The authors thank S. Tausch for excellent technical assistance. The work was supported by BMBF (Bernstein Focus 01GQ0923) and DFG (FOR1738).

NOTE: All procedures involving living animals were carried out in accordance with the EC directive 86/609/EEC guidelines on the care and use of laboratory animals and approved by the local ethics committee (Th&#252;ringer Landesamt f&#252;r Lebensmittelsicherheit und Verbraucherschutz).
1. Intraperitoneal Injection of Thymidine Analogs
<ol>
	<li>Weigh animals the day before injection. Calculate the amount of thymidine analog required for all injections planned on the next day as well as individual weight-adjusted injection volumes of a 10 mg/ml stock solution.</li>
	<li>Prepare 10 mg/ml thymidine stock solution.&#160;(CAUTION! Thymidine analogs are toxic. Follow the specific Material Safety Data Sheets (MSDS) provided by the suppliers, i.e., wear lab coat and gloves, use a chemical fume hood).
	<ol>
		<li>Take thymidine analog from the freezer and bring it to RT,&#160;approx. 21 &#176;C. Weigh 10 mg and add sterile saline (for BrdU and CldU) or 0.04 N NaOH (in sterile saline; for IdU), vortex. Place for at least 10 - 15 min in a 50 &#176;C water bath and vortex every 2 - 3 min to dissolve the powder. 
		NOTE: Use solutions for up to 24 hr when stored at RT and for several weeks at -20 &#176;C. Protect solutions from light (cover with aluminum foil). Always check for precipitates and re-dissolve if necessary.</li>
	</ol>
	</li>
	<li>Restrain the mouse by the scruff and intraperitoneally inject an appropriate, weight-adjusted volume of stock solution (at RT) using a fine dosage syringe with a 30 G needle. 
	NOTE: In case CldU and IdU have to be administered sequentially within the same animal, consider to inject equimolar concentrations (e.g., 42.5 mg/kg CldU and 57.5 mg/kg IdU, which correspond to 50 mg/kg BrdU). Therefore, adjust the injection volumes of the 10 mg/ml stock solutions accordingly.</li>
</ol>
2. Tissue Preparation
<ol>
	<li>Prepare 4% formaldehyde in 0.1 M phosphate buffer pH 7.4 on the day before perfusion. Store at 4 &#176;C.</li>
	<li>Transcardially perfuse the deeply anesthetized mice (3.5% isoflurane) via the left ventricle with 10 ml ice-cold PBS, then with 40 ml ice-cold formaldehyde (flow rate 5 ml/min). Dissect the brain and post-fix in the same fixative for 24 hr at 4&#176;C.</li>
	<li>Transfer the brains consecutively into 10% (24 hr at 4&#176;C) and 30% sucrose (until the brain sinks, approx. 48 hr). For freezing, slowly submerge the cryoprotected brains into -25 &#176;C isopentane until no bubbles emerge from the tissue. Store at -80 &#176;C.</li>
	<li>Cut coronal sections of 40 &#181;m thickness on a freezing microtome (block temperature at -25 to -16 &#176;C). Sequentially transfer sections into antifreeze solution containing wells of a 24-well cell culture plate (see Figure 1). Store at -20 &#176;C.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/52551/52551fig1.jpg" /> 
Figure 1. Schematic illustration of transferring microtome slices into a 24-well plate. Start at A1 and place subsequent slices into row A, after A6 go to the next row B and so forth. When reaching D6, go back to A1 and continue. This arrangement of slices allows for quantification of every nth section of an entire brain. For quantification of newborn cells take every 6th brain section (equivalent to the content of one column), for immunofluorescence phenotyping take every 12th section (equivalent to the content of 2 alternating rows of one column).
3. Immunostaining
NOTE: Sections are processed free floating, usually in 6-well plates equipped with a carrier plate and mesh inserts. As an exception, blocking, antibody incubations and ABC reaction are done in 12- or 24-well plates without mesh inserts (0.5 to 1 ml per well is sufficient, depending on the number of slices that have to be stained). During these steps, transfer sections with the help of a fine brush (rinse with each new solution). All incubations are done with continuous agitation (max 150 rpm).
<ol>
	<li>Immunohistochemistry (ABC method)
	<ol>
		<li>Transfer sections from antifreeze into TBS and rinse thoroughly (once O/N&#160;at 4 &#176;C, 5 times at RT for 10 min each) to completely remove antifreeze.</li>
		<li>Incubate for 30 min in 1.5% H2O2 in TBS-T to quench endogenous peroxidase activity. Pay attention to bubbling and re-submerge sections if necessary. Rinse 3 times in TBS for 15 min each.</li>
		<li>Optional: Meanwhile preheat a heating cabinet and 2 N HCl to 37 &#176;C. Incubate sections for 30 min at 37 &#176;C in 2 N HCl to denature DNA. Gently separate sections with the help of a brush.</li>
		<li>Optional: Neutralize sections for 10 min in 0.1 M borate buffer pH 8.5, RT. While transferring, briefly swab the mesh inserts containing the sections on a paper towel to remove HCl leavings. Rinse 2 times in TBS for 15 min each.</li>
		<li>Incubate in TBSplus to permeabilize the tissue and to block unspecific antibody binding sites, 1 hr at RT.</li>
		<li>Incubate in primary antibody diluted in TBSplus, O/N at 4 &#176;C. Rinse 3 times in TBS for 15 min each.</li>
		<li>Incubate in biotinylated secondary antibody diluted in TBSplus, 3 hr at RT. Rinse 3 times in TBS for 15 min each. Meanwhile...</li>
		<li>Prepare ABC complex according to manufacturer&#39;s protocol (1% A + 1% B in TBS-T). Allow to stand for 30 min at RT before use. Incubate sections in AB reagent for 1 hr at RT. Rinse 3 times in TBS for 15 min each.</li>
		<li>Prepare 50 ml 0.5 mg/ml DAB in TBS-T per 6- or 12-well plate, split into two halves and pipette 4 ml or 2 ml per well, respectively. Transfer sections into DAB solution (CAUTION! DAB is toxic. Follow the specific MSDS provided by the supplier, i.e., wear lab coat and gloves, use a chemical fume hood).</li>
		<li>Add 0.5 ml 1% H2O2 to the remaining 25 ml DAB solution, mix and pipette equivalent volumes as above to each well to start peroxidase reaction. Incubate for 12 min. Rinse 3 times in TBS for 15 min each.</li>
		<li>Mount sections to slides in gelatin, air dry O/N. Coverslip with permanent mounting medium.</li>
		<li>Optional: Counterstain before placing the coverslip (see section 3.5). 
		NOTE: If the signal-to-noise ratio is low because of high background, repeat H2O2 treatment after the incubation with AB reagent (step 3.1.8).</li>
	</ol>
	</li>
	<li>Multiple-immunofluorescence
	<ol>
		<li>Single or simultaneous multiple immunofluorescence
		<ol>
			<li>Transfer sections from antifreeze into TBS and rinse thoroughly (once O/N at 4 &#176;C, 5 times at RT for 10 min each) to completely remove antifreeze.</li>
			<li>Optional: as steps 3.1.3 - 3.1.4.</li>
			<li>Incubate in TBSplus to permeabilize the tissue and block unspecific antibody binding sites, 1 hr at RT.</li>
			<li>Incubate in primary antibody cocktail (e.g., rat &#945;-BrdU, guinea pig &#945;-DCX, goat &#945;-GFP) diluted in TBSplus, O/N at 4&#176;C. Rinse 3 times in TBS for 15 min each.</li>
			<li>Incubate in cocktail of fluorochrome-conjugated secondary antibodies (e.g., Rhodamine Red &#945;-rat, Alexa-647 &#945;-guinea pig, Alexa-488 &#945;-goat; all derived in donkey) diluted in TBSplus, 3 hr at RT or O/N at 4 &#176;C. From now on protect sections from light. Rinse 3 times in TBS for 15 min each.</li>
			<li>Mount sections to slides in gelatin, air dry O/N. Coverslip with aqueous mounting medium.</li>
		</ol>
		</li>
		<li>Sequential multiple immunofluorescence with primary antibodies from same host species
		<ol>
			<li>As steps 3.2.1.1 - 3.2.1.3.</li>
			<li>Incubate in first primary antibody (e.g., rabbit &#945;-antigen A), O/N at 4 &#176;C. Rinse 3 times in TBS and once in TBS-T for 10 min each.</li>
			<li>Incubate in first fluorochrome-conjugated secondary antibody (e.g., Rhodamine Red-conj. donkey &#945;-rabbit), 3 hr RT. From now on protect sections from light. Rinse 3 times in TBS and once in TBS-T for 10 min each.</li>
			<li>Incubate in 10% normal serum from same host as the primary antibodies (e.g., rabbit serum) for 3 hr RT to saturate open paratopes on the first secondary antibody. Rinse 3 times in TBS and once in TBS-T for 10 min each.</li>
			<li>Incubate in TBSplus with 50 &#181;g/ml unconjugated monovalent Fab fragments directed against the host of the primary antibodies (e.g., &#945;-rabbit IgG (H+L)) to cover epitopes that could be recognized by the second secondary antibody, O/N at 4 &#176;C.</li>
			<li>Rinse at least 3 times in TBS and once in TBS-T for 10 min each. While transferring, briefly swab the mesh inserts containing the sections on a paper towel to remove any Fab leavings.</li>
			<li>Incubate in second primary antibody (e.g., rabbit &#945;-antigen B), O/N at 4 &#176;C. Rinse 3 times in TBS and once in TBS-T for 10 min each.</li>
			<li>Incubate in second fluorochrome-conjugated secondary antibody (e.g., Alexa-488-conj. donkey &#945;-rabbit), 3 hr RT. Rinse 3 times in TBS for 15 min each, mount and coverslip as above. 
			NOTE: To label antigens with antibodies from different host species add them to step 3.2.2.2 and the respective secondary antibodies to step 3.2.2.3.</li>
		</ol>
		</li>
		<li>One of the secondary antibodies from same species as one of the primary antibodies 
		NOTE: This protocol is suitable for quadruple-staining against BrdU or Ki67 together with GFAP, nestin-GFP and DCX.
		<ol>
			<li>Follow strictly protocol steps 3.2.1.1 - 3.2.1.5. Until this point use only donkey serum in TBSplus.</li>
			<li>Incubate in TBSplus containing 3% goat serum for 1 hr at RT. This covers open paratopes on the &#945;-goat secondary antibody. Rinse 3 times in TBS and once in TBS-T for 10 min each.</li>
			<li>Incubate in AMCA-conj. goat &#945;-rabbit diluted in TBSplus, 3 hr RT or O/N 4 &#176;C. Rinse three times in TBS for 15 min each, mount and coverslip as above.</li>
		</ol>
		</li>
		<li>CldU, IdU co-staining
		<ol>
			<li>Rinse, denature and neutralize sections as described in section 3.2.1 (steps 3.2.1.1 - 3.2.1.2).</li>
			<li>Incubate with 20 &#181;g/ml unconjugated Fab fragments &#945;-mouse IgG (H+L) in TBSplus, 1 hr at RT. Rinse 4 times in TBS and once in TBS-T for 10 min each.</li>
			<li>Incubate in primary antibody cocktail containing rat &#945;-BrdU (1:400; purified IgG2) and mouse &#945;-BrdU (1:350) diluted in TBSplus, O/N at 4&#176;C. Rinse 3 times in TBS and once in TBS-T for 10 min each.</li>
			<li>Incubate in secondary antibody cocktail containing biotinylated donkey &#945;-rat (1:500) and FITC-conj. donkey &#945;-mouse Fab fragments (1:100). Rinse 3 times in TBS and once in TBS-T for 10 min each.</li>
			<li>Incubate in Rhodamine Red-conj. Streptavidin, 2 hr at RT. Rinse 3 times in TBS for 15 min each, mount and coverslip as above.</li>
		</ol>
		</li>
		<li>Amplification of fluorescence signal in nestin-GFP mice
		<ol>
			<li>Add goat &#945;-GFP to the primary antibody cocktail.</li>
			<li>Add a fluorochrome conjugated secondary antibody with spectral properties similar to GFP (such as Alexa 488 conj. donkey &#945;-goat) to the secondary antibody cocktail.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Epitope retrieval
	<ol>
		<li>Carry out epitope retrieval after rinsing out antifreeze. Preheat steamer with 6- or 12-well plates containing 0.1 M citrate buffer pH 6.0 to 95 - 99 &#176;C (approximately 25 min).</li>
		<li>Transfer the sections into the hot citrate buffer and steam for 30 min (cover the plates with aluminum foil as plastic lids are not heat-resistant).</li>
		<li>Immediately place the plates in an ice bath to cool down. This helps to protect tissue morphology, which is of particular importance when working with brain sections of postnatal animals.</li>
		<li>Rinse 3 times in TBS for 10 min each to rinse out and neutralize the citrate and continue staining.</li>
	</ol>
	</li>
	<li>Mouse antibodies on mouse tissue
	<ol>
		<li>Add 20 &#181;g/ml monovalent Fab fragments &#945;-mouse IgG (H+L; same host-species than secondary antibody) to the first blocking step (e.g., step 3.1.5 or 3.2.1.3).</li>
		<li>Rinse 4 times in TBS and once in TBS-T for 10 min each, and proceed with respective protocol.</li>
	</ol>
	</li>
	<li>Cresyl violet counterstaining
	<ol>
		<li>Preheat cresyl violet solution to 60 &#176;C (in glass jar). Incubate slides in the hot solution for 3 min.</li>
		<li>Rinse in aq. dest. and dehydrate 2 times for 1 min each in 70%, 96% and 100% isopropanol.</li>
		<li>Clear for 5 - 6 min in xylene or a xylene substitute and coverslip with a compatible permanent mounting medium.</li>
	</ol>
	</li>
</ol>
4. Data Analysis
<ol>
	<li>Count peroxidase stained newborn cells in every 6th section along the entire rostrocaudal extent of the dentate gyrus. Use a light microscope at 400X&#160;magnification.</li>
	<li>Multiply the resulting cell numbers with the intersection interval to obtain an estimate of total numbers of newborn cells.</li>
	<li>Image the fluorescence labeled sections with a confocal laser microscope equipped with appropriate lasers and filter systems. Take image stacks at random positions along the entire extent of the dentate gyrus at 400X magnification and analyze at least 50 randomly selected cells of interest per hemisphere for co-labeling with other markers.</li>
	<li>Multiply the resulting percentages of co-labeled cells (%/100) with the total numbers of newborn cells to calculate absolute numbers of specific newborn cell populations.</li>
</ol>

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The authors declare that they have no competing financial interests.

Quantification and identification of subpopulations of newborn cells is a central issue in adult neurogenesis research. Combining proliferation markers and antibodies against proteins expressed during specific stages of adult neurogenesis allows immunohistochemical detection of these subpopulations. Some of the antibodies or antibody combinations require specific staining conditions.
Labeling of dividing cells with synthetic thymidine analogs is still the gold standard for studying adult hippo...

Two brain regions constitutively generate new neurons throughout life, the subventricular zone of the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). The newborn neurons derive from neural progenitor cells and go through different stages of morphological and physiological development before reaching maturity1,2. From a slowly dividing radial glia-like stem cell (type 1) consecutive stages of transit amplifying intermediate progenitor cells arise. The more undifferentiated subtypes (type 2a and type 2b) have an irregular shape with short, tangential processes. They generate neuroblasts (type 3) that gradually exit the cell cycle to become immature neurons (with dendrites extended towards the molecular layer) and finally integrate into the hippocampal network as mature granule cells. Due to their particular physiological characteristics these cells provide the circuitry with enhanced plasticity3 suggesting a unique role in hippocampal function. Actually, studies of the last decade generated substantial evidence that adult neurogenesis contributes to spatial memory, pattern separation and emotional behavior4,5.
Adult neurogenesis can be studied using different approaches. Thymidine analogs incorporate into DNA during the S-phase of the cell cycle and allow birth dating, quantification and fate analysis of newborn cells6-8. Sequential application of different thymidine analogs (e.g., CldU, EdU or IdU) can be used to study cell turnover or cell populations born at different time points during the course of an experiment9. An alternative, endogenous marker for cell proliferation is Ki67. It is expressed in dividing cells during all phases of the cell cycle (G1, S, G2, M) except the resting phase (G0) and the beginning of G1&#160;10,11. To analyze the phenotype of newborn cell populations in the adult dentate gyrus several stage-specific molecular markers can be used such as GFAP, nestin, DCX and NeuN1,6. GFAP is a marker of mature astrocytes but is also expressed in radial glia-like cells in the adult forebrain. Nestin is an intermediate filament specific for radial glia-like cells and early intermediate progenitor cells. DCX is a microtubule-associated protein expressed in intermediate progenitors, neuroblasts and immature neurons. Based on the (co-) expression of these three markers and the morphological features of the labeled cells four distinct progenitor cell subtypes can be identified: type 1 (GFAP+, nestin+, DCX-), type 2a (GFAP-, nestin+, DCX-), type 2b (GFAP-, nestin+, DCX+) and type 3 (GFAP-,nestin-, DCX+)1. Co-labeling of DCX together with NeuN, which is expressed in postmitotic neurons, allows the differentiation of immature (DCX+, NeuN+) and mature (DCX-, NeuN+) granule neurons.
The above mentioned markers are frequently used for immunofluorescent co-labeling and subsequent confocal microscopy to analyze the number and identity of newborn cells. This typically requires antibodies from different host species to prevent undesired antibody cross-reactivity. However, the majority of primary antibodies suitable for neurogenesis research are raised either in rabbits or mice (e.g., mouse &#945;-BrdU, mouse &#945;-NeuN, rabbit &#945;-Ki67, rabbit &#945;-GFAP). This leads to serious limitations in the number and combination of antigens that could be evaluated in a single slice. This in turn not only increases the staining effort, as multiple stainings have to be performed, but might also compromise the reliability of results. Furthermore, some antigens are susceptible to formalin fixation-induced epitope masking (e.g., Ki67, nestin). We herein describe modifications from the classical single- and multiple immunolabeling protocols (e.g., epitope retrieval, multiple sequential immunostaining, use of nestin-GFP transgenic mice12) that overcome many of these issues. In particular, the sequential multiple immunofluorescence protocol allows staining against up to four different antigens even if part of the antibodies is derived from the same host. This enables the simultaneous detection of type 1, type 2a, type 2b and type 3 progenitor cells, as well as their proliferative activity within a single section.

<table><tbody><tr><td colspan="4">&lt;em&gt;Thymidine analog administration&lt;/em&gt;</td></tr><tr><td>5-Bromo-2&amp;prime;-deoxyuridine, BrdU</td><td>Sigma-Aldrich</td><td>B9285</td><td>toxic (mutagenic, teratogenic)</td></tr><tr><td>5-Chloro-2&amp;prime;-deoxyuridine, CldU</td><td>Sigma-Aldrich</td><td>C6891</td><td>toxic (mutagenic, teratogenic)</td></tr><tr><td>5-Chloro-2&amp;prime;-deoxyuridine, CldU</td><td>MP Biomedicals</td><td>2105478</td><td>toxic (mutagenic, teratogenic)</td></tr><tr><td>5-Iodo-2&amp;prime;-deoxyuridine, IdU</td><td>MP Biomedicals</td><td>2100357</td><td>toxic (mutagenic, teratogenic)</td></tr><tr><td colspan="4">&lt;em&gt;Tissue preparation&lt;/em&gt;</td></tr><tr><td>Isoflurane-Actavis</td><td>Piramal Healthcare</td><td>700211</td><td></td></tr><tr><td>Paraformaldehyde powder (PFA)</td><td>Riedel-De H&amp;auml;en</td><td>16005</td><td>toxic, flammable</td></tr><tr><td>Perfusion pump PD5206</td><td>Heidolph Instruments</td><td>523-52060-00</td><td></td></tr><tr><td>Masterflex Tygon lab tubing, &amp;Oslash; 0.8 mm</td><td>Thermo Fischer Scientific</td><td>06409-13</td><td></td></tr><tr><td>Feeding needle, straight, 21 G, 1.75 mm olive tip, 40 mm</td><td>Agnthos</td><td>1036</td><td></td></tr><tr><td>Freezing microtome Microm HM 400</td><td>Thermo Fischer Scientific</td><td></td><td></td></tr><tr><td>24-well Cell culture multiwell plates</td><td>Greiner Bio-One</td><td>662160</td><td></td></tr><tr><td colspan="4">&lt;em&gt;Immunohistochemistry&lt;/em&gt;</td></tr><tr><td>Tefal vitacuisine steamer</td><td>Tefal</td><td>VS 4001</td><td></td></tr><tr><td>Netwell 24 mm polyester mesh membrane inserts</td><td>Corning</td><td>3479</td><td>pre-loaded in 6-well culture plates</td></tr><tr><td>Netwell 15 mm polyester mesh membrane inserts</td><td>Corning</td><td>3477</td><td>pre-loaded in 12-well culture plates</td></tr><tr><td>Netwell plastic 6-well carrier kit</td><td>Corning</td><td>3521</td><td>for 24 mm polyester mesh membrane inserts</td></tr><tr><td>Netwell plastic 12-well carrier kit</td><td>Corning</td><td>3520</td><td>for 15 mm polyester mesh membrane inserts</td></tr><tr><td>Vectastain Elite ABC kit</td><td>Vector Laboratories</td><td>PK-6100</td><td></td></tr><tr><td>DAB (3,3&amp;prime;-Diaminobenzidine tetrahydrochloride hydrate)</td><td>Sigma-Aldrich</td><td>D-5637</td><td>carcinogenic, light sensitive</td></tr><tr><td>Fluoromount-G</td><td>SouthernBiotech</td><td>0100-01</td><td></td></tr><tr><td colspan="4">&lt;em&gt;Primary antibodies&lt;/em&gt;</td></tr><tr><td>Rabbit IgG1 &amp;alpha;-Ki67</td><td>Novocastra/ Leica Biosystems</td><td>NCL-L-Ki67MM1</td><td>DAB 1:400/IF 1:100; requires epitope retrieval</td></tr><tr><td>Rabbit &amp;alpha;-GFAP, AS-3-GF</td><td>Synaptic Systems</td><td>173 002</td><td>1:500</td></tr><tr><td>Goat IgG (H+L) &amp;alpha;-GFP</td><td>Acris Antibodies</td><td>R1091P</td><td>1:300</td></tr><tr><td>Mouse IgG1 &amp;alpha;-nestin</td><td>Abcam</td><td>ab6142</td><td>1:200; requires epitope retrieval</td></tr><tr><td>Guinea pig IgG (H+L) &amp;alpha;-Doublecortin</td><td>Merck Millipore</td><td>AB2253</td><td>1:500</td></tr><tr><td>Rat IgG2a &amp;alpha;-BrdU (ascites)</td><td>AbD Serotec/ Bio-Rad</td><td>OBT0030CX</td><td>for detection of BrdU; DAB 1:500/IF 1:400</td></tr><tr><td>Rat IgG2a &amp;alpha;-BrdU (purified)</td><td>AbD Serotec/ Bio-Rad</td><td>OBT0030&amp;nbsp;&amp;nbsp;</td><td>for detection of CldU; DAB 1:500/IF 1:250-400</td></tr><tr><td>Mouse IgG1 &amp;alpha;-NeuN</td><td>Merck Millipore</td><td>MAB377</td><td>1:500</td></tr><tr><td>mouse IgG1&amp;#954; &amp;#945;-BrdU</td><td>BD Biosciences</td><td>347580</td><td>For detection of IdU; DAB 1:500/IF 1:350</td></tr><tr><td colspan="4">&lt;em&gt;Secondary antibodies&lt;/em&gt;</td></tr><tr><td>Donkey &amp;alpha;-guinea pig IgG (H+L)-Biotin</td><td>Dianova</td><td>711-065-152</td><td>1:500</td></tr><tr><td>Donkey &amp;alpha;-rat IgG (H+L)-Biotin</td><td>Dianova</td><td>712-065-150</td><td>1:500</td></tr><tr><td>Donkey &amp;alpha;-mouse IgG (H+L)-Biotin</td><td>Dianova</td><td>715-065-151</td><td>1:500</td></tr><tr><td>Goat &amp;alpha;-rat IgG (H+L)-Alexa Fluor 488</td><td>Molecular Probes</td><td>A11006</td><td>1:250</td></tr><tr><td>Donkey &amp;alpha;-goat IgG (H+L)-Alexa Fluor 488</td><td>Molecular Probes</td><td>A11055</td><td>1:250</td></tr><tr><td>Donkey &amp;alpha;-mouse IgG (H+L)-FITC, Fab-Fragment</td><td>Dianova</td><td>715-097-003</td><td>1:100</td></tr><tr><td>Donkey &amp;alpha;-mouse IgG (H+L)-Alexa Fluor 647</td><td>Dianova</td><td>715-605-151</td><td>1:250</td></tr><tr><td>Donkey &amp;alpha;-guinea pig IgG (H+L)-Alexa Fluor 647</td><td>Dianova</td><td>706-605-148</td><td>1:250</td></tr><tr><td>Donkey &amp;alpha;-rat IgG (H+L)-Rhodamine Red-X</td><td>Dianova</td><td>712-295-150</td><td>1:250</td></tr><tr><td>Donkey &amp;alpha;-rabbit IgG (H+L)-Rhodamine Red-X</td><td>Dianova</td><td>711-295-152</td><td>1:250</td></tr><tr><td>Donkey &amp;alpha;-guinea pig IgG (H+L)-Rhodamine Red-X</td><td>Dianova</td><td>706-296-148</td><td>1:250</td></tr><tr><td>Streptavidin-rhodamine Red-X</td><td>Dianova</td><td>016-290-084</td><td>1:500</td></tr><tr><td>Goat &amp;alpha;-rabbit IgG (H+L)-AMCA</td><td>Dianova</td><td>111-155-144</td><td>1:250, works only with rabbit &amp;alpha;-GFAP</td></tr><tr><td>Hoechst 33342</td><td>Molecular Probes</td><td>H3570</td><td>1:1000</td></tr><tr><td>DAPI</td><td>Molecular Probes</td><td>D1306</td><td>1:1000</td></tr><tr><td colspan="4">&lt;em&gt;Blocking&lt;/em&gt;</td></tr><tr><td>Fab-fragment donkey &amp;alpha;-mouse IgG (H+L)</td><td>Dianova</td><td>715-007-003</td><td>1:20</td></tr><tr><td>Fab-fragment donkey &amp;alpha;-rabbit IgG (H+L)</td><td>Dianova</td><td>711-007-003</td><td>1:20</td></tr><tr><td>Normal donkey serum</td><td>Merck Millipore</td><td>S30</td><td></td></tr><tr><td>Normal rabbit serum</td><td>Dianova</td><td>011-000-010</td><td></td></tr><tr><td>Normal goat serum</td><td>Dianova</td><td>005-000-001</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A7906</td><td></td></tr><tr><td colspan="4">&lt;em&gt;Histology&lt;/em&gt;</td></tr><tr><td>Cresyl violet</td><td>Sigma-Aldrich</td><td>C5042</td><td></td></tr><tr><td>Neo-Clear</td><td>Merck Millipore</td><td>109843</td><td>non-toxic xylene substitute</td></tr><tr><td>Neo-Mount</td><td>Merck Millipore</td><td>109016</td><td>permanent mounting medium</td></tr><tr><td colspan="4">&lt;em&gt;Microscopy&lt;/em&gt;</td></tr><tr><td>Axioskop 2</td><td>Carl Zeiss Microscopy</td><td></td><td></td></tr><tr><td>LSM 710</td><td>Carl Zeiss Microscopy</td><td></td><td></td></tr></tbody></table>

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.

We applied the methods described above to quantify and characterize newborn cells in the postnatal and adult hippocampus. Therefore, we used wildtype and neurogenesis-deficient cyclin D2 knock out (Ccnd2KO) mice housed under conditions known to affect the rate of neurogenesis (i.e., enriched environment, EE)13,14. Immunohistochemical DAB staining against either Ki67, BrdU, CldU or IdU consistently revealed differences in newborn cell numbers between wildtype and Ccnd2KO mice (<strong...

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

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.

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

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Research

JoVE Journal

Neuroscience

26.5K Views.  Jena University Hospital. 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.

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

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

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.

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 ...

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.

We describe an immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. This protocol can be applied to both genetic or pharmacological mouse models characterized by cognitive deficits.

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

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.

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 ...

Double Labeling Immunofluorescence using Antibodies from the Same Species to Study Host-Pathogen Interactions

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to obtain commercial monoclonal or polyclonal antibodies that recognize specific cell structures and proteins in parasites. Besides, there are few commercial antibodies available to label trypanosomatids. Usually, polyclonal antibodies against parasites are prepared in-house and could be more challenging to use in combination with other antibodies produced in the same species. Here, the protocol demonstrates how to use polyclonal and monoclonal antibodies raised in the same species to perform double labeling immunofluorescence to study host cell and pathogen interactions. To achieve the double labeling immunofluorescence, it is crucial to incubate first the mouse polyclonal antibody and then follow the incubation with the secondary mouse IgG antibody conjugated to any fluorochrome. After that, an additional blocking step is necessary to prevent any trace of the primary antibody from being recognized by the next secondary antibody. Then, a mouse monoclonal antibody and its specific IgG subclass secondary antibody conjugated to a different fluorochrome are added to the sample at the appropriate times. Additionally, it is possible to perform triple labeling immunofluorescence using a third antibody raised in a different species. Also, structures such as nuclei and actin can be stained subsequently with their specific compounds or labels. Thus, these approaches presented here can be adjusted for any cell whose sources of primary antibodies are limited.

Here, the protocol describes how to perform double labeling immunofluorescence using primary antibodies raised in the same species to study host-pathogen interactions. Also, it can include the third antibody from a different host in this protocol. This approach can be made in any cell type and pathogens.

Nowadays, it is possible to find a wide range of molecular tools available to study parasite-host cell interactions. However, some limitations exist to ...

Immunology and Infection

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.

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

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

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&#39;-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.

This paper presents four of the most common techniques for visualizing BrdU positive cells to measure adult neurogenesis in rats. This work includes instructions for reagent preparation, thymidine analog administration, transcardial perfusion, tissue preparation, peroxidase immunohistochemical reaction, immunofluorescence, signal amplification, counterstaining, microscopy imaging, and cell analysis.

One of the most important things in the field of adult hippocampal neurogenesis (AHN) is the identification of the newly generated cells. The ...

Biology

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

Source: Quintanilla, L. J., et al. <a href="https://app.jove.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.

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

	...

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://app.jove.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.

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

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

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