neuronale qui est exprimé dans le type 2ab, Type-2b et de type 3 progéniteurs neuronaux. En utilisant cette combinaison de trois marqueurs, nous pouvons étiqueter sous-types distincts de cellules progénitrices neurales. Il s&#39;agit notamment du type 1 (MCM2 + nestine + TBR2-), de type 2a (MCM2 + nestine + TBR2 +), Type-2ab (MCM2 partielle + nestine-TBR2 + et partielle MCM2 + TBR2 + DCX-), Type-2b ( MCM2 + TBR2 + + DCX) et de type 3 (MCM2 + TBR2-DCX +). Cellules post-mitotiques tels que les neurones immatures et fonctionnellement intégrés peuvent être identifiés par l&#39;utilisation de DCX et le marqueur neuronal mature, NeuN. Les méthodes traditionnelles d&#39;IHC utilisent des anticorps pour identifier des antigènes de types de cellules spécifiques sur la base de leur expression des gènes. Après visualisation grâce à des techniques d&#39;imagerie à haute résolution telle que la microscopie confocale peut être utilisée pour leur identification. Toutefois, actuellement, il ya des problèmes qui existent avec ces méthodes qui ne permettent pas l&#39;identification efficace desles cellules gliales comme radiaux et progénitrices début types de cellules. Il est difficile d&#39;identifier ces différents types cellulaires particuliers, car l&#39;anticorps n&#39;est pas appliqué capable de pénétrer et de se lier de manière efficace la protéine nestine. La nestine est la protéine de filament intermédiaire qui comprend les processus radiales produites par la cellule de cellules gliales en forme radiale. La liaison inefficace d&#39;un anticorps à l&#39;antigène peut être le résultat de nombreux facteurs, y compris le temps de fixation, de la température et de la technique utilisée 7.6. Pour aider à résoudre ces problèmes, nous avons développé une réaction antigène-extraction, ou de méthode &quot;d&#39;ébullition&quot;. En plus de la nestine, ce procédé de récupération d&#39;antigène permet également d&#39;améliorer la coloration pour les autres marqueurs tels que Ki67, BrdU, la protéine acide fibrillaire gliale (GFAP), TBR2 et MCM2. Notre méthode combine ensemble des approches chimiques et physiques. Elle implique la fixation chimique du tissu dans du paraformaldéhyde et ultérieur à haute température d&#39;ébullition de minces sections coronales de l&#39;hippocampe dans un tampon d&#39;un dénaturant et chaotroptraitement ic. Cela améliore l&#39;accessibilité de l&#39;antigène à l&#39;anticorps, permet d&#39;améliorer la spécificité des anticorps et permet une meilleure identification des types de cellules progénitrices. Notre approche est simple à utiliser nécessitant des outils et des réactifs pour la plupart déjà disponibles dans le laboratoire. Nous avons utilisé intensivement pour étudier le développement des cellules souches neurales et leur régulation par des facteurs environnementaux génétiques ou extrinsèques intrinsèques 8.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <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

Les méthodes traditionnelles de l&#39;immunohistochimie (IHC) suivantes fixation du tissu permettent de visualiser différents types de cellules. Ceux-ci se déroulent généralement avec l&#39;application de l&#39;anticorps à lier des antigènes et identifier les cellules dont les caractéristiques sont fonction de la biologie et de développement inhérente. Neurogenèse hippocampique adulte est un processus séquentiel, dans lequel une cellule souche neurale de repos peut être activée et de passer par les étapes de prolifération, la différenciation, la maturation et l&#39;intégration fonctionnelle. Chaque phase se distingue avec une morphologie caractéristique et la régulation positive des gènes. L&#39;identification de ces phases est important de comprendre les mécanismes de régulation en jeu et toute modification dans ce processus qui sous-tendent la physiopathologie des troubles débilitants. Notre approche d&#39;extraction de l&#39;antigène induit par la chaleur permet d&#39;améliorer l&#39;intensité du signal qui est détecté et permet une identification correcte du type de cellules progénitrices. Comme on le verra dans cepapier, il permet surtout de nous de contourner les problèmes actuels dans la détection de certains types de cellules souches.

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.

La chaleur induite par un antigène de récupération: une méthode efficace pour détecter et identifier les types de cellules progénitrices adultes au cours de l&#39;hippocampe neurogenèse

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

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12.4K vues.  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.