immunofluorescence staining for visualizing proliferated neural stem cells in mouse brain sections

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

To stain the tissue sections with thymidine analog, remove the tissue sections from the citrate buffer. Let the tissue sections dry and completely adhere to the slides before drawing a border with a hydrophobic pen. Next, permeabilize the sections with permeabilization buffer for 20 to 30 minutes. Wash the sections twice using TBS Triton for 5 minutes each time. Then, prepare an EdU reaction solution.
Incubate the sections in EdU reaction solution for 30 minutes to an hour. Subsequently, wash them three times in TBS Triton for 5 minutes each time. At this stage, check if the EdU reaction works by using a fluorescent microscope. EdU-labeled cells should be observed if the reaction works. Now, block the mounted tissue sections using blocking buffer raised in the same animal as the secondary antibody for 30 minutes to an hour. Then, wash them twice in TBS Triton for 5 minutes each time.
During the blocking step, prepare primary antibody solution by mixing the primary antibody in blocking buffer. Subsequently, add 250 microliters of primary antibody solution per slide to ensure the tissue sections are completely submerged, and incubate them overnight at room temperature. The next day, wash the tissue sections three times in TBS Triton for 5 minutes each to remove excess primary antibody.
Then, incubate the tissue sections in fluorophore-conjugated secondary antibody prepared in blocking buffer solution for two hours at room temperature. Wash the tissue sections three times in TBS Triton for 5 minutes each to remove excess secondary antibody. Next, apply 300 micromolar DAPI solution at 1-to-100 in PBS for 15 minutes at room temperature. Afterward, wash the tissue sections three times in PBS for five minutes each to remove excess DAPI, and remove the PAP pen circle around the tissue using a cotton swab. Let the sections dry before applying mounting media and covering them with coverslips.

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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Immunohistochemistry
<ol>
	<li>Thymidine analog staining&#8203;
	<ol>
		<li>Place tissue sections in PBS (phosphate buffered saline) and mount 5&#8722;8 sections on a positively charged slide, maintaining serial order from anterior to posterior and the same orientation. Let tissue sections dry to completely adhere to slides, and then draw a border using a hydrophobic pen or PAP pen.</li>
		<li>Permeabilize sections with permeabilization buffer (0.5% TBS (tris buffered saline) -triton) for 20&#8722;30 min. Then wash sections 2x using TBS-triton for 5 min each. 
		&#8203;NOTE: Permeabilization aids intracellular antibody penetration. Permeabilization time can be adjusted depending on tissue thickness and antibody efficiency. Alternatively, one can increase detergent concentration to increase permeabilization potency. However, care should be taken to not permeabilize for too long since tissue fragility increases with prolonged permeabilization time.</li>
		<li>Prepare Edu (5-ethynyl-2&#39;-deoxyuridine) reaction solution according to Table 1. The final concentration of Alexa488-azide is 15 &#181;M in 1 mL of Edu reaction solution.</li>
		<li>Incubate sections in the Edu reaction solution for 30 min to 1 h and then wash 3x in TBS-triton for 5 min each. After this step, cover slides in aluminum foil to protect them from light. At this stage, check if the Edu reaction works by using a fluorescent microscope. Edu-labeled cells will fluoresce under an epifluorescence microscope.</li>
	</ol>
	</li>
	<li>Mounted tissue section protocol
	<ol>
		<li>Block tissue sections mounted on a slide from step 1.1.4 using blocking buffer raised in the same animals as the secondary antibody, e.g., donkey serum, for 30 min to 1 h and then wash 2x in TBS-triton for 5 min each.</li>
		<li>Prepare primary antibody (i.e., chicken anti-nestin) solution during the blocking step by mixing primary antibodies in blocking buffer solution at 1:200. 250 &#181;L per slide is sufficient to ensure that tissue is completely submerged in solution.</li>
		<li>Incubate tissue sections in a primary antibody solution overnight at RT after blocking washes. Modify this step depending on the primary antibody used. 
		NOTE: If antibodies have a high background or non-specific binding, incubating at 4 &#176;C instead of RT (room temperature) may improve results. Antibodies with poor tissue penetration can be left to incubate for 2 days if needed.</li>
		<li>Incubate tissue sections 3x in TBS-triton for 5 min to remove excess primary antibody. Then, incubate tissue sections in fluorophore-conjugated secondary antibodies (e.g., Alexa 647 anti-chicken at 1:200) prepared in blocking buffer solution for 2 h at RT.</li>
		<li>Incubate tissue sections 3x in TBS-triton for 5 min to remove excess secondary antibody and apply 300 &#181;M DAPI (4&#8242;,6-diamidino-2-phenylindole) solution at 1:100 in PBS for 15 min at RT.</li>
		<li>Incubate tissue sections 3x in PBS for 5 min to remove excess DAPI and remove the pap pen circle from around the tissue using a cotton swab or a delicate task wipe. Let sections dry, and then apply mounting media and coverslip. Let mounting media dry before imaging slides.</li>
	</ol>
	</li>
</ol>
Table 1: Solutions utilized for immunohistochemistry
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antifreeze Solution</td>
			<td>Ethylene-glycol 150 mL + sucrose 150 g + fill to 500 mL 0.1 M PB for 500 mL solution</td>
		</tr>
		<tr>
			<td>Citrate Buffer</td>
			<td>9 mL of citric acid stock + 41 mL of tri-sodium citrate buffer + 450 mL of ddH2O</td>
		</tr>
		<tr>
			<td>Citric acid stock</td>
			<td>[0.1 M] Citric Acid 21 g/1 L ddH2O</td>
		</tr>
		<tr>
			<td>Tri-sodium citrate stock</td>
			<td>[0.1 M] Tri-sodium Citrate 29.4 g/1 L ddH2O</td>
		</tr>
		<tr>
			<td>Tris Buffered Saline -Triton (TBS -Triton)</td>
			<td>0.05% 100-x Triton in TBS</td>
		</tr>
		<tr>
			<td>Permeabilization Buffer</td>
			<td>0.5% 100-x Triton in TBS</td>
		</tr>
		<tr>
			<td>Blocking Buffer</td>
			<td>0.33 mL Donkey Serum in 10 mL TBS-Triton</td>
		</tr>
		<tr>
			<td>Edu Reaction Solution</td>
			<td>Make a CuSO4&#183;5H2O solution by adding 1 mg of CuSO4&#183;5H2O in 4 mL solution of [0.1 M] Tris pH 8.5. Then add 1:40 of a 600 &#181;M Alexa488-azide solution and 10 mg/mL of L-Na+ ascorbate to the CuSO4&#183;5H2O solution before applying it to tissue.</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>5-Ethynyl-2&#39;-deoxyuridine (Edu)</td>
			<td>Carbosynth</td>
			<td>NE08701</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa-488 Azide</td>
			<td>Thermo Fisher Scientific</td>
			<td>A10266</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Chicken Nestin</td>
			<td>Aves</td>
			<td>NES; RRID: AB_2314882</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Goat DCX</td>
			<td>Santa Cruz</td>
			<td>Cat# SC_8066; RRID: AB_2088494</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Mouse Tbr2</td>
			<td>Thermo Fisher Scientific</td>
			<td>14-4875-82; RRID: AB_11042577</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Software (Zen Black)</td>
			<td>Zeiss Microscopy</td>
			<td>Zen 2.3 SP1 FP1 (black)</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper (II) Sulfate Pentahydrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>AC197722500</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Denville Scientific</td>
			<td>M1100-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Plus Charged Slide</td>
			<td>Denville Scientific</td>
			<td>M1021</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Solution (PBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr>
			<td>Super PAP Pen 4 mm tip</td>
			<td>PolySciences</td>
			<td>24230</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Buffered Solution (TBS)</td>
			<td>Sigma-Aldrich</td>
			<td>T5912</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>93443</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

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

	...

Take fixed mouse brain sections containing neural cells, including proliferated neural stem cells labeled with EdU, a thymidine analog.
Outline the sections with a hydrophobic marking to prevent solution spread.
Add a detergent to permeabilize the tissue. Wash to remove excess detergent.
Add a fluorophore to label the EdU. Remove the excess fluorophores.
Using a fluorescence microscope, confirm proper staining of the EdU-labeled cells.
Apply a blocking buffer containing proteins to prevent non-specific antibody binding. Remove the excess proteins.
Incubate with primary antibodies that bind to specific proteins in neural stem cells. Remove the unbound antibodies.
Add fluorophore-conjugated secondary antibodies to bind the primary antibodies. Wash off the unbound antibodies.
Stain the nuclei with a dye and remove the excess dye.
Remove the hydrophobic marking, apply mounting media, and place a coverslip.
Using a confocal microscope, observe the sections to visualize the labeled neural stem cells.

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Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

Cell cycle analysis in eukaryotes frequently utilizes chromosome morphology, expression and/or localization of gene products required for various phases of the cell cycle, or the incorporation of nucleoside analogs. During S-phase, DNA polymerases incorporate thymidine analogs such as EdU or BrdU into chromosomal DNA, marking the cells for analysis. For C. elegans, the nucleoside analog EdU is fed to the worms during regular culture and is compatible with immunofluorescent techniques. The germline of C. elegans is a powerful model system for the studies of signaling pathways, stem cells, meiosis, and cell cycle because it is transparent, genetically facile, and meiotic prophase and cellular differentiation/gametogenesis occur in a linear assembly-like fashion. These features make EdU a great tool to study dynamic aspects of mitotically cycling cells and germline development. This protocol describes how to successfully prepare EdU bacteria, feed them to wild-type C. elegans hermaphrodites, dissect the hermaphrodite gonad, stain for EdU incorporation into DNA, stain with antibodies to detect various cell cycle and developmental markers, image the gonad and analyze the results. The protocol describes the variations in the method and analysis for the measurement of S-phase index, M-phase index, G2 duration, cell cycle duration, rate of meiotic entry, and rate of meiotic prophase progression. This method can be adapted to study the cell cycle or cell history in other tissues, stages, genetic backgrounds, and physiological conditions.

Cell Cycle Analysis in the C. elegans Germline with the Thymidine Analog EdU

An imaging-based method is described that can be used to identify S-phase and analyze cell cycle dynamics in the C. elegans hermaphrodite germline using the thymidine analog EdU. This method requires no transgenes and is compatible with immunofluorescent staining.

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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.

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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.

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

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