Name: immunohistochemistry techniques to analyze cellular proliferation and neurogenesis in rats using the thymidine analog brdu
Uploaded: 2020-09-09T14:30:00
Duration: 8 min 37 s
Description: Watch this Scientific Journal Video about Immunohistochemistry Techniques to Analyze Cellular Proliferation and Neurogenesis in Rats Using the Thymidine Analog BrdU at JoVE.com

We would like to thank Mr. Miguel Burgos, and Gustavo Lago for providing technical assistance. We also want to thank Dr. Clorinda Arias, Dr. Karla Hernandez and Dr. Oscar Galicia for their kind support in providing reagents and material. We also thank Divisi&#243;n de Investigaci&#243;n y Posgrado of the Universidad Iberoamericana Ciudad de M&#233;xico for providing funding for the performance of this work and for covering video production expenses.

<ol>
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W., Urbach, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+and+multiple+labeling+with+antibodies+from+the+same+host+species+to+study+adult+hippocampal+neurogenesis.">Immunohistochemistry and multiple labeling with antibodies from the same host species to study adult hippocampal neurogenesis.</a> Journal of Visualized Experiments. (98), e52551 (2015).</li><li>Tuttle, A. H., 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=Immunofluorescent+detection+of+two+thymidine+analogues+(CldU+and+IdU)+in+primary+tissue.">Immunofluorescent detection of two thymidine analogues (CldU and IdU) in primary tissue.</a> Journal of Visualized Experiments. (46), e2166 (2010).</li><li>Tischler, A. 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R., Gould, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thymidine+analog+methods+for+studies+of+adult+neurogenesis+are+not+equally+sensitive.">Thymidine analog methods for studies of adult neurogenesis are not equally sensitive.</a> Biosystems. 517 (2), 123-133 (2010).</li><li>Magavi, S. S., MacKlis, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+newborn+cells+by+BrdU+labeling+and+immunocytochemistry+in+vivo.">Identification of newborn cells by BrdU labeling and immunocytochemistry in vivo.</a> Methods in Molecular Biology. 438, 335-343 (2008).</li><li>Kempermann, G., Gast, D., Kronenberg, G., Yamaguchi, M., Gage, F. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+immunohistochemical+methods+using+two+different+antigen+retrieval+methods+on+formalin-fixed,+paraffin-embedded+tissues:+Experience+with+63+markers.">Optimization of immunohistochemical methods using two different antigen retrieval methods on formalin-fixed, paraffin-embedded tissues: Experience with 63 markers.</a> Journal of Veterinary Diagnostic Investigation. 12 (4), 307-311 (2000).</li><li>Ramos-Vara, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+and+methods+of+immunohistochemistry.">Principles and methods of immunohistochemistry.</a> Methods in Molecular Biology. 1641, 115-128 (2017).</li><li>Wojtowicz, J. 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The authors have nothing to disclose.

Adult neurogenesis is a process that occurs most frequently in niches of adult neural precursor cells that have the potential to generate new neurons throughout their lifespans. Bromodeoxyuridine (BrdU) labeling is widely used in immunology to characterize the number of newly generated cells in an adult brain. BrdU will be mainly incorporated into cells of discrete brain regions (neurogenic zones). These cells are located in the sub-ventricular zone (SVZ), the dentate gyrus of the hippocampus&#8212;between the hilus and granular cells know as sub-granular zone (SGZ)1,2,18. Moreover, there are different brain regions characterized by a lower proliferative capacity in adulthood, including the hypothalamus, striatum, neocortex, and amygdala19. As mentioned before, BrdU staining is the commonly used method for adult neurogenesis research to detect cell proliferation. However, the use of BrdU as a marker has limitations and pitfalls. The first one is that BrdU is a cell cycle marker. Therefore, double or triple staining must be performed to identify the cell fate and include cell markers to detect the specific developmental stage of the cells labeled. One more concern about BrdU is that it is a toxic and mutagenic solution that modifies DNA stability may alter cellular function and cell cycles. Consideration should be given to the previous information when deciding to follow an administration protocol and administration doses (50&#8210;600 mg/kg). Another crucial feature is that BrdU is a DNA synthesis marker, not a cell proliferation marker14. Therefore, it is relevant to distinguish cell proliferation from other events such as a DNA repair, abortive cell cycle re-entry, and gene duplication. Researchers must follow appropriate controls to ensure the appropriate use of BrdU. For a more detailed discussion about these problems and limitations, we recommend reviewing Taupin&#39;s work14. The standardization process of an immunohistochemistry protocol could be slow and challenging. In this work, we have presented all the general steps to manage a successful IHC protocol. However, we recommend that every research group test and evaluate tissue, antibodies, and conditions in advance. Tests and evaluations must be carried out with at least three different levels of incubations, washing steps, and strengths for each antibody and tissue tested. We also recommend that researchers review additional protocols to be able to choose the best one that meets specific needs and requirements20,21,22,23,24,25.
As mentioned previously, the procedure involves several steps and methodological considerations that are commonly used and mentioned in scientific articles, which will be later discussed. We recommend that researchers choose the antibodies carefully and correctly in terms of technique, budget, equipment, setup, and main research goal. Antibodies must be tested with the same type of tissue that will be later tested in the experiment. We also recommend the use of an antibody that was tested for the same purpose (IHC) (i.e., not just in western blot or flow cytometry techniques) to test its compatibility with the fixation technique. Different routes could be used to administer the BrdU staining such as intraperitoneal injection, intraperitoneal infusion, oral ingestion, or intraventricular infusion (for a more detailed description of each technique, see reference26). If the intraperitoneal injection is selected, make sure that BrdU is administrated into the peritoneal cavity avoiding the intestine area. Since the intestine has several cells in duplication that can exhaust the BrdU before it gets to the brain which will affect the number of labeled cells. It is crucial to obtain thin sections since they allow a better penetration of solutions. Coronal slices of 40 &#181;m thick were cut rostro-caudally and were transferred into a 24-well cell culture plate, following the stereological procedure proposed by Kempermann et al.27. The immunohistochemistry can be carried out with tissue mounted on slides or as free-floating sections. Since BrdU is located deep in cells nuclei, it allows the penetration of solutions in free-floating sections which provides better results and better access to the area of interest. It is important to open DNA bonds (DNA denaturation) to allow the primary anti-BrdU antibody access. In this work, we carried out these specific procedures with the use of HCI incubation. On the other hand, the process of blocking unspecific epitopes allowed a more accurate identification of cell signal.
The good membrane permeabilization allows the antibodies to properly penetrate the interest area. Adding a permeabilizer such as Triton X-100 to PBS++ and PBS+ solutions improves membrane permeabilization. Both PBS and Tris-buffered saline (TBS) reagents can be used in this protocol. In terms of budget, the TBS could be relativity cheaper than the PBS. However, PBS could interfere with anti-phosphate antibodies and inhibit alkaline phosphatase-conjugated antibodies, so avoid the use of PBS if the target is post translationally modified by phosphorylation (i.e., phosphorylated). We used the PBS for this work, and we found out that tissue washing steps gave a more specific signal. We also recommend researchers to carry out at least three washing cycles using either TBS o PBS. The solutions must be freshly prepared. Antigen retrieval (AR) is a method intended to reduce the loss of antigenicity caused by the fixation which modifies the tertiary and quaternary antigens&#39; structure. This reduction makes antigens undetectable by antibodies28,29. The heat-induced epitope retrieval (HIER) used in this protocol attempted to reverse the chemical reactions between formaldehyde and proteins by high temperature or strong alkaline hydrolysis (with other buffer solutions as EDTA pH 8.5 or Tris pH 9.5). It is essential to test new antibodies with different AR protocols to compare results and choose the best one for the protocol. This last step might be optional in a regular protocol; however, we treated tissues with an antigen retrieval protocol to provide better results for this protocol.
It is crucial to select the correct final contrasting color and the counterstain technique in consideration of the primary staining color and method used to make a non-staining structure visible and avoid masking the primary staining color from the immune reaction. For the fluorescence microscopy, the DAPI (4&#39;, 6-diamidino-2-phenylindole) is a very popular nuclear and chromosome counterstain that emits blue fluorescence (absorption: 360 nm, emission: 460 nm) upon binding to AT regions of DNA. DAPI-containing mounting medium is available and is easy to use; this provides excellent signal retention for image acquisition. For the peroxide reaction, IHC was available in different options such as cresyl violet, hematoxylin, neutral red, or methyl green staining. For multiple immunostaining techniques, it is crucial to choose a compatible antibody with the fixation technique used to avoid cross-reactivity30. When issues and complications with single staining are solved, administer another color staining as deemed necessary. It is crucial to control the non-specific binding between the secondary antibodies. This could be done by saturating the primary antibodies before using a secondary antibody produced in the same host species of the primary antibodies. For example, when using anti-mouse produced in rabbit and anti-rabbit produced in goat secondary antibodies, the anti-rabbit produced in goat antibody must be used before the anti-mouse produced in rabbit antibody. When the sequential method is dominated completely, then the simultaneous immunostaining process can be initiated. In this method, it is essential to choose secondary antibodies appropriately. Ideally, all those antibodies must come from the same host animal to avoid cross-reactivity. We recommend running a positive control to confirm that the staining method works accurately in postnatal hippocampus tissue (abundant neurogenesis around this age). If the positive control tissue shows staining problems, review and go over the procedure, make corrections and adjustments, and repeat until a good staining is produced. Then, run a negative control to test that the antibody works correctly by omitting or replacing a particular primary antibody with normal serum (same species as the primary antibody). As mentioned in the introduction, image deconvolution is a powerful tool and provides an alternative when a confocal microscope is not available. It can apply the image deconvolution to all images gained using transmitted light bright-field, wide-field fluorescence, and confocal fluorescence microscopy. The ultimate purpose of image deconvolution is to reconstruct the original signal that the acquisition system deteriorates10.
In summary, the identification of the newly generated cells visualized by the immunodetection of thymidine analog BrdU is a complicated but powerful technique. This work is an attempt to help scientists, particularly in the field of adult hippocampal neurogenesis, to quantify new cells more accurately. We hope that this effort has been helpful to the scientific community and makes it easier to fine-tune the study of cell proliferation by the immunohistochemistry technique.

The idea that new neurons are generated in the adult human brain throughout the lifespan has fascinated the scientific community for decades. The knowledge that the brain generates new neurons throughout its lifespan was attained through the detection of cells under division1,2. Detection of newly generated neurons in the adult brain was first identified by intracranially injecting tritiated thymidine (thymidine-H3) in rats and detecting cells in the cell cycle by autoradiograms1,2. Cell division of glia and the presence of neuroblasts was reported, which was the first promising data on the postnatal neurogenesis1. Nevertheless, the use and detection of thymidine-H3 implied the use of radioactivity, which can be harmful to the people who manage it. The first effort examining the suitability of BrdU immunohistochemistry in the study of proliferation, migration, and origin of cells in the nervous systems appeared in 1988 by Miller and Nowakowski3. In 1998, a paper published by Eriksson and colleagues showed that new neurons were visualized postmortem in the human adult brain of patients injected with 5-Bromo-2&#8242;-deoxyuridine (BrdU)4. These patients received the BrdU injection (250 mg intravenous) to label the growth of tumors4. This technique was adopted into animal models. The introduction of these methods marked a milestone for the field since this allowed detection of newly generated cells without the use of radioactive compounds. This procedure became the gold standard to measure cell proliferation in adult brain niches to promote further research in the field.
The limitation of the thymidine analog technique is that it does not allow the determination of cellular identity for the newly generated cells. However, immunohistochemistry allows us to carry out double- or triple- labeling technique of the same cell, which validates the cellular fate of the newly generated cells and even their stages of maturation, leading to further evolution of the field. This method was characterized to differentiate newly generated cells into glia, undifferentiated neurons, or a fully mature granular cell, and even to determine if they are participating actively in the circuitry. Another breakthrough in the field was the use of transgenic models to identify undifferentiated cells under the domain of nestin. The nestin-GFP transgenic mice express an enhanced green fluorescent protein (GFP), which is under the control of the nestin promoter. Nestin is an intermediate filament characterized by progenitor cells5. The nestin-GFP transgenic mice allowed to establish early developmental steps involved in neurogenesis6. However, a significant limitation is to be able to maintain a nestin-GFP transgenic mice colony under special conditions in a laboratory facility that becomes cost-effective for some scientific groups, especially those from developing countries.
The techniques mentioned above have advantages and disadvantages. However, identification of proliferating cells by immunohistochemistry (IHC) and the possibility to carry out double- or triple- labeling technique by immunofluorescence to identify cell maturation stage or cell fate represents the most feasible way to measure adult neurogenesis, so far. The identification process using immunohistochemistry consists of labeling proteins, protein domain, or nucleotides with a specific antibody that allows their recognition known as primary antibody. The latter is recognized by the secondary antibody, which is marked with a chromogen (e.g., horseradish peroxidase) or a fluorochrome (e.g., FITC) coupled with the secondary antibody. Microscopes can detect both chromogens and fluorochromes signals. Using IHC, it is possible to identify membrane proteins, cytoskeleton proteins, or nuclear components such as BrdU. On the other hand, BrdU can be found in the cellular nucleus since it is incorporated into the DNA during S-phase by competition. Therefore, a crucial step is the DNA denaturation with HCl, which opens DNA bonds to allow the BrdU antibody access to BrdU within the DNA. It is essential to know that BrdU is present in a saturated concentration in mice and rat serum for 15 and 60 min respectively, after intraperitoneal administration, then drops rapidly to undetectable levels at 60 and 120 min respectively7.
Here, we describe four different but closely related IHC techniques: chromogenic indirect detection using horseradish peroxidase (HRP) reaction with DAB (3,3&#39;-diaminobenzidine) sans signal amplification (step 4.1), avidin-biotin complex (ABC) amplification (step 4.1), indirect immunofluorescence detection without signal amplification (step 4.4) and labeled streptavidin-biotin (LSAB) amplification (step 4.3). Each method has advantages and disadvantages and could be useful for specific tissue requirements (see Table 1). We decided to follow indirect ICH methods due to their affordability and simplicity to make changes from chromogenic to fluorescent detection methods when using unconjugated primary antibodies. The HRP approach is a commonly used IHC method due to its affordability, high stability, high turnover rate, and substrates&#39; full availability. Nevertheless, we recommend using a positive control to confirm that the staining method works accurately and the use of negative control to test the antibody function effectively. Multiple immunostainings or multiplex IHC methods (see step 6) are potent tools to acquire large amounts of data from the tissue section in a single experiment. This technique is particularly important when the availability of samples is limited. Another advantage is the possibility to simultaneously identify specific proteins co-expressed in the same cellular space while preserving tissue integrity. Multiplex allows to stain different markers expressed during specific proliferative stages (e.g., nestin, GFAP, DCX, Ki-67), enabling us to reach a more detailed proliferation and differentiation research8. It is crucial to choose antibodies compatible with the fixation technique used to avoid cross-reactivity. We recommend testing each new antibody (including BrdU) individually to adjust and refine the method. Then, introduce the double sequential staining and, finally, start the simultaneous immunostaining process when the sequential method is entirely dominated. It is crucial to choose appropriate secondary antibodies for this method.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Method</td>
			<td>Specific Method</td>
			<td>Advantages</td>
			<td>Disadvantages</td>
		</tr>
		<tr>
			<td rowspan="2">Indirect Detection Method</td>
			<td>Peroxidase reaction with DAB</td>
			<td>1. Higher sensitivity than the direct detection and indirect fluorescence method. 
			2. Higher resistance to Photobleaching than fluorochromes. 
			3. Lower cost than fluorescence detection method</td>
			<td>1. Difficult for Multiplexing with fewer color dyes. 
			2. Complicated for Co-expressed targets in the same cellular space. 
			3. Reduced Dynamic Range for simultaneous scarce and high abundant targets on the same tissue.</td>
		</tr>
		<tr>
			<td>Fluorescence</td>
			<td>1. Best and easiest for Multiplexing with more color dyes. 
			2. Best for Co-expressed targets in the same cellular space. 
			3. Better Dynamic Range for simultaneous scarce and high abundant targets on the same tissue. 
			4. No Additional Steps.</td>
			<td>1. Lower sensitivity than the indirect peroxidase reaction with DAB method. 
			2. Weak resistance to Photobleaching over time. 
			3. More expensive.</td>
		</tr>
		<tr>
			<td rowspan="3">Signal Amplification Method</td>
			<td>Avidin-Biotin Complex (ABC)</td>
			<td>1. Higher sensitivity than the direct and indirect detection method. 
			2. Reduce background</td>
			<td>1. Additional Steps. 
			2. More expensive than not amplification.</td>
		</tr>
		<tr>
			<td>Labeled Streptavidin-Biotin (LSAB)</td>
			<td>1. Higher sensitivity than the direct and indirect detection method. 
			2. More substantial tissue penetration than the ABC method. 
			3. Reduce background</td>
			<td>1. Additional Steps. 
			2. More expensive than the ABC method.</td>
		</tr>
		<tr>
			<td>Not additional amplification method</td>
			<td>1. Lower cost. 
			2. No additional steps. 
			3. Ideal for high abundant targets.</td>
			<td>1. Lower sensitivity: problematic with no abundant targets.</td>
		</tr>
	</tbody>
</table>
Table 1: Advantages/disadvantages of IHC techniques. This table shows the advantages/disadvantages for indirect detection methods: Peroxidase reaction with (3,3&#39;-diaminobenzidine) DAB and fluorescence; and signal amplification methods: avidin-biotin complex (ABC), labeled streptavidin-biotin (LSAB), and not additional amplification method.
A high-resolution image is fundamental to perform proper analysis and present the outcomes. There are two approaches to improve the resolution: 1) use of a better microscope design (e.g., confocal, multiphoton) or 2) numerically inverting the blurring process to enhance images using deconvolution9. Unfortunately, confocal microscopy is not affordable due to the high costs of equipment and its servicing10. A wide-field epifluorescence microscope and the subsequent deconvolution of the z-stack images provide a suitable, low-cost alternative to confocal microscopy8,9. As noted above, deconvolution goal is to restore the original signal that was degraded by the acquisition system9, by reducing blur, out-of-focus haze and distortion shown in the image obtained by an epifluorescence or confocal microscope using mathematical removal algorithms10. The acquired blurred image can be mathematically modeled as the result of convolving the observed objects with a 3D point-spread function (PSF). PSF is a theoretical diffraction pattern of the points of light emitted by the tissue sample and collected by the microscope. PSF file is created with the specific conditions of each image, such as the CCD cell spacing of the camera, refractive index of the media used, the numerical aperture of the objective lens, the emission wavelength of the fluorophore, image sizes, number of images in the z-stack processing method and the space between them (see technical specification in Table 2). In other words, the PSF file summarizes the effects of the imaging setup on the microscope observations9. However, we use the diffraction PSF 3D Plugin (https://imagej.net/Diffraction_PSF_3D) to create our own specific PSF file for each z-stack image. Z-stack images are a series of digitized optical sections from defined depths (z-axis) at the same XY location of the slide. A computer compiles the information obtained from the focus plane by reassigning signals which have originated from objects located in other focal planes. To create z-stack images, it is necessary to take images from different focused layers of the slides (e.g., ten different images of the same XY area every 1 &#956;m depth). Then, we use microscopy software provided by the manufacturer or Fiji to create a z-stack or 3D image. The result will be a single stack image file (e.g., ten images with different focuses). There are several customer-specific tools and software solutions, such as open-source software for deconvolution microscopy. We will show the outputs of the deconvolution process using DeconvolutionLab29 which is a Fiji11 plugin (distribution of ImageJ12). Deconvolution will help improve the resolution of final micrographs (see Figure 1B,C ). For further information and instruction, we strongly recommend reading reference13.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61483/61483fig01.jpg" /> 
Figure 1: Representative image of 3D deconvolution for multiple color channels.&#160;(A) DG at low magnification. (B) The original z-stack images for each channel and the merged image. (C) 3D deconvoluted z-stack images for each channel and the merged image. This brain was from the rat that was the part of physical activity group. Labeled streptavidin-biotin (LSAB) amplification method was used. It showed Cy3 streptavidin conjugated antibody for indicating BrdU (red), DAPI as a counterstaining (blue), and glial fibrillary acidic protein (GFAP) as an astroglial marker (green). ML = molecular layer; GCL = granular cell layer; SGZ = subgranular zone. <a href="https://www.jove.com/files/ftp_upload/61483/61483fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The purpose of this work is to provide a detailed description of the steps to obtain positive and successful outcomes with immunostaining and to list commonly used steps in BrdU-based studies, without the use of a confocal microscope. BrdU staining is a technique that requires several steps that must be carefully followed to achieve a successful stain. Standardizing these staining techniques typically takes months and is time and resource intensive. We anticipated that this article could provide information to the groups starting out within this field by reducing time and errors.

<table>
	<tbody>
		<tr>
			<td>REAGENT PREPARATION AND SETUP</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Donor Horse Serum</td>
			<td>BioWest</td>
			<td>S0900</td>
			<td>Blocking and incubation solutions in PBS</td>
		</tr>
		<tr>
			<td>Paraformaldehyde reagent grade, crystalline (PFA)</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>toxic, flammable</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>746436-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate, monobasic</td>
			<td>J.T Bker</td>
			<td>3246-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sacarose</td>
			<td>J.T Baker</td>
			<td>4072-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Meyer</td>
			<td>2365-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>S5881-500G</td>
			<td>Corrisive, to calibrate pH</td>
		</tr>
		<tr>
			<td>Sodium Phophate Dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>S9763-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-x 100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>THYMIDINE ANALOGUE BRDU ADMINISTRATION</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-2&#8242;-deoxyuridine, BrdU</td>
			<td>Sigma-Aldrich</td>
			<td>B9285</td>
			<td>toxic (mutagenic, teratogenic)</td>
		</tr>
		<tr>
			<td>23&#8211;27G hypodermic needle</td>
			<td>BD PrecisionGlide</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Saline solution</td>
			<td>PiSA</td>
			<td>30130032</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringes 1 mL</td>
			<td>NIPRO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TISSUE PREPARATION</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15-ml polypropylene conical tube</td>
			<td>Thermo Scientific</td>
			<td>339650</td>
			<td></td>
		</tr>
		<tr>
			<td>50-ml polypropylene conical tube</td>
			<td>Thermo Scientific</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting tools</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Guillotine</td>
			<td>Stoelting</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome Cryostat</td>
			<td>MICROM</td>
			<td>HM525</td>
			<td></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 12-well carrier kit</td>
			<td>Corning</td>
			<td>3520</td>
			<td>for 15 mm polyester mesh membrane inserts</td>
		</tr>
		<tr>
			<td>Netwell plastic 6-well carrier kit</td>
			<td>Corning</td>
			<td>3521</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion pump</td>
			<td>Cole-Parmer</td>
			<td>7553-70</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>IKA</td>
			<td>ROKCER 3D Digital</td>
			<td></td>
		</tr>
		<tr>
			<td>IMMUNOSTAINING</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cresyl violet</td>
			<td>Sigma-Aldrich</td>
			<td>C5042</td>
			<td>1%, Light sensitive</td>
		</tr>
		<tr>
			<td>DAB Peroxidase (HRP) Substrate Kit (with Nickel), 3,3&#8217;-diaminobenzidine</td>
			<td>Vector Laboratories</td>
			<td>SK-4100</td>
			<td>carcinogenic, light sensitive</td>
		</tr>
		<tr>
			<td>Hydochloric Acid</td>
			<td>J.T.Baker</td>
			<td>9535-05</td>
			<td>Corrosive, to calibrate pH</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide, 50%</td>
			<td>Meyer</td>
			<td>5375-1L</td>
			<td>Toxic, oxidative</td>
		</tr>
		<tr>
			<td>Permount Mounting Medium</td>
			<td>Fisher Chemical</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASHIELD Antifade Mounting Medium with DAPI</td>
			<td>Vector Laboratories</td>
			<td>H-1200</td>
			<td>Light sensitive</td>
		</tr>
		<tr>
			<td>VECTASTAIN&#174; Elite&#174; ABC Kit Peroxidase (HRP)</td>
			<td>Vector Laboratories</td>
			<td>PK-6100</td>
			<td>enzymatic, avidin/biotin based amplification system</td>
		</tr>
		<tr>
			<td>Primary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-GFAP antibody produced in rabbit</td>
			<td>Sigma-Aldrich</td>
			<td>HPA056030</td>
			<td>1:500</td>
		</tr>
		<tr>
			<td>Monoclonal Anti-BrdU antibody produced in Mouse</td>
			<td>Sigma-Aldrich</td>
			<td>B2531</td>
			<td>1:250</td>
		</tr>
		<tr>
			<td>Secondary antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-SP (long spacer) AffiniPure Donkey Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-065-151</td>
			<td>1:250</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, HRP</td>
			<td>Invitrogen</td>
			<td>G-21040</td>
			<td>1:1000</td>
		</tr>
		<tr>
			<td>Goat Anti-Mouse IgG (whole molecule), TRITC</td>
			<td>Sigma-Aldrich</td>
			<td>T5393</td>
			<td>1:250</td>
		</tr>
		<tr>
			<td>Goat Anti-Rabbit IgG (H+L) Cross-Adsorbed, FITC</td>
			<td>Invitrogen</td>
			<td>F-2765</td>
			<td>1:250</td>
		</tr>
		<tr>
			<td>Streptavidin, Cy3</td>
			<td>Vector Laboratories</td>
			<td>SA-1300</td>
			<td>1:250</td>
		</tr>
		<tr>
			<td>IMAGING AND ANALYSIS</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td>Dell Computer Company</td>
			<td>T8P8T-7G8MR-4YPQV-96C2F-7THHB</td>
			<td>For controlling and monitoring protocols&#8217; processes</td>
		</tr>
		<tr>
			<td>CCD-camera</td>
			<td>Olympus</td>
			<td>UC50</td>
			<td></td>
		</tr>
		<tr>
			<td>CellSens Standard software</td>
			<td>Olympus</td>
			<td>cellSens Standard Edition</td>
			<td>Acquisition Software</td>
		</tr>
		<tr>
			<td>Epifluorescent microscope</td>
			<td>Olympus</td>
			<td>BX53</td>
			<td></td>
		</tr>
		<tr>
			<td>High-pressure 130 W mercury arc lamp</td>
			<td>Olympus</td>
			<td>U-HGLGPS</td>
			<td></td>
		</tr>
		<tr>
			<td>low autofluorescence immersion oil</td>
			<td>Olympus</td>
			<td>MOIL-30 Type F</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro cover-glasses (VWR, cat no 48404 454; 24&#160; 60 mm)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides (VWR, cat no 48323-185; 76&#160; 26 mm)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>U-FBW filter cube</td>
			<td>Olympus</td>
			<td>U-FBW</td>
			<td>excitation 460 - 495 nm, dichroic mirror 505 nm, suppression 510 nm</td>
		</tr>
		<tr>
			<td>U-FGW filter cube</td>
			<td>Olympus</td>
			<td>U-FGW</td>
			<td>excitation 530 - 550 nm, dichroic mirror 570 nm, suppression 575 nm</td>
		</tr>
		<tr>
			<td>U-FUW filter cube</td>
			<td>Olympus</td>
			<td>U-FUW</td>
			<td>excitation 340 - 490 nm, dichroic mirror 410 nm, suppression 420 nm</td>
		</tr>
	</tbody>
</table>

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.

The methods described above were applied to quantify newborn cells in adult rat hippocampus after voluntary physical activity, in contrast with a control group without any extra physical activity. We used the postnatal rat hippocampus as a positive control. Male rats 3 months of age were under a voluntary physical activity protocol (endless wheel) for seven days. On day 6, rats were injected with BrdU (section 2), and every 12 h after until three complete injections. To complete three cell cycle divisions, the animals were transcardially perfused (section 3) 8 h after the last BrdU injection. The same procedure was used on three-month old rats which did not undergo physical activity to be used as a comparative control. As a positive control, one-day-old rat pups (postnatal day 1) were injected with BrdU one time, as described in section 2 above. One day after the injection (postnatal day 2), pups were euthanized, and their heads were immersed in PFA solution, as described in step 3.4. Adult rats were deeply anesthetized (step 3.1), transcardially perfused, as described in step 3.2. Brains were dissected and post-fixated (step 3.4). Brains were cut into 40 &#956;m coronal sections (step 3.5). Sections were processed for BrdU immunohistochemistry, as described in step 4.
We used horseradish peroxidase reaction with DAB IHC for staining (step 4.1) and counting BrdU-positive cells in DG. Figure 5 shows a DG section with BrdU-labeled cells. Figure 5C,D shows a representative part of the DG section at higher magnification. Labeled cells showed intense dark staining, which were marked with arrows. The inset shows the average numbers of labeled cells in the experimental and control groups (counted positive cells multiplied by six as described in step 8.1). A Student&#8217;s t-test revealed significance differences between the numbers of BrdU-positive cells (t(10) = 2.704, p = 0.0222). The control group that did not undergo physical activity showed 2,040 &#177; 314 cells (n&#8202;=&#8202;6 rats). In comparison, the physical activity group showed, on average, 3,606 &#177; 486 (n&#8202;=&#8202;6 rats) BrdU-positive cells. As observed, physical activity exposure increases BrdU-positive cells. Therefore, these results are consistent with other reported results that show that physical activity increased cellular proliferation in the adult dentate gyrus17.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61483/61483fig03.jpg" /> 
Figure 3: Examples of different morphology of BrdU-labeled cell nucleus.&#160;BrdU is a DNA synthesis marker which labels the nucleus. In the hippocampus region, the BrdU-positive nuclei had a semi-oval shape located in the dentate gyrus sub-granular zone. Since BrdU is incorporated by competition, the amount incorporated for every cell will have a variation that will later be reflected in how the nucleus will be visualized. (A) Immunofluorescence image. (B) An image using peroxidase reaction without an additional amplification method is presented. Yellow arrows show artifacts and non-specific signals. Black or white arrows show BrdU+ cells. 1 &#8211; Fully filed nucleus, semi-oval nuclei highly colored. 2 &#8211; Nuclei with dots, the border of the nuclei is marked and has inside several dots. 3 &#8211; Nuclei with few dots, the border of the nuclei are marked and have a small number of dots inside. 4 &#8211; Small nucleus is possible cells in a different differentiation stage but still part of the niche. 5 &#8211; Clusters are precursor cells under division, therefore several cells together in condensed groups can be observed. Within these groups, the counting must be done especially carefully to avoid mislabeling positive cells. Red arrows show the nucleus under division that can be confused to be a single cell. Each cell is enclosed in a box and can be distinguished in a Z-axis plane in real-time. <a href="https://www.jove.com/files/ftp_upload/61483/61483fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61483/61483fig04.jpg" /> 
Figure 4: Representative RGB image for single and merged channels.&#160;The top image shows the original z-stack image, and the lower image shows the 3D deconvoluted z-stack image. (A) Low magnification of the DG. (B) RGB image for each channel, and (C) RGB merged image. This was a brain from the control group. Immunofluorescence was used without an additional amplification method. BrdU (red), DAPI as a counterstaining (blue), and GFAP (glial fibrillary acidic protein) as an astroglial marker (green). ML = molecular layer; GCL = granular cell layer; SGZ = subgranular zone. <a href="https://www.jove.com/files/ftp_upload/61483/61483fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61483/61483fig05.jpg" /> 
Figure 5: Representative DG section with BrdU labeled cells (intense dark) for each experimental group.&#160;The peroxidase reaction was used with the avidin-biotin-peroxidase complex amplification method. (A, B) Show a low magnification of the DG, and (C, D) show the box area at higher magnification. Panels A and C are tissues from the physical activity group, panels B and D are from the control group. The inset shows the average numbers of labeled cells in the physical activity and control groups (counted positive cells multiplied by six as described in step 8.1). ML = molecular layer; GCL = granular cell layer; SGZ = subgranular zone; arrows indicate BrdU+ cells. <a href="https://www.jove.com/files/ftp_upload/61483/61483fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Video showing a different focus of positive cells along the z-axis distributed in different layers. <a href="https://cloudflare.jove.com/files/ftp_upload/61483/VIDEO_1.avi" target="_blank">Please click here to download this video.</a>
Video 2: Video showing a different focus of positive cell clusters along the z-axis distributed in different layers. Move slowly over the z-axis to quantify all several nuclei that integrate a cluster. <a href="https://cloudflare.jove.com/files/ftp_upload/61483/VIDEO_2.mp4" target="_blank">Please click here to download this video.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Microscope Type:</td>
			<td colspan="5">Epifluorescence Microscope Olympus BX53</td>
		</tr>
		<tr>
			<td>Light Source:</td>
			<td colspan="5">High-pressure 130 W mercury arc lamp (U-HGLGPS)</td>
		</tr>
		<tr>
			<td>Acquisition Software:</td>
			<td colspan="5">CellSens Standard</td>
		</tr>
		<tr>
			<td rowspan="4">Filters Sets:</td>
			<td colspan="2">Catalog Number</td>
			<td>Excitation Range</td>
			<td>Dichromatic mirror</td>
			<td>Suppression Range</td>
		</tr>
		<tr>
			<td colspan="2">U-FUW</td>
			<td>340 - 490 nm</td>
			<td>410 nm</td>
			<td>420 nm</td>
		</tr>
		<tr>
			<td colspan="2">U-FBW</td>
			<td>460 - 495 nm</td>
			<td>505 nm</td>
			<td>510 nm</td>
		</tr>
		<tr>
			<td colspan="2">U-FGW</td>
			<td>530 - 550 nm</td>
			<td>570 nm</td>
			<td>575 nm</td>
		</tr>
		<tr>
			<td rowspan="4">Camera:</td>
			<td colspan="2">Model:</td>
			<td colspan="3">CCD-camera UC50</td>
		</tr>
		<tr>
			<td colspan="2">Spectral range:</td>
			<td colspan="3">290 &#8211; 1000 nm</td>
		</tr>
		<tr>
			<td colspan="2">CCD chip size:</td>
			<td colspan="3">2/3 in, 2588 (Width *7) X 1960 (Height *8) pixels</td>
		</tr>
		<tr>
			<td colspan="2">Pixel Size:</td>
			<td colspan="3">3.4 X 3.4 &#181;m</td>
		</tr>
		<tr>
			<td rowspan="5">Fluorochrome:</td>
			<td colspan="2">Name</td>
			<td>Excitation Wavelength (nm)</td>
			<td>Emission *3 Wavelength (nm)</td>
			<td>Emission Color</td>
		</tr>
		<tr>
			<td colspan="2">4, 6-diamidino-2-phenyl-indole HCI (DAPI)</td>
			<td>345</td>
			<td>455</td>
			<td>Blue</td>
		</tr>
		<tr>
			<td colspan="2">Tetramethylrhodamine-isothyocyanate (TRITC)</td>
			<td>541</td>
			<td>572</td>
			<td>Red</td>
		</tr>
		<tr>
			<td colspan="2">Fluorescein-isothiocyanate (FITC)</td>
			<td>494</td>
			<td>519</td>
			<td>Green</td>
		</tr>
		<tr>
			<td colspan="2">Cy3</td>
			<td>552</td>
			<td>565</td>
			<td>Red</td>
		</tr>
		<tr>
			<td rowspan="5">Mounting Medium and immersion oil:</td>
			<td colspan="3">Name</td>
			<td colspan="2">Index of refraction of the media *1</td>
		</tr>
		<tr>
			<td colspan="3">Air (nothing between the slide and the lens)</td>
			<td colspan="2">1.00029</td>
		</tr>
		<tr>
			<td colspan="3">Antifade Mounting Medium with DAPI</td>
			<td colspan="2">1.45</td>
		</tr>
		<tr>
			<td colspan="3">Permount Mounting Medium</td>
			<td colspan="2">1.519</td>
		</tr>
		<tr>
			<td colspan="3">Low autofluorescence immersion oil (MOIL-30 Type F)</td>
			<td colspan="2">1.518</td>
		</tr>
		<tr>
			<td rowspan="6">Magnification Lens (Plan Fluorite)</td>
			<td>Magnification</td>
			<td>Numerical Aperture (NA) *2</td>
			<td>Resolution (&#181;m)</td>
			<td>Image pixel spacing (nm) *5</td>
			<td>Slice spacing Z-axis (nm) *6</td>
		</tr>
		<tr>
			<td>4X</td>
			<td>0.13</td>
			<td>2.12</td>
			<td>850</td>
			<td>3000</td>
		</tr>
		<tr>
			<td>10X</td>
			<td>0.3</td>
			<td>0.92</td>
			<td>340</td>
			<td>3000</td>
		</tr>
		<tr>
			<td>20X</td>
			<td>0.5</td>
			<td>0.55</td>
			<td>170</td>
			<td>2000</td>
		</tr>
		<tr>
			<td>40X</td>
			<td>0.75</td>
			<td>0.37</td>
			<td>85</td>
			<td>1000</td>
		</tr>
		<tr>
			<td>100X</td>
			<td>1.3</td>
			<td>0.21</td>
			<td>34</td>
			<td>1000</td>
		</tr>
	</tbody>
</table>
Table 2: Microscope setup specifications and point spread function (PSF) file creation requirements. There are 11 slots in the diffraction PSF 3D plugin window to create the PSF file. Each slot is described as follows: *1 - Index of refraction of the media: index of refraction for the medium between the slide and the lens (e.g., air = 1.00029). *2 - Numerical Aperture: NA of lens used (it must be corrected when a different immersion media is used and lens was assigned to). *3 - Wavelength: Fluorochrome maximum emission wavelength (nm). *4 - Longitudinal Spherical Aberration: 0.00. *5 - Image pixel spacing: CCD pixel size (nm)/Magnification (e.g., 3.4 &#181;m and 100X lens, 3400/100 = 34 nm). *6 - Distance between images Z-axis. *7 - Width: Enter the width of the image to be deconvolved in pixels. *8 - Height: Enter the height of the image to be deconvolved in pixels. *9 - Depth, slices: the number of images in the z-stack. *10 - Normalization: Sum of pixel values = 1. *11 - Title: Desired name for the PSF file. The file should match up with the unique given z-stack image.

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