Copyright ©The Histochemical Society, Inc.

Light and Electron Microscopic Immunohistochemical Detection of Bromodeoxyuridine-labeled Cells in the Brain : Different Fixation and Processing Protocols

Laura B. Ngwenya, Alan Peters and Douglas L. Rosene

Department of Anatomy & Neurobiology, Boston University School of Medicine, Boston, Massachusetts (LBN,AP,DLR), and Yerkes National Primate Research Center, Emory University, Atlanta, Georgia (AP,DLR)

Correspondence to: Laura B. Ngwenya, Department of Anatomy & Neurobiology, Boston University School of Medicine, 715 Albany St., Boston, MA 02118. E-mail: ngwenya{at}bu.edu


    Summary
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Literature Cited
 
Bromodeoxyuridine (BrdU) immunohistochemistry is the method of choice for labeling newly generated cells in the brain. Most BrdU studies utilize paraformaldehyde-fixed brain tissue because of its compatibility with both BrdU and other immunohistochemical methods. However, stronger fixation is required for electron microscopic studies, and unfixed tissue is needed for biochemical and molecular studies. Because there are no systematic studies comparing the effects of different fixatives on BrdU immunohistochemistry in brain tissue, we compared BrdU immunohistochemical methods in brain tissue fixed with 4% paraformaldehyde, a mixed glutaraldehyde–paraformaldehyde fixative for electron microscopy, and unfixed tissue from brains perfused only with buffer and flash frozen. After optimizing immunostaining protocols, qualitative assessments of light microscopic diaminobenzidine labeling and of double-label immunofluorescence with confocal microscopy demonstrated excellent BrdU labeling in each of the three groups. Quantitative stereological assessment of the number of BrdU-labeled cells in rat dentate gyrus showed no significant difference in the number of labeled cells detected with each perfusion protocol. Additionally, we developed a protocol to visualize BrdU-labeled cells in the electron microscope with adequate preservation of fine structure in both rat and monkey brain.

(J Histochem Cytochem 53:821–832, 2005)

Key Words: bromodeoxyuridine • ultrastructure • electron microscopy • glutaraldehyde • dentate gyrus • adult neurogenesis


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Literature Cited
 
BROMODEOXYURIDINE (BrdU) is a thymidine analog that labels newly generated cells so that they can be identified immunohistochemically, rather than with autoradiography (Gratzner 1982Go). Like thymidine, BrdU is incorporated into the DNA of cells during the synthesis stage of the cell cycle, passed along to any daughter cells, and retained within the chromosomal material for the life of the cell. BrdU has become the current standard for labeling mitotically active cells and has many advantages over tritiated thymidine. First, BrdU can be given in larger systemic doses than tritiated thymidine without risk to the animal. Second, in contrast to autoradiography, BrdU detection by immunohistochemistry takes only a few days rather than months. Third, antibodies to BrdU can identify labeled nuclei throughout the thickness of the section (at least up to 30 µm), whereas tritiated thymidine can only identify labeled nuclei present in the top 3 or 4 µm of the section. Finally, BrdU allows the use of fluorescence immunohistochemical methods to double or even triple label newly generated cells.

The use of BrdU to investigate developmental neurogenesis was first demonstrated by Miller and Nowakowski (1988)Go, and more recently BrdU has been used to demonstrate the birth of new neurons in the adult brain. Indeed, adult neurogenesis has now been well documented in the olfactory bulb and in the dentate gyrus of the hippocampus in mammals (Eriksson et al. 1998Go; Gould et al. 1999bGo; Kornack and Rakic 1999Go; Pencea et al. 2001Go; van Praag et al. 2002Go).

One caveat to the use of BrdU is that because commercial antibodies can only recognize BrdU in single-stranded DNA, the immunohistochemistry protocol requires treatment of the section with hydrochloric acid (HCl) to break double-stranded DNA into single strands to expose the BrdU. Despite this harsh treatment of the tissue, standard BrdU immunohistochemical methods can accurately and reliably detect labeling of cells in tissue that is well preserved with paraformaldehyde (PF).

For this reason, studies using BrdU in brain tissue have almost exclusively utilized PF-fixed tissue. PF provides adequate fixation of brain tissue, binds proteins, which permits immunohistochemical detection of most antigens, and stabilizes the tissue enough for the 2 N HCl treatment not to compromise tissue integrity. A few studies have utilized BrdU immunohistochemistry on tissues preserved with fixatives other than 4% paraformaldehyde (4PF) (Harms et al. 1986Go; Schutte et al. 1987Go; Hayashi et al. 1988Go; Cottell et al. 1989Go), but these studies have been on non-neural tissues. Consequently, very little is known about how other fixatives might affect the efficiency of BrdU immunohistochemistry to identify labeled nuclei in brain tissue.

One situation requiring alternative fixation is the application of electron microscopy (EM), as this requires a stronger fixation than PF alone to obtain adequate preservation of cellular membranes and intracellular structures. Sabatini et al. (1963)Go introduced glutaraldehyde (G) for biological use, and Karnovsky introduced a mixture of G and PF for EM (Karnovsky 1965Go), but thus far the effects of these stronger fixatives on BrdU immunohistochemistry have not been determined.

In contrast to the needs of EM, fresh-frozen tissue is required for a number of different antigens, the demonstration of some enzyme activities, and other techniques such as receptor binding and biochemical or molecular assays. In these situations, no fixation can be utilized, but thus far little is known about how BrdU immunohistochemistry in unfixed tissues compares with standard PF-fixed tissue.

To assess the efficiency of BrdU immunohistochemistry on strongly fixed or on unfixed tissue, we compared BrdU immunohistochemical labeling on rat brain tissues perfused with 4PF, to those perfused with a PF–G mixture for EM, and to those from unfixed, fresh-frozen tissue that was flushed with buffer and flash frozen. Qualitative assessment of the form and distribution of BrdU labeling was done in tissue prepared for light and confocal microscopy after slight modifications of the immunostaining protocol used for 4PF-fixed tissue. Because G-fixed tissues exhibit autofluorescence due to unbound aldehyde groups, common methods for reducing autofluorescence were assessed to determine which method was most effective in allowing confocal laser scanning microscopy analysis on tissue fixed for EM. Additionally, the use of BrdU usually necessitates a determination of the total number of labeled cells in a subject. Therefore, a quantitative evaluation of the number of BrdU-labeled cells was carried out using unbiased stereological counting methods for each perfusate group to reveal any differences that might by attributed to the fixation protocols.

Finally, because the fine structure of BrdU-labeled cells in the brain has not been adequately described, we investigated how to optimally prepare BrdU-labeled tissues for EM in brain tissue from both rats and monkeys. Others have combined BrdU immunohistochemistry with EM (Migheli et al. 1991Go; Tamatani et al. 1995Go; Kondo and Makita 1996Go; Momma et al. 2002Go), but their protocols using postembedding BrdU immunohistochemistry have been done on 1 µm or thin sections. Though these postembedding methods are adequate when there are numerous BrdU-labeled cells, they are not well suited for adult brain tissues in which BrdU-labeled cells are rare. Consequently, we developed a preembedding immunohistochemical method for examining BrdU-immunolabeled cells by EM.

These qualitative, quantitative, and electron microscopic studies were performed on the dentate gyrus of the hippocampal formation, which is an important region for adult neurogenesis. This study will show that BrdU immunohistochemistry can reliably be done on tissues perfused with 4PF, mixed PF and G, or buffer perfused and fresh frozen, allowing the application of multiple techniques within a subject group, or even within a single subject.


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Literature Cited
 
Subjects
Twenty-eight adult male rats, 60 days of age, were obtained from Charles River Laboratories (Wilmington, MA) and used in this study. Additionally, two rhesus monkeys (7.8 and 12.5 years old) obtained from the Yerkes National Primate Research Center (Atlanta, GA) were used for EM. Both rats and monkeys were housed in a 12-hr light–dark cycle with food and water ad libitum in the Laboratory Animal Science Center of Boston University Medical Center, which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. All procedures were approved by the Boston University Institutional Animal Care and Use Committee, and followed National Institutes of Health guidelines and the Institute of Laboratory Animal Resources Commission on Life Sciences (1996)Go, Guide for the Care and Use of Laboratory Animals.

BrdU Administration and Perfusion
Bromodeoxyuridine (BrdU; Sigma, St Louis, MO) was administered IP to the rats at a dose of 200 mg/kg from a 15 mg/ml solution made in warm Tris-buffered saline at pH 7.4. Monkeys received either a single IP dose of 200 mg/kg or twice-daily intravenous injections of 50 mg/kg for 3 consecutive days.

Three weeks after the BrdU injection, animals were deeply anesthetized with sodium pentobarbital and then killed by exsanguination during transcardial perfusion of the brain with one of three perfusates. 1. A 4PF solution was made in 0.1 M phosphate buffer at pH 7.4 and warmed to 37C for perfusion. 2. A mixed aldehyde perfusate for EM (PF + G) that consisted of 1% PF and 1.25% G (EM grade; TedPella, Redding, CA) was made in 0.1 M phosphate buffer at pH 7.4 and warmed to 37C for perfusion. The monkeys received injections of 5000 U of heparin and 5 ml of 1% sodium nitrite into the heart just prior to perfusion with fixative to prevent coagulation of the blood and to dilate blood vessels. All animals were respired with a mixture of 95% oxygen and 5% carbon dioxide immediately before the chest cavity was opened until the fixative was flowing, in order to prevent any anoxia and to further dilate blood vessels. 3. For unfixed tissue (Krebs), animals were perfused with a modified Krebs–Henseleit buffer (Krebs and Henseleit 1932Go) cooled to 4C to rapidly reduce autolysis and to firm the brain for subsequent dissection. This balanced salt solution consisted of 6 mM Na2HPO4, 2 mM NaH2PO4, 0.14 M NaCl, 3 mM KCL, 6 mM D-glucose, 0.3 mM CaCl2, and 1 mM MgCl2 in dH2O at pH 7.4.

Tissue Processing
Fixed rat brains for light microscopy (LM) were cryoprotected in graded glycerol solutions with 2% DMSO and flash frozen in 2-methyl-butane at –70C (Rosene et al. 1986Go). Unfixed tissue was flash frozen immediately at –70C in 2-methyl butane. All frozen tissue was stored at –80C until cut. Fixed rat brains for immunohistochemistry were cut on a sliding microtome in the horizontal plane into six interrupted series of 30-µm-thick sections spaced 180 µm apart. These sections were stored free floating at –80C in a cryoprotectant solution consisting of 15% glycerol in 0.1 M phosphate buffer until processed for BrdU immunohistochemistry. Fresh-frozen tissue was cut in the horizontal plane on a cryostat into 12 interrupted series of 15-µm sections spaced at 180-µm intervals. These were thaw mounted onto poly-L-lysine-coated slides, dried rapidly on a warming plate, and stored at –20C until processed for BrdU immunohistochemistry. Brains fixed with PF + G for EM were stored in 2% PF with 2.5% G in 0.1 M phosphate buffer at pH 7.4 at 4C until used. Two-mm blocks of dentate gyrus from PF + G–fixed brains were cut with a Vibratome 1000 (Vibratome; St Louis, MO) into 50-µm-thick sections and stored in 2% PF with 2.5% G at 4C until processed for BrdU immunohistochemistry.

Standard BrdU Immunohistochemistry of Sections from Different Fixation Protocols
Immediately before processing, frozen sections from all three perfusion conditions were removed from the freezer and warmed to room temperature. Free-floating sections fixed with PF + G were removed first and pretreated in a 1% sodium borohydride solution at room temperature for 1 hr to reduce free aldehydes. This sodium borohydride solution was prepared prior to use and stored at 4C for 16–20 hr. Krebs-perfused slide-mounted cryostat sections were removed next and immediately placed in cold 4PF for 10 min to fix soluble proteins in place and inactivate proteases that might degrade the tissue. Sections from cases perfused with 4PF were not pretreated. Sections from all brains, regardless of perfusate protocol, were then processed for BrdU immunohistochemistry simultaneously, using aliquots of solutions that were prepared as single batches. This "batch processing" minimized the possibility that differences between fixation groups would be confounded by between-group differences in solutions or reaction times.

Although pretreatments differed, all sections were processed following a standard BrdU immunohistochemistry protocol, as described below. All sections were washed in 0.1 M phosphate buffer and then placed in 3% hydrogen peroxide for 1 hr to quench endogenous peroxidases. They were then incubated in 2 N HCl with 0.5% Triton-X at room temperature for 1 hr to create single-stranded DNA and to expose the incorporated BrdU. Sections were washed in buffer at pH 8.4 to neutralize the HCl and then blocked in 3% normal horse serum (NHS) in 0.1 M phosphate buffer. Sections were incubated overnight at 4C in primary antibody solution containing mouse anti-BrdU (1:200; Chemicon, Temecula, CA), 3% NHS, and 0.3% Triton-X in 0.1 M phosphate buffer. Negative controls that did not contain primary antibody were run in parallel. Following primary antibody incubation, sections were washed in phosphate buffer, then incubated in secondary antibody solution containing biotinylated horse anti-mouse antibody (1:200; Vector Labs, Burlingame, CA), 3% NHS, and 0.3% Triton-X in 0.1 M phosphate buffer for 1 hr at room temperature. Sections were subsequently washed and treated with an avidin–biotin complex (Vector Elite kit; Vector Labs) for 1 hr at room temperature. After washing, sections were processed for visualization with 0.2% diaminobenzidine (Sigma) in 0.1 M phosphate buffer containing 0.025% hydrogen peroxide for approximately 5 min and then washed in 0.1 M phosphate buffer. Free-floating sections were mounted on slides and dried alongside the cryostat sections. All sections were lightly counterstained with a 0.05% thionin solution at pH 4.8, dehydrated through a graded series of alcohols, cleared with xylene, and coverslipped using Permount.

Suppression of Glutaraldehyde Autofluorescence
G-fixed tissue exhibits autofluorescence due to unbound aldehyde groups. To diminish the autofluorescence in PF + G–fixed tissue to be processed for immunofluorescence, the following pretreatments were tried prior to immunohistochemical staining: (a) 1% or 3% sodium borohydride for either a 30-, 60-, or 90-min incubation to reduce free aldehydes (Clancy and Cauller 1998Go; Baschong et al. 2001Go); (b) a 30-min incubation in a pH 7.4 solution of 0.1 M glycine with 1 M Tris base to bind and block aldehyde groups; (c) a 5-min incubation in 50 mM ammonium chloride also to bind and block aldehyde groups. These three pretreatments were tried alone and in combination. An alternative treatment that can be applied after immunohistochemistry is staining with Sudan Black B, which has been recommended for autofluorescence of tissue components (Romijn et al. 1999Go; Schnell et al. 1999Go). This was tried alone and on select sections that also received the pretreatments listed above.

Fluorescence Immunohistochemistry of Sections with Different Fixation Protocols
The double-label fluorescence immunohistochemistry protocol was similar to that described above for BrdU alone. The best reduction of autofluorescence in PF + G–fixed tissue was obtained by pretreatment with 1% sodium borohydride for 1 hr at room temperature. Therefore, PF + G–fixed tissue received this borohydride pretreatment and Krebs tissue was placed in cold 4PF for 10 min prior to immunohistochemistry. Subsequently, these sections were run in parallel with 4PF-fixed tissue. All sections were washed in 0.1 M phosphate buffer and then treated with HCl as described above. Sections were blocked in 10% normal goat serum (NGS) and incubated in a pooled primary antibody solution containing rat anti-BrdU (1:200; Accurate Chemical, Westbury, NY) and mouse anti-Neuron-Specific Nuclear Protein (NeuN, 1:200; Chemicon) in 10% NGS and 0.3% Triton-X for 16–20 hr at 4C. Negative controls lacking primary antibodies were run in parallel. Sections were washed in phosphate buffer containing 3% goat serum, followed by incubation for 2 hr in the dark, at room temperature, in pooled secondary antibodies (AlexaFluor-488 goat anti-rat, 1:250 and AlexaFluor-568 highly cross-absorbed goat anti-mouse, 1:250; Molecular Probes, Eugene, OR). Sections remained in the dark and were washed, mounted on slides, and coverslipped using a polyvinyl alcohol and glycerol solution with diazabicyclooctane for antifade (PVA-DABCO; Sigma). Slides were stored in the dark at 4C until examined with either standard episcopic fluorescence microscopy or confocal fluorescence microscopy using the multi-track setting of an inverted Zeiss LSM 510 confocal microscope (Carl Zeiss; Thornwood, NY).

Stereological Estimation of Total Number of BrdU+ Cells
To compare the efficacy of BrdU labeling in frozen sections of the three different perfusion protocols, in one randomly selected hemisphere of immunostained dentate gyrus per rat, the number of BrdU-labeled nuclei present was counted using a modified version of the optical fractionator method (West et al. 1991Go). Counts were done with a Nikon E600 series microscope (Nikon; Melville, NY), motorized stage, and the StereoInvestigator software (MicroBrightField; Williston, VT). The dentate gyrus was defined as the region of interest including a combined molecular layer, granule cell layer, and hilus (Figure 1). Because BrdU-immunostained (BrdU+) cells are relatively rare, accurate estimates of numbers required implementation of an exhaustive sampling scheme. For this, all BrdU-labeled cells in an average of 16 sections, which systematically covered the entire dorsal–ventral extent of the dentate gyrus of each animal, were counted with a x60 objective.



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Figure 1

Region of interest used for stereological counting of BrdU+ cells. (A) The region of interest included all three subareas of the dentate gyrus as outlined in this representative section from 4% paraformaldehyde (4PF)-fixed tissue immunostained for BrdU+ cells and counterstained with thionin. (B) A nearby thionin-stained section from the same brain, which was used to ensure accurate outlining of the region of interest. Bar = 200 µm.

 
All three types of tissue sections shrink significantly in the z-dimension when mounted on slides, dried, dehydrated, and coverslipped. Specifically, it was found that 30-µm sections from the 4PF-fixed brains shrink to approximately 6 µm in thickness; 30-µm sections from PF + G–fixed brains shrink to an average of 9 µm; and the 15-µm cryostat sections from Krebs-perfused brains shrink to approximately 3 µm. As a result, guard volumes at the top and bottom of the disector-counting frame could not be effectively implemented, but for the 4PF and PF + G sections, a bottom z-axis exclusionary plane was used to avoid overcounting. The limitations that result from not being able to implement guard volumes is addressed more fully in the Discussion. For Krebs tissue, the 3-µm thickness did not allow an exclusionary plane to be established because the entire thickness of the section was within the depth of focus. Consequently, counts were effectively two-dimensional. Labeled cells were only counted if more than one-third of the nucleus was darkly labeled and if the labeled nucleus did not belong to a cell within a blood vessel. This counting method omitted lightly labeled cells and endothelial cells from the count.

The estimated total number of BrdU-labeled cells was calculated from the mean cell count of each brain using the following formula:

N = {Sigma}Q x (t/h) x (1/asf) x (1/ssf)

where {Sigma}Q is the total number of cells counted, t represents the mean section thickness, h is the height of the optical disector, asf is the area sampling fraction (area of counting frame divided by area of x,y step), and ssf is the section sampling fraction (1/section interval) (West et al. 1991Go). Because guard volumes were not used, t was equivalent to h, and because an exhaustive sampling scheme was implemented, asf was equal to one. This simplified the formula for estimated total number of BrdU+ cells to the following:

N = {Sigma}Q x (1/ssf)

which is the total number of BrdU+ cells counted, multiplied by the reciprocal of the section sampling fraction.

For Krebs tissue, the inability to apply a z-axis exclusionary plane resulted in overcounting cells. To correct for this overcount, we applied the Abercrombie correction factor (Abercrombie 1946Go). The correction factor involves the number of objects counted, a measure of section thickness (t), and a measure of object diameter (d): n = count x [t/(t x d)]. The measure of object diameter was obtained using the nucleator method. The nuclear sizes of 25 randomly selected BrdU+ cells were estimated by using four rays and their points of intersection with the boundaries of the nucleus to obtain an area. The formula used for this calculation was as follows: a = {pi}l2 with l being equal to the length of the intercept. From the averaged areas, a composite diameter was determined from area = {pi}r2 with diameter equal to twice the radius (r). This composite diameter was used in the correction factor. Although the shortcomings of this and similar corrections are well documented (Sterio 1984Go; Hedreen 1998bGo), we applied the Abercrombie correction to our data to provide numbers from the Krebs tissue, which allowed comparison with numbers obtained from the 4PF- and PF + G–fixed tissues. The limitations and the extent of the bias involving the use of the Abercrombie correction are addressed more fully in the Discussion.

The coefficient of error (CE) for each animal was calculated using Gundersen's equation, which states that the CE is equal to the square root of the total variance divided by the sum of counts (Gundersen et al. 1999Go). CE values <=0.1 were considered to indicate that the sampling method was adequate for this study.

Differences between groups were assessed with a one-way ANOVA. A Scheffe post-hoc test was done to further explore the relationship between the perfusates used; p values <0.05 were considered to indicate the presence of a difference between groups.

BrdU Immunohistochemistry for EM
The procedure described above for light microscopic BrdU immunohistochemistry was modified to seek the best preservation of ultrastructure. Investigation revealed that both HCl and Triton-X disrupt cellular membranes and compromise ultrastructure. To minimize this effect, the concentrations of these reagents were varied. To better improve the preservation of ultrastructure, some sections were subjected to a 1-hr postfixation in 4PF with 5% G, made in 0.1 M phosphate buffer at pH 7.4, prior to the immunohistochemistry protocol. As in the standard BrdU immunohistochemistry, select sections were run in the absence of primary antibody as negative controls.

Fifty-µm-thick sections with adequate BrdU immunohistochemical labeling from protocols that showed the promise of well-preserved ultrastructure were osmicated in 0.5% OsO4, dehydrated through a series of alcohols, embedded in araldite, and mounted on plastic slides with aclar coverslips. These sections were examined under the light microscope for the presence of BrdU+ cells, and the region of interest containing BrdU+ cells was cut out of the plastic slide and adhered to a blank Beem capsule of Araldite. One-µm sections were taken and stained with 1% toluidine blue until the first BrdU+ cell was seen. The face of the block was then thin sectioned and sections collected on thin-bar grids. Additional 1-µm sections were cut and stained until the next BrdU+ cell was reached, whereupon more thin sections were cut. This cutting procedure was repeated until all labeled cells in the selected region had been sectioned. All thin sections collected onto thin-bar grids were stained with lead citrate followed by uranyl acetate and observed on a JEOL 100 Electron Microscope (JEOL; Peabody, MA).

Additional 3-mm-thick blocks of hippocampus were processed for routine EM. These blocks were osmicated, sectioned, stained, and viewed in the electron microscope.

As illustrated in Table 2, a total of 12 different immunohistochemical protocols were tried and assessed qualitatively for the presence of BrdU staining as well as for preservation of ultrastructure. Light microscopic examination was used to assess the relative intensity of BrdU labeling as compared with labeling on frozen PF + G sections processed for standard immunohistochemistry. Evaluation of the preservation of the ultrastructure of labeled cells on the electron microscope was compared with the ultrastructure of the normal tissue, which had not been processed for immunohistochemistry.


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Table 2

Assesment of protocols tried for electron microscopya

 
Image Processing
Adobe Photoshop version 7.0 (Adobe Systems; San Jose, CA) was used to adjust brightness and contrast, to minimize scratch marks on electron micrographs, and for the general compilation of the figures.


    Results
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Literature Cited
 
Qualitative Assessment of Standard BrdU Immunohistochemistry
BrdU immunohistochemical techniques on all three of the perfusate types yielded clearly detectable BrdU-labeled cells, visible by LM, as shown in Figures 2A–2C. PF + G– and Krebs-perfused tissues appeared similar to 4PF-fixed tissue, and the intensity of labeling and lack of interfering background staining was similar in all three groups.



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Figure 2

Light and confocal laser scanning microscopy images of BrdU immunohistochemistry on rat brain tissue with different fixation protocols. 4PF-fixed tissue (A,D,G,J), mixed aldehyde [paraformaldehyde (PF) + glutaraldehyde (G)]–fixed tissue (B,E,H,K), and fresh-frozen Krebs tissue (C,F,I,L) showed adequate diaminobenzidine labeling of BrdU+ cells as shown with light microscopy (LM) (A–C) and double immunofluorescence labeling as shown with confocal laser scanning microscopy (D–L). Examples of labeled cells seen in LM are indicated by arrows in A–C. Arrowheads illustrate examples of light or speckled labeling that would not have been counted stereologically. Double-label immunofluorescence with NeuN (red, D–F) and BrdU (green, G–I) show that double-labeled cells can be detected in tissue from the different fixation protocols by confocal microscopy. Arrows in D–F indicate the BrdU+ cells seen in G–I, and arrows in J–L show the same cells in the merged images. Bars: A–C = 20 µm; D–L = 5 µm.

 
Suppression of Glutaraldehyde Autofluorescence
Of the protocols assessed for the suppression of autofluorescence in PF + G–fixed tissue, none completely abolished the autofluorescence. Incubation for 1 hr with 1% sodium borohydride, the identical pretreatment used prior to standard immunohistochemistry, proved to be the most effective. Higher concentrations or longer incubation times with sodium borohydride made the tissue fragile and unsuitable for immunohistochemistry. Tris-glycine and ammonium chloride solutions diminished the autofluorescence but were not as effective as sodium borohydride. Combinations of Tris-glycine, ammonium chloride, and sodium borohydride were effective, but it was found that this effectiveness was mainly due to the sodium borohydride treatment alone. Sudan Black B staining after the fluorescence immunohistochemistry was also effective in reducing autofluorescence. Sudan Black B in combination with 1% sodium borohydride resulted in markedly diminished autofluorescence with standard fluorescence microscopy. However, the Sudan Black B produced an overall dimming of all fluorescence—both the tissue autofluorescence and the specific immunofluorescence. On the confocal laser scanning microscope, in sections stained with Sudan Black B, immunolabeled cells could not be visualized without the interference of background. As shown in Figures 2D–2L the 1% sodium borohydride treatment yielded tissue for the PF + G group that was comparable in background to the two other groups.

Qualitative Assessment of Fluorescence Immunohistochemistry
Sections from all three perfusate types were amenable to fluorescence confocal imaging. Tissue from each of the different perfusate types exhibited labeling of both BrdU-labeled nuclei and of NeuN-labeled neurons in the granule cell layer. In all sections examined, cells that double-labeled for BrdU and NeuN were demonstrated by confocal slice imaging (Figures 2D–2L). Cells that were BrdU positive and NeuN negative were also seen in sections from each of the different perfusate types (not shown), demonstrating the specificity of the immunofluorescence staining.

Estimated Total Number of BrdU-labeled Cells Following Different Perfusion Protocols
Individual results for the stereological estimates of the number of BrdU+ cells in the dentate gyrus are given in Table 1, whereas Figure 3 shows the mean estimated total for each group and the standard error of the mean. One-way ANOVA yielded a probability of p=0.18, showing that there were no differences among the fixation types (F2,19 = 1.90). Scheffe posthoc analysis more specifically demonstrated that the 4PF- and mixed aldehyde- fixed tissue were similar with p=0.69. The probability of a difference between the numbers of BrdU-labeled cells in Krebs- and PF + G–perfused tissue was p= 0.65. In contrast, the PF and the Krebs tissue were the most dissimilar, but the numbers of cells labeled was still not significantly different at p=0.18. Therefore, no quantitative differences in the numbers of BrdU-labeled cells were seen among the three groups.


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Table 1

Estimated total number of BrdU+ nuclei per dentate gyrus

 


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Figure 3

Mean estimated total number of BrdU+ cells in brain tissues prepared with different perfusates. No significant difference was seen among the number of labeled cells in 4PF-fixed-, PF + G–fixed-, or Krebs-perfused tissue (corrected with the Abercrombie correction). Error bars represent standard error of the mean.

 
EM of Immunolabeled BrdU+ Cells
The effect of variations in the BrdU immunohistochemistry protocol yielded different results in rat and monkey tissue. The protocols tried on each tissue and the outcomes are listed in Table 2. In this Table, the notation "excellent labeling" means that the labeling resembled the relative intensity seen on frozen PF + G sections processed for standard BrdU immunohistochemistry, whereas a notation of "poor labeling" indicates a visibly diminished, unsuitable intensity of labeling. A notation of "poor preservation" indicates tissue in which cell membranes were broken and overall tissue quality was not sufficient to provide useful fine structural information.

Labeled cells were seen in both rat and monkey tissue when Triton-X was removed from the antibody incubations. However, variation of the HCl concentration and length of incubation was not well tolerated by rat tissue and resulted in very faint or non-existent labeling. Postfixation in a strong mixed aldehyde solution consisting of 4PF with 5% G for 1 hr prior to the start of the immunohistochemistry significantly improved the ultrastructural preservation of the tissue. The best results for rat tissue were obtained by postfixation followed by a 1-hr incubation with 2 N HCl. In contrast, the best results for monkey tissue were obtained by postfixation followed by a 30-min incubation in 1 N HCl. Examples of BrdU-labeled nuclei stained with these preferred protocols and visualized on EM are shown in Figure 4.



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Figure 4

Electron micrographs of BrdU-labeled nuclei in rat and monkey dentate gyrus. Inset in (A) shows a low-magnification electron micrograph of a labeled cell at the bottom of the granule cell layer in rat hippocampus. An electron-dense reaction product is present around the perimeter of the nucleus. This cell is also seen in B (marked with an asterisk), which is the 1-µm section just prior to sectioning. At higher magnification (A), this cell is seen to have an axon extending beyond the granule cell layer (labeled Ax). The asterisk in C shows a labeled cell within the granule cell layer of monkey hippocampus in a 1-µm section just prior to thin sectioning. This labeled cell seen in D shows robust diaminobenzidine reaction product mostly on the perimeter of the nucleus. N = labeled nucleus, n = unlabeled granule cell nucleus, Ax = axon. Bars: A = 1 µm; inset = 2 µm; B,C = 10 µm; D = 4 µm.

 
General ultrastructural characteristics of BrdU-labeled cells seen 3 weeks after BrdU injection are of immature cells. BrdU labeled nuclei tend to be indented or lobulated and the DAB reaction product, which indicates the location of BrdU, is often at the perimeter of the nucleus, or clustered around clumps of heterochromatin. The cytoplasm of these immature cells is often scant and primitive, with few organelles. Hence, it was common to find BrdU labeled cells with few mitochondria and free polyribosomes in the cytoplasm. Though immature, in the rat some of the BrdU labeled cells examined showed ultrastructural features of neurons, including emerging axons (Figure 4A) or synapses on their cell bodies. However, no immature neurons were seen among the BrdU labeled cells examined in monkey tissue three weeks after BrdU injection.


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Literature Cited
 
The results from this study demonstrate that BrdU immunohistochemistry for LM can be effectively applied to brain tissue perfusion fixed with 4PF or mixed aldehydes (PF + G), as well as unfixed tissue perfused with Krebs buffer and flash frozen. Qualitatively, no differences were observed among groups, and quantitatively the three perfusate groups showed similar numbers of labeled cells. Additionally, it was shown that when PF + G fixatives are used, and the standard BrdU immunohistochemical protocol is modified, BrdU-labeled cells can be visualized by EM with adequate preservation of ultrastructure.

Comparison of BrdU Immunohistochemistry on Tissue with Different Fixation Protocols
The similarities among the three perfusate groups in standard and fluorescence immunohistochemistry can be attributed to the pretreatments used for Krebs- and PF + G–perfused tissues. Without postfixation with 4PF, unfixed tissue is likely degraded during BrdU immunohistochemistry. Without pretreatment with 1% sodium borohydride, antigenic sites in PF + G–fixed tissue are blocked by unbound free aldehyde groups; additionally, these aldehyde groups exhibit autofluorescence that interferes with specific immunofluorescence (Kiernan 1999Go). Although we found 1% sodium borohydride to be effective for diminishing autofluorescence, others have suggested alternative methods to abolish G autofluorescence. For example, Neumann and Gabel (2002)Go describe a photobleaching method and Tagliaferro et al. (1997)Go demonstrate a Schiff-quenching method. Neither of these methods was feasible for our study, but they are alternatives that may be useful in some circumstances.

Whereas the different fixation protocols produce similar labeling, fixation could potentially affect the creation of single-stranded DNA, which is necessary to expose the BrdU label and allow antibodies to access all antigenic sites. Comparison between the numbers of BrdU+ cells obtained in this study and results obtained in other studies in which the animals received similar amounts of BrdU show that the results in this study fall within the range of other published studies (Table 3). The exception is the study by Merrill et al. (2003)Go, which reports numbers of labeled cells four times greater than the results obtained by others. There is no apparent explanation for the large discrepancy, although a different strain of rat was used.


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Table 3

Estimated total number of BrdU+ cells in dentate gyrus: comparison among studies

 
Stereological Considerations
The stereological methods applied here are optimal when relatively thick sections are used, so that guard volumes in the z-axis can be implemented. However, sections thicker than ~30 µm are not feasible for immunohistochemistry because reagents cannot penetrate through more than 10–20 µm per side. The degree of shrinkage on these immunostained and slide-mounted sections does not allow a guard volume thicker than 1 µm to be easily implemented. Whereas a 1-µm guard volume can be applied in instances where the cell nucleolus or another similar small structure is to be counted, the BrdU+ nucleus averages 8.7 µm in diameter. Hence, implementation of a 1-µm guard volume would not be effective against lost caps or torn-out nuclei near the section surface. The implementation of a z-axis exclusionary plane in the absence of guard volumes, as used in our study, assures that nuclei were not overcounted. However, this may have yielded results that were a slight undercount because of the inability to compensate for lost caps or cells damaged at the inclusion surface (for a more extensive discussion, see Hedreen 1998aGo; Lister et al. 2005Go). Nonetheless, the total number of BrdU+ nuclei counted in 4PF tissue and in PF + G–fixed tissue was not significantly different. This suggests that a modified optical fractionator can be applied to accurately estimate the total number of BrdU+ cells in PF + G–fixed tissue, the same as in 4PF-fixed tissue.

Because the Krebs-perfused tissue could not be analyzed with an unbiased optical fractionator probe, the numbers counted could not be directly compared with the 4PF- and PF + G–fixed-tissue cell counts. Whereas the Abercrombie correction adjusts for the split-cell overcounts in the Krebs tissue, this is not an unbiased estimate of the total number. To accurately apply the Abercrombie correction, the true diameter of the counted object in the plane of sectioning is required. In the z-axis where the tissue shrinks, this diameter is impossible to obtain; hence, the diameter was measured in the x-y plane. By measuring the diameter in the x-y plane, the assumption is made that the counting objects are spherical and have not been distorted during the shrinkage process, but there is no way to assure this and obtain an unbiased estimate. Nonetheless, the number of labeled cells estimated in Krebs-perfused tissue after using the Abercrombie correction yielded numbers that were in the same range seen in 4PF- and PF + G–fixed tissue. Even if we applied a more liberal posthoc analysis (Fisher least significant difference: PF + G and 4PF, p=0.39; PF + G and Krebs, p=0.36; 4PF and Krebs p=0.07), there was still no significant difference in the number of BrdU-labeled cells seen among groups. The number of labeled cells estimated in 4PF- and Krebs-perfused tissue approached significance; however, we believe this was a reflection of the inability to use an unbiased optical fractionator as discussed above.

Comparative Uses for BrdU Studies of Different Fixation Protocols
The advantage of PF + G–fixed tissue mainly lies in the ability to do EM, which requires strong fixation to allow successful visualization of fine structure. Using PF + G–fixed tissue, areas of interest (or a hemisphere) can be serially sectioned to allow standard BrdU immunohistochemistry and stereological counts. Blocks of tissue can then be reserved for routine or immunoelectron microscopy. The methods presented here have demonstrated the feasibility of such procedures although, interestingly, successful BrdU immunohistochemistry for EM required different protocols for monkey and rat tissues. Rat tissue required a more rigorous HCl treatment to allow access to BrdU sites on DNA, suggesting that PF + G fixation of rat tissue is more effective at cross-linking nuclear proteins than the identical fixation in monkey tissue.

Recently, Momma et al. (2002)Go described a postembedding BrdU immunohistochemistry method recommended for instances in which BrdU cell labeling is rare. Their methodology involved placing 1-µm plastic sections on glass slides. Standard BrdU immunohistochemistry using 4 M HCl was used, and sections of interest were then removed from the glass by hydrofluoric acid for thin sectioning. This method requires not only the making of numerous 1-µm sections with the hope that some of the sections will show labeled cells, but once sections containing cells are found, it requires careful thin sectioning of a 1-µm section. In our approach, a preembedding BrdU immunohistochemistry was done on 50-µm-thick sections, which could then be mounted on plastic slides and examined to allow the easy selection of labeled cells for EM. Such preembedding BrdU immunohistochemistry has also been used in two recent studies (Liu et al. 2003Go; Yamashima et al. 2004Go). In these studies, the amount of HCl and Triton-X was not modified from the high amounts normally used in BrdU immunohistochemistry. In our tissue, standard amounts of HCl and Triton-X were found to compromise fine structure (Table 2).

The present study has also shown that it is possible to do BrdU immunohistochemistry on unfixed tissue. But because slide-mounted cryostat sections prohibit application of unbiased stereology, the numbers of labeled cells obtained must be treated with caution and probably should not be directly compared with those obtained from the 4PF and PF + G protocols (as discussed above). However, the ability to apply BrdU immunohistochemistry techniques to unfixed tissue is useful, because unfixed tissue is required for ligand-binding studies of receptors, biochemical studies, and molecular studies. This study demonstrates that BrdU immunohistochemistry can be applied to slide-mounted fresh-frozen tissue and will yield numbers of labeled cells that are similar to those seen with 4PF.


    Conclusion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Literature Cited
 
The ability to apply BrdU immunohistochemistry to PF + G fixed tissue and to fresh-frozen tissue provides a means to explore adult neurogenesis in ways not possible with PF fixation alone. This study reveals that use of these different fixation protocols can be accomplished without significant consequences for BrdU immunohistochemistry. This could be of particular use in studies where few animals are available, such as with transgenic animals or with primates, because it allows for a combination of techniques, such as stereological counts of BrdU+ cells and EM, to be applied to an individual animal.


    Acknowledgments
 
This research was supported by NIH Grants P01 AG-00001, P51 RR-00165, and F31 AG-21873.

We wish to thank Dan Kim, James Lister, and Debra Roberts for technical assistance with perfusion of the rats, and Claire Sethares for helpful discussions regarding the design of the electron microscopy portion of this investigation.


    Footnotes
 
Received for publication December 21, 2004; accepted January 7, 2005


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
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