Copyright ©The Histochemical Society, Inc.

Histological Analysis of GFP Expression in Murine Bone

Xi Jiang, Zana Kalajzic, Peter Maye, Alen Braut, Justin Bellizzi, Mina Mina and David W. Rowe

Department of Genetics and Developmental Biology (XJ,ZK,PM,JB,DWR) and Department of Pediatric Dentistry (AB,MM), University of Connecticut Health Center, Farmington, Connecticut

Correspondence to: David W. Rowe, Department of Genetics and Development Biology, University of Connecticut Health Center, Farmington, CT 06030. E-mail: rowe{at}neuron.uchc.edu


    Summary
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
The power for appreciating complex cellular interactions during embryonic development using green fluorescent protein (GFP) as a visual histological marker has not been applied to adult tissues due to loss of GFP signal during paraffin embedding and a high autofluorescent background, particularly in section of bone and bone marrow. Here we demonstrate that the GFP signal is well preserved in frozen sections of adult decalcified bone. Using a tape-transfer system that preserves histological relationships, GFP expression can be related to standard histological stains used in bone biology research. The choice of a dual-filter cube and a strong GFP signal makes it possible to readily distinguish at least four different GFP colors that are distinctly different from the autofluorescent background. An additional advantage of the frozen sections is better preservation of immunological epitopes that allow colocalization of an immunostained section with an endogenous GFP and a strong lacZ signal emanating from a ß-gal marker gene. We present an approach for recording multiple images from the same histological section that allows colocalization of a GFP signal with subsequent stains and procedures that destroy GFP. Examples that illustrate the flexibility for dual imaging of various fluorescent signals are described in this study. The same imaging approach can serve as a vehicle for archiving, retrieving, and sharing histological images among research groups. (J Histochem Cytochem 53:593–602, 2005)

Key Words: green fluorescent protein (GFP) • transgenic mice • CryoJane frozen section • murine bone • entire femur image scanning • colocalization • histology


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
GREEN FLUORESCENT PROTEIN (GFP) is a visual reporter that has received wide use in early embryonic development (Gong et al. 2003Go; Tam and Rossant 2003Go) and in cell culture studies (Maruvada et al. 2003Go). It provides a technically unbiased assessment of transgene activity and can be viewed as a real-time image in living tissues. For example, cell-specific promoter-GFP reporter constructs have been used to identify cells within a defined lineage either in primary cell culture (Kalajzic et al. 2003Go) in cells entering apoptosis (Harvey et al. 2001Go) or in developing embryos (Grant et al. 2000Go; Ma et al. 2002Go; Mignone et al. 2004Go). This success is based on the ability to visualize GFP in living tissues or in optical sections of developing embryo using confocal microscopy. However, this approach has not proven to be useful in histological sections of developing or mature tissues because the GFP signal was not sufficiently preserved in paraffin sections for standard epifluorescent microscopy.

We have utilized the promoter-GFP reporter strategy to assess progression of cells within the osteoblast lineage in primary cultures derived from transgenic mice harboring a variety of type I collagen-driven reporter constructs (Kalajzic et al. 2002Go). In part, because the type I promoter drives high expression of GFP and because of the availability of improved GFP variants (GFP-topaz and emerald; Aurora Biosciences, San Diego, CA), we are able to visualize GFP expression in paraffin sections of developing and mature bone using standard epifluoresence microscopy. By relating expression of the reporter in primary culture with its expression in intact bone, greater confidence was gained in the assignment of the reporter to a level of cellular development. However, we came to appreciate the limitations of this approach because there was variability of GFP expression introduced by the process of paraffin embedding related to the temperature oscillation and different solvents used in this procedure. Recent studies suggest that GFP is well preserved in frozen sections of spleen that have been fixed either with formaldehyde vapor (Jockusch et al. 2003Go) or by standard emersion fixation (Kusser and Randall 2003Go); however, there are no reports of imaging GFP in fixed sections from adult skeletal tissue.

This paper reports the progress that has been made using an adhesive tape system for preserving histological features and GFP expression in sections of mature murine bone. There were two objectives underlying this work. First was the ability to detect various colors of GFP distinct from the autofluorescent background inherent in bone and bone marrow and to multiplex various fluorescent or visual histological stains that can reveal cellular relationships to GFP positive cells. Second was to develop a method for recording, archiving, and retrieving a comprehensive image of the histological section such that the cellular and architectural relationships of the GFP can be appreciated in an unbiased manner. The progress made to date indicates that preservation of the GFP signal in high-quality frozen sections of bone and other tissues can greatly increase the power of a histological study that utilizes GFP reporter constructs.


    Materials and Methods
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Transgenic Mice
A variety of transgenic mice, expressing one of four colors of GFP, have been used in these studies. Figure 3 summarizes the construct name, provides the map of the construct, and illustrates its expression pattern in intact bone at low and high power of the type I-collagen-derived expression constructs. In most cases the transgenes have been developed in a CD1 background and have remained stable and consistent in their expression for multiple generations. A Tie2-eGFP transgenic mouse [STOCK Tg(TIE2GFP)287Sato/J; Jackson Laboratory, Bar Harbor, ME] to mark vascular endothelial cells was also examined. In one set of experiments, the cre conditional ß-gal mice, Gt(ROSA)26Sor (Jackson Laboratory), were crossed with a Col2.3CreERT2 transgenic mouse under development. The F1 double, transgenic mice were injected with tamoxifen to induce activation of the ß-gal transgene to test for the ability of the histological methods to detect lacZ activity in a histological section of bone.



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Figures 1–3

Figure 1 Comparison of a scanned image of paraffin (A) and frozen section of a femur (B) from a pOBCol2.3GFPemd transgenic mouse. The weaker signal coming from the paraffin section is responsible for the higher fluorescent background from the bone marrow. The frozen section was prepared without the use of the CryoJane tape method. Bc, cortical bone; Bm, bone marrow.

Figure 2 Scanning routine used to produce a composite image of a whole bone section. (A) The cartoon illustrates the steps that are taken to define the boundaries (black dots) of the set of adjacent images (4 x 8 grid) that the Openlab macro will use to generate a file list. The list is exported to Graphic Converter to assemble the composite image where it can be rotated, cropped, and saved as a primary image. (B) A x5 scanned image of an entire femur section from a pOBCol3.6GFPsaph mouse produced with the CryoJane tape system showing enhanced activity in the primary spongiosa of the distal epiphysis (top), head, and greater trochanter. The distal femur was rescanned at x20 and a region was selected (white box) that is shown in (C). Bc, cortical bone; Bm, bone marrow.

Figure 3 Comparison of four different colors of GFP imaged in cryosections of femur. The map of each construct is placed above the x10 scanned image of the distal femur. A region of the same image is selected to show the resolution of the x10 image. (A) pOBCol2.3GFPemd; (B) pOBCol3.6GFPtpz; (C) pOBCol3.6GFPsaph; (D) pOBCol3.6GFPcyan. Bc, cortical bone; Bm, bone marrow; arrow, endosteum; arrowhead, periosteum.

 
Tissue Fixation and Embedding
For most of the studies reported here, mice were 2–3 months of age when bone formation is still present, although most somatic growth has been achieved. These study procedures were reviewed and approved by UCHC Animal Care Committee and met National Institutes of Health Guidelines for the Use of Animals in Research. Animals were killed with CO2 asphyxiation and bones were quickly dissected free of most adherent tissue without scraping the surface of the cortical bone. The bone was placed in a biopsy cassette (Thermo Shandon, Pittsburgh, PA) and immersed for 2 days in freshly prepared 4% paraformadehyde dissolved in PBS and adjusted to pH 7.4 with NaOH. After fixation, the bones were decalcified in daily changes of 14% EDTA (ED; Sigma, St Louis, MO) (pH 7.1) for 4–7 days. The bones were then soaked in 30% sucrose in PBS for 1 day. All processes of fixation, decalcification, and cryoprotection were performed at 4C under gentle agitation.

In preparation for embedding, a 200-ml plastic beaker containing 2-methylbutane was pre-chilled over dry ice. Disposable base molds (Thermo Shandon) were filled with frozen embedding medium (Thermo Shandon), with care to avoid the introduction of bubbles. Bone samples were immersed in embedding medium with their posterior surface against the floor of the mold. The embedding media was flash frozen by holding the mold with forceps in a solution of 2-methylbutane taking care to keep the embedding mold on a horizontal level. Once the medium was frozen, the mold was allowed to sink to the bottom of the beaker for a longer time until it was completely frozen. The molds were removed from the methyl butane solution and wrapped in a square of aluminum foil, placed in a plastic container, and stored at –20C or –80C.

CryoJane Frozen Section
Cryosectioning was performed on a Leica CM1900 Cryostat (D-69226; Leica, Inc., Nussloch, Germany) equipped with CryoJane Frozen Sectioning Kit (Instrumedics Inc.; Hackensack, NJ). The CryoJane process is designed to capture a frozen cryostat section on a special cold adhesive tape to assist in transferring the section to a cold glass microscope slide coated with an ultraviolet light-curable, pressure-sensitive adhesive. The section is permanently bonded to the adhesive on the slide with a flash of ultraviolet light. Subsequently, the transfer tape is removed from the slide leaving the still-frozen section adherent to the microscope slide. The block containing the decalcified femur was oriented in the block holder to obtain a 5-µm longitudinal central section that includes the central vein. The slides were air dried in the dark and kept in slides box at 4C or –20C before examination and staining.

Paraffin Embedding and Sectioning
The 4% paraformadehyde-fixed and decalcified femurs (see above) were dehydrated in gradual alcohol from 50% to 100% and cleared by xylene and impregnated in four changes of paraffin at 58C. The bones were sectioned at 5-µm thickness and deparaffined before examination with fluorescent microscope.

Fluorescent Imaging
The slides holding the frozen or paraffin sections were placed in PBS with 1 mM MgCl2 for 30 min at room temperature and then mounted with cover slide using 50% glycerin in PBS. GFP was examined with a Zeiss Axioplan 200 inverted microscope equipped with epifluoresence and a Zeiss AxioCam color digital camera. The average exposure time was 0.5–1 sec without binning or cosite sampling but was adjusted to obtain the GFP signal without exceeding the capacity of the camera to capture the full dynamic range of image. Table 1 shows the filter combinations that were used to distinguish the GFP signal from the autofluorescent background of bone marrow.


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Table 1

Fluorescent filter sets used to image GFP in mouse bone.

 
A Zeiss-Improvision microscope workstation was assembled that includes a motor-controlled mechanical stage (Ludl Electronics; Hawthorne, NY), filter wheel, objective turret, and shutters, which in turn are addressed by Openlab software (Improvision; Lexington, MA) using the Macintosh platform. A macro language was utilized to assemble a series of steps for imaging a region of a histological section in a reproducible manner. The macro was used to generate a 10x composite image of the entire longitudinal bone section. It instructs the microscope to take a series of adjacent 10x images from a start and finish point that is determined by the operator. At each stop point the operator sets the focus that is recorded for subsequent recall. A composite image was generated from one or more fluorescent images recorded at each focused stop point. Subsequently the slide was removed from the stage for further histological procedures. The image of the treated section was again recorded using the same location settings as the fluorescent image.

In both cases a stack of labeled image files of the adjacent optical sections were generated within the Improvision software. The file set was exported into Graphic Converter (http://www.lemkesoft.com) that assembles the individual files into a composite image. This was imported into Photoshop where it was rotated to a vertical position (distal femur up), and extraneous background was cropped. The full unaltered image (~60 MB) is saved as the primary source file from which smaller subsections were derived for overlapping or merging images of the same region recorded under different optical conditions. The macro can be downloaded and more details of the conversion process can be obtained at http://skeletalbiology.uchc.edu/30_ResearchProgram/304_gap/index.htm (click the link, Lineage in Vivo followed by the link, image acquisition).

Histological Staining and Immunohistochemstry
A variety of light- and fluorescent-based staining techniques were investigated to assess their compatibility for colocalization with the endogenous GFP signal. Because some chemical and immunological staining steps can destroy the GFP signal, the sections should be prepared with a non-permanent mounting medium (e.g., 50% glycerin) to obtain the GFP images. Subsequently, the coverslip can be removed by soaking the slide in PBS until it spontaneously detaches, after which the slide can be used in the following staining procedures. Details of each procedure can be found at our web site at http://skeletalbiology.uchc.edu/30_ResearchProgram/304_gap/index.htm (click the link, Lineage in Vivo followed by the link, histological protocols).

Hematoxylin and Eosin (HE) Staining
Slides of frozen section were rinsed in water and stained in hematoxylin for 2 min and then washed well in tap water. After rinsing the slides in 95% alcohol, the slides were stained with eosin for 10 sec and rinsed with tap water, then dehydrated with alcohol, cleared with xylene, and mounted with polymount mounting media.

Fluorescent Counterstaining
The cell nuclei of the sections were stained with complementary orange color to provide a fluorescent counterstain of the GFP. SYTO Orange (S-11368; Molecular Probes, Eugene, OR) was diluted to 50 nM in 50% glycerin/PBS and used to mount the coverslip.

Tartrate-resistant Acid Phosphatase (TRAP) Staining for Osteoclasts
Sections were incubated for 30 min at 37C in preheated sodium acetate buffer, pH 4.9, with naphthal AS-BI phosphate (Sigma). The slide was transferred to a filtered solution of pararosanilline and sodium nitrate for ~2–4 min at room temperature. The sections were rinsed with distilled water, counterstained with hematoxylin for 30 sec, dehydrated, and mounted with a coverslip. As controls for TRAP activity, sections were incubated in substrate-free solution.

TUNEL Staining for Cellular Apoptosis
In situ detection of apoptosis was performed using the TMR red in situ cell-death detection kit (Roche, Penzberg, Germany). The sections were washed with PBS and placed in a permeabilization solution containing 0.1% Triton X-100 and 0.1% sodium citrate for 2 min on ice. Subsequently, the slides were incubated with TUNEL reaction mixture that contains the enzyme (TdT) and fluorescent label (tetramethlyrhodamine-conjugated nucleotides) in a humidified chamber for 1 hr at 37C in the dark. After washing with PBS, the slides were mounted with 50% glycerin in PBS.

LacZ Staining for Transgenic ß-Galactosidase Activity
Sections were incubated in X-gal reaction buffer (1 mg/ml of 5-bromo-4-chloro-3-indolyl-ß-D-galactoside in PBS, pH 7.4, 5 mmol/L of potassium ferrocyanide, 5 mmol/L of potassium ferricyanide, 2 mmol/L of MgCl2, and 0.1% Triton X-100) for 2 hr at 37C in the dark. After washing with PBS-MgCl2 x 10 min, slides were postfixed with 4% paraformaldehyde and washed with distilled water. Slides were rinsed with 95% ethanol for 2 min and stained with eosin for 10 sec. Sections were dehydrated, cleared, and mounted in permount.

CD31 Immunostaining
Slides were rinsed with PBS and incubated for 30 min at room temperature with 3% hydrogen peroxide in distilled water to block endogenous peroxidase activity. After washing in PBS the slides were blocked for 20 min in 1x Universal Blocking Reagent (BioGenex; San Ramon, CA) and rinsed with PBS. The frozen sections were incubated overnight at 4C with a 1:100 dilution of purified rat anti-292 mouse CD31 monoclonal antibody (BD Biosciences; San Diego, CA). After washing in PBS, the sections were incubated with 1:200 dilution of biotinylated rabbit anti-rat IgG (Vector Laboratories Inc.; Burlingame, CA) for 1 hr at room temperature, then washed and incubated for 30 min at room temperature with Vector ABC reagent. The slides were rewashed and developed with DAB kit (Vector Laboratories Inc.). Slides were mounted with 50% glycerin in PBS.

Bromodeoxyuridine (BrdU) Immunostaining
BrdU was prepared at 6 mg/ml in PBS and injected intraperitoneally at 60 µg/g body weight, two times in 1 day before sacrificing. CryoJane frozen sections were cut and air dried. The slides were rinsed with PBS and incubated for 50 min at 37C with 0.1% pepsin in 0.1 N HCl in PBS. After washing, the slides were placed in 2 N HCl in distilled water at 37C for 30 min to denature DNA. The slides were rinsed with PBS five times and blocked with 5% normal donkey serum in PBS. The sections were incubated overnight at 4C with a 1:1000 dilution of mouse monoclonal anti-BrdU antibody (Sigma) in 1% normal donkey serum and 0.1% bovine serum albumin (BSA) in PBS. After washing, the sections were incubated with 1:100 dilution of FITC- or TRITS-conjugated donkey anti-mouse IgG at room temperature for 40 min. The slides were then washed and mounted with 50% glycerin in PBS for fluorescent microscopy.

In Situ Hybridization
The patterns of expression of dentin sialophosphoprotein (DSPP) in frozen sections were examined by in situ hybridization. The protocol was essentially the same as that described for paraffin sections (Braut et al. 2003Go). Briefly, slides were rinsed with PBS, fixed, acetylated, and hybridized to a 32P-labeled RNA probe. A 1445-bp fragment of mouse DSPP cDNA corresponding to nucleotides 1442–2887 (kindly provided by Dr. M. MacDougall) in Bluescript was digested with EcoRI or SacI and transcribed with T3 or T7 RNA polymerase for antisense and sense probes, respectively. The silver grains were pseudocolored red to contrast with the endogenous GFP signal.


    Results
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
This work began as a comparison of GFP expression in long bone prepared by paraffin vs frozen embedding and sectioning method. Although the paraffin processing did allow certain colors of GFP (emerald and topaz) to persist, the intensity of the GFP signal was lower, which became evident when the paired femurs from a single animal were prepared by the two methods (Figure 1). Although the quality of the histological image from the frozen section was poor as anticipated, the strength of the GFP signal was much more intense than in the paraffin section. Furthermore, the signals from eGFP, GFPcyan, and GFPsaph were retained in the frozen section but completely lost in the paraffin section (data not shown).

Imaging GFP in Frozen Sections of Adult Bone
The feasibility of using frozen embedding for the standard analysis of GFP expression in adult bones became apparent when sections were prepared using the CryoJane tape system. The adhesive tape captures the section as it is cut from the block and allows the section to be transferred and bonded directly to the slide while still frozen. Because the transfer process occurs while the sample remains frozen, distortion due to melting is minimized. Furthermore, the bonding of the section to the slide stabilizes the histological relationships through multiple staining and imaging procedures. With experience, the quality of the image rivals a paraffin section, and the time necessary to prepare a section is significantly reduced. The tape-transfer process is sufficiently reliable to routinely produce serial 5-µm sections. An additional advantage of retaining a stronger GFP signal is a significant reduction in the autofluorescent background of the bone section, although it is still necessary to use the dual bandpass filters to shift the background activity away from the GFP spectrum (see Materials and Methods).

One of the objectives of the histological analysis was the ability to record, archive, and retrieve a full high-power digital image of the section that would be available for detailed visual inspection. The image file would serve as the primary data source obtained under standardized optical conditions and could be retrieved and compared with similar sections from test and control. One solution was provided by the Openlab software package that contains a macro programming language and flexible image management options to capture and process multiple images of a histological section. Figure 2 illustrates the process for recording and generating the primary composite image file and the ability to view any portion of the section as a high-power image. It is from the primary file that the higher power images are obtained and processed for optimal visual effects. However, the original file is maintained and can be retrieved for verification or reanalysis. Examples of source files that were used in this report can be downloaded from the web site at http://skeletalbiology.uchc.edu/index_program.htm (link to Image Center). The images can be viewed either directly in the browser by clicking the view button, or the original high-resolution file can be downloaded to the computer for examination in Photoshop by a right click (PC) or option click (Mac). The latter step will allow viewing the image at the same high-power setting as originally recorded.

Spectrum of GFP Reporters that Are Visible in Bone
Key to the analysis of GFP in bone cells is a strong promoter capable of driving high expression that exceeds the autofluorescent background. Promoters that drive extracellular matrix genes as opposed to transcription factor promoters appear to be optimal choices. The Col1a1 promoter has proven to be useful in part because different fragments appear to be preferentially expressed at different stages of osteoblast differentiation. Two fragments that have been extensively evaluated are pOBCol3.6GFP and pOBCol2.3GFP. They contain either 3.6 kb or 2.3 kb of upstream flanking sequence of the rat Col1a1 gene, the first exon containing a mutated AUG start codon, and the majority of the first intron. It is joined to an expression cassette containing a splice acceptor, triple translation stop codon, the GFP reporter containing a Kozak box, and translation start codon within the GFP cDNA, as well as a growth-hormone-derived polyadenylation sequence. Four versions of GFP (GFPcyan, GFPemd, GFPsaph, and GFPtpz) were utilized in this report, and the maps of the constructs are shown in Figure 3.

Femurs from 2- to 3-month-old transgenic-carrying mice derived from the four constructs are shown in Figure 3. A low-power image of the entire bone and high-power image of trabecular and cortical bone are shown to illustrate the quality of the image and clarity of the GFP signal relative to the background. Irrespective of the color of GFP, the pOBCol3.6 constructs were active in the periosteum and endosteal bone cells with minimal extension into the osteocytic layer, whereas the pOBCol2.3 construct was limited to endosteal osteoblasts and osteocytes.

Because eGFP is the most widely used version of GFP found in transgenic mouse studies, it was important to demonstrate that it can be visualized in the cryostat sections. Figure 4 illustrates the expression of eGFP driven by the Tie2 promoter, which is active in vascular endothelial cells. The pattern demonstrates the utility of this transgene for visualizing endothelial cells in adult lung and the endothelial network that is part of the bone marrow.



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Figures 4–8

Figure 4 Detection of eGFP. The Tie2eGFP mouse generates a GFP signal from vascular endothelium that is lost in paraffin sections but is preserved in frozen sections. (A) Lung section showing GFP positive vascular endothelium (arrow). (B) Expression of GFP in the endothelial cells (arrow) of the bone marrow. The weaker signal of GFP is responsible for the higher (red tinged) background of the hematopoietic elements of the marrow. Note that the osteoblasts lining the endosteal layer are GFP negative (arrowhead). Bv, blood vessel; Br, bronchus; Bc, cortical bone; Bm, bone marrow.

Figure 5 Colocalization of GFP with chemical and fluorescent counterstains of bone. (A) x10 scanned image of pOBCol 2.3GFPemd. (B) The image after the same section was processed for HE. (C) High-power overlay of the two images using Photoshop. The area of the original used for this image is boxed in (B). (D) Simultaneous dual-filter imaging of pOBCol2.3GFPemd and SYTOX Orange fluorescent dye counterstaining of bone section. Gp, growth plate; Bc, cortical bone; Bm, bone marrow; arrow, osteoblasts; arrowhead, osteocytes.

Figure 6 Enzymatic staining of cryostat sections of bone. In both cases shown, the endogenous GFP signal is destroyed by the enzymatic procedure. (A) TRAP-stained (red color) section from the metaphyseal region of the femur. The section is counterstained with hematoxylin. The length of time the section is exposed to the color substrate needs to be reduced from paraffin conditions due to better preservation of enzymatic activity in the frozen section. (B) LacZ expression (blue color) in osteoblasts in the primary spongiosa of a 7-day-old double transgenic mouse with an activated conditional Rosa26 ß-gal transgene. The intermittent number of positive cells reflects the transient activation of the Col2.3CreERT2 transgene after a single injection of tamoxifen. The section is counterstained with eosin. Gp, growth plate; Bm, bone marrow.

Figure 7 Simultaneous imaging of GFP and immunostained section of bone. The figure shows examples in which the GFP signal is not destroyed by an immunological procedure. (A) TUNEL staining of normal mouse bone. The rhodamine-linked nucleotides label cells red (apoptosis) within the bone marrow and in osteocytes. Presumably, failure to colocalize pOBCol3.6GFPtpzwith the red-labeled cells is due to loss of promoter activity in cells undergoing apoptosis. (B–D) Individual images (B,C) and overlay (D) of a section stained with antiCD31 antibodies followed by detection with HRP-labeled secondary antibody. The section is prepared from a Col2.3{Delta}tk – pOBCol3.6GFPtpz double transgenic mouse recovering from gangciclovir-induced osteoblast ablation that secondarily removes the hematopoietic elements from the marrow space (Visnjic et al. 2001Go,2004Go). This section was obtained during the early recovery phase when new bone cell differentiation develops in the marrow space prior to the repopulation of the marrow space with hematopoietic elements. Bc, cortical bone; Bm, bone marrow; M, muscle; arrow, vascular endothelium; arrowhead, endosteum.

Figure 8 Sequential imaging of GFP followed by in situ hybridization of teeth and immunostaining of bone. (A–C) In situ labeling of DSPP in the developing tooth of a pOBCol2.3GFPemd transgenic mouse. After the section is imaged for GFP (A), the slide is processed for hybridization with the cRNA to DSPP that includes incubation with proteinase K that destroys the GFP signal (B). The GFP signal is extracted from A and is placed over image B to obtain image C. (D) The incorporation of BrdU into DNA of dividing cells is detected by a TRITC-conjugated secondary antibody after the nuclear chromatin is denatured in HCl. The GFP image obtained prior to the BrdU detection step is overlaid to reveal cells within the osteoblast lineage that were undergoing cell division when the animal was exposed to BrdU. The interval between BrdU exposure and tissue acquisition is 3 days to decrease the strong signal within the bone marrow. O, terminally differentiated odontoblasts; Gp, growth plate; Bt, trabecular bone; arrow, functional odontoblasts.

 
Histochemical Stains of Bone
Standard histological stains of bone such as HE or Masson trichrome (not shown) destroy the GFP signal. The stains reveal the quality of the histological preparation but are not routinely used to evaluate the location of GFP expression because there is sufficient detail of the bone revealed by the autofluorescent background. When it is necessary to align the GFP signal with a standard histological stain, the GFP image can be recorded prior to staining (Figure 5A), and the visual image can be repeated after the staining is performed (Figure 5B). The composite image is produced by extracting the GFP signal from its black background and placing it over the HE image (Figure 5C). Another approach is to use a fluorescent counterstain for DNA of the entire cellular population of the section using a color that can be simultaneously contrasted with the GFP expression of the section (see Figure 5D). The nuclei of cells that are GFP negative are a solid red, whereas the GFP signal from strongly positive cells overpowers the red counterstain. Cells that are weaker in GFP expression show a yellow nuclear color and a green color in the cytoplasm.

Enzymatic Staining of Bone
Because frozen sections maintain the enzymatic activities of a tissue section to a greater extent than a paraffin section, a number of commonly used histological stains were investigated. TRAP stain (Figure 6A) was used to identify osteoclastic cells on the bone surface. In the bone sections prepared with the tape system, it was possible to identify intensely red stained cells with sufficient retention of morphology to appreciate the multinucleated appearance of an osteoclast. However, the incubation conditions for paraffin sections need to be modified to prevent mononuclear cells from staining. Alkaline phosphatase staining also gave a strong red signal that was particularly strong in the periosteal layer of cortical bone (not shown). The reaction conditions of each enzymatic stain destroys the GFP signal. Many transgenic mice contain the lacZ reporter construct, which has proven to be very useful for developmental studies but of limited utility in adult tissues. Young embryos allow diffusion of the lacZ substrate more readily and therefore stain a uniform blue color either in whole mount preparations or in frozen sections. However, substrate diffusion into adult bone is limited to surface cells, and the activity is unreliably retained in paraffin sections. The tape-prepared bone frozen sections from neonatal or early adult bone harboring the conditional Rosa reporter activated by a tamoxifen-induced Cre transgene shows that lacZ activity can be visualized when the cut section is exposed to the lacZ substrate. Figure 6B shows scattered blue-stained cells throughout the primary metaphysis. Because the activating Cre is driven by a Col2.3 promoter, the positive cells are limited to newly developed osteoblasts and are not seen in cartilage of growth plate. This experiment shows the feasibility of planning experiments with marker transgenes that can include GFP and lacZ. The conditions for lacZ staining employed here destroyed the GFP.

Molecular and Immunological Procedures of Bone
Two types of stains were employed to show relationships between a histological feature and the pattern of GFP expression. The first type is a stain that does not interfere with endogenous GFP expression and allows the two signals to be simultaneously imaged. TUNEL staining is a widely used method for identifying apoptotic cells. Standard kits supply the reaction components to end-label fragmented DNA with a red fluorescently labeled deoxynucleotide. The procedure maintains the endogenous GFP signal because the enzyme can act on its target without prior DNA denaturation. Figure 7A illustrates the coexpression of pOBCol2.3GFPemd and the red fluorescence of cells entering apoptosis. It is unusual to find cells that coexpress both markers indicating that GFP expression is probably lost early in the apoptotic process. Other examples are immunostains that perform without an antigen retrieval step such as the CD31 cell surface marker of endothelial cells (Figures 7B–7D). In this example, the pigment produced by the peroxidase reaction requires the sample to be sequentially imaged under light and fluorescent conditions and the two images fused. However, if a rhodamine-labeled secondary antibody is used to visualize the CD31 antibody, the two colors can be simultaneously imaged (not shown). Antigens are well preserved in the paraformaldehyde-frozen sections and appear to give a stronger signal than a corresponding paraffin-embedded section. GFP expression is not lost after membranes are permeabilized by Triton, suggesting that intracellular antigens should also be coimaged with GFP.

The second class of molecular stains requires protein denaturation to expose nuclear DNA or cytoplasmic RNA. In situ hybridization also requires a vigorous deproteination and denaturation step to expose the RNA target. Figures 8A–8C also show the fluorescent image before (Figure 8A) and light images after of a developing tooth that was hybridized with a 32P-labeled cRNA probe to DSPP, pseudocolored red, and counterstained with hematoxylin (Figure 8B). The GFP signal from the image in Figure 8A is placed over the image in Figure 8B and shows coexpression of GFP and the DSPP (Figure 8C, yellow). The strength of the in situ labeling procedure appears to be equivalent to paraffin-embedded tissue. Mice or cultured cells exposed to BrdU incorporate this modified nucleotide into cells actively synthesizing DNA. The nuclear DNA needs to be denatured in HCl for the fluorescently labeled anti-BrdU antibody to recognize its target, and this step destroys GFP. Figure 8D shows the overlay of the two images before and after denaturation and incubation with rhodamine-labeled anti-BrdU labeling. Colocalization of the antibody and GFP signal (yellow) is observed on endosteal osteoblasts while non-osteoblastic BrdU-labeled cells within the bone marrow are red.


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
The intersection of modern molecular genetics and classical histology has greatly enhanced our understanding of tissue and cell interactions in a complex environment that is not possible to reproduce in cell culture or in vitro setting. Expressing lacZ or ß-lactamase (Spergel et al. 2001Go; Knapp et al. 2003Go) in cell culture and the developing embryo has made it possible to observe protein trafficking (Maruvada et al. 2003Go), perform fate mapping of a cell lineage (Chai et al. 2000Go; Tidhar et al. 2001Go; Durcova-Hills et al. 2003Go), and define the autonomous or non-autonomous nature of gene function (Rivera-Perez et al. 1999Go; Tam and Rossant 2003Go). To overcome the difficulty of substrate diffusion in more mature tissues, promoters that drive a unique epitope have been used to identify transgene expression by immunohistochemical techniques (Luby-Phelps et al. 2003Go; Zagzag et al. 2003Go). GFP has significant advantages over both approaches primarily because the image is generated independent of experimental manipulation and the ease of correlating expression in primary culture with expression within intact tissue. Realizing this potential required developing methods that preserve a distinct GFP signal in histological sections with a significant autofluorescent background.

Although this paper reviews the success we have experienced in applying multiple colors of GFP in bone and teeth, the signals are equally preserved in soft tissues. Key to the analysis is the cryostat sections using a tape system that preserves the integrity of the histological section that is permanently attached to the glass slide (Figure 1 vs Figure 3). The air-drying step prior to mounting avoids the appearance of distorting water droplets, and the intensity of the GFP does not noticeably decline until 1–2 months after preparation. This allows the section to be imaged under fluorescent conditions and subsequently to be treated with standard histological techniques, allowing the relationship between the fluorescent images to be maintained. An additional advantage of the tape method is the ease to acquire full-length longitudinal bone sections without distortion and collapse of the tissue structure. Using strong promoters to drive GFP is essential in obtaining a cellular image distinct from an autofluorescent background. Because the GFP signal is better preserved in frozen section compared with a paraffin section may partially explain the lower autofluorescent background. In addition, the dual bandpass filters optimized for a specific GFP and a far-red channel allows the GFP to be recognized as distinct from the bone marrow autofluoresence. Without the red-shifted filter, the GFP signal is only appreciated as a different intensity of background. We have demonstrated that transgenic mice can be produced to express three distinctly different colors of GFP (cyan, saph, topaz) that have the potential to be combined for a multiplex analysis. Unfortunately, eGFP is only useful when combined with a red-shifted transgene because its spectrum cannot be adequately separated from the other colors.

The frozen section is superior at preserving immunological epitopes and endogenous enzymatic activity. Wherever possible, it is advantageous to use fluorescent-based indicator systems because of the possibility of simultaneously localizing the GFP signal with the stain signal. Thus, immunostaining with a fluorescently tagged antibody or fluorescent chemical stains for cell nuclei are examples where the image can be simultaneously compared. In the case of signals that are optimized with dual filters, the two signals can be examined directly. The cyan, saph, and topaz colors require recording separate images that can be optically overlapped. Procedures that denature the proteins on the slide and stains that precipitate a visual pigment appear to damage the GFP signal requiring a sequential procedure of image capture, staining procedure, and second image capture. Accurate alignment is more difficult, particularly after chemical staining due to the dehydration steps that can induce tissue shrinkage. The process of alignment of the assembled images is facilitated with a mechanical stage and computer system that can store and retrieve the position of the original image. Final alignment is done manually in Photoshop by merging and adjusting the layers.

The use of a computer-controlled microscope permits the development of image recording strategies with the potential of archiving and distributing a raw microscopic image that could serve as a reference standard for the expression of a GFP transgene in various genetic backgrounds. The raw image could be subjected to digital analysis or a specific feature could be recorded in a queriable database. The images that have been placed on our website illustrate the potential of this technology as a raw dataset not unlike the deposition of an unprocessed microarray dataset. The distribution to the bone biology community of the GFP transgenes with established expression patterns in bone may provide a means for comparing bone cell dynamics in intact bone based on age, sex, genetic background (see image center at our home page for appended figures, http://skeletalbiology.uchc.edu/index_program.htm), and underlying bone abnormality independent of the site where the research is being performed.


    Acknowledgments
 
Supported by NIH Grants AR-43457 and DK-63478, NASA grant NAG5-6316 to DWR, and a Michael Geisman fellowship from the Osteogenesis Imperfecta Foundation to ZK.


    Footnotes
 
Received for publication May 17, 2004; accepted September 30, 2004


    Literature Cited
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 Summary
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 Materials and Methods
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 Literature Cited
 

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