Journal of Histochemistry and Cytochemistry, Vol. 47, 949-958, July 1999, Copyright © 1999, The Histochemical Society, Inc.


ARTICLE

Specificity of the Localization of Transforming Growth Factor-{alpha} Immunoreactivity in Colon Mucosa

Cory J. Xiana, Carolyn E. Mardella, and Leanna C. Reada
a Child Health Research Institute and Co-operative Research Centre for Tissue Growth and Repair, North Adelaide, Australia

Correspondence to: Cory J. Xian, Child Health Research Inst., 72 King William Road, North Adelaide 5006, South Australia, Australia.


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Transforming growth factor-{alpha} (TGF-{alpha}) plays an important role in gastrointestinal pathophysiology. However, the exact location of its expression in the intestine is still controversial. This study systematically compared the localization of TGF-{alpha} immunoreactivity in frozen or fixed human colon using three different antibodies and examined specificity of antibodies by using tissues from TGF-{alpha} knockout mice and by Western blotting. Consistent with the mRNA distribution revealed by in situ hybridization, a similar staining pattern was obtained in frozen sections by all three antibodies, localizing on the surface and along the crypt epithelium. In paraffin sections, although the polyclonal antibodies (raised against recombinant human or rat TGF-{alpha}) gave minimal staining, the monoclonal antibody (against C-terminal peptide of human TGF-{alpha}) still gave intense staining on the surface and upper crypt epithelium. By using specimens from TGF-{alpha} knockout mice in immunostaining and Western blotting, the polyclonal antibodies were shown to be specific. In contrast, specificity of the monoclonal antibody was in doubt in rodent tissues because it gave similar detection between wild-type and knockout mice in both analyses, indicating its crossreaction to non-TGF-{alpha} molecules. In conclusion, frozen sections and antibodies raised from recombinant TGF-{alpha} should be used for TGF-{alpha} immunohistochemistry in the colon. (J Histochem Cytochem 47:949–957, 1999)

Key Words: TGF-{alpha}, immunohistochemistry, colon, TGF-{alpha} knockout mice, localization, specificity


  Introduction
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PREVIOUS STUDIES have shown that TGF-{alpha} is expressed in the mucosa throughout the gastrointestinal tract at all ages (Beauchamp et al. 1989 ; Koyama and Podolsky 1989 ; Malden et al. 1989 ; Thomas et al. 1992 ; Miettinen 1993 ) and appears to play important roles in promoting proliferation and migration of intestinal epithelial cells and in stimulating gut growth, maturation, and repair (Konture et al. 1992 ; Polk et al. 1992 ; Romano et al. 1992 ; Karnes 1994 ; Podolsky 1994 ; Barnard et al. 1995 ). Despite these known actions in the gut, the site-specific expression of TGF-{alpha} immunoreactivity and mRNA is still uncertain or controversial. Most of the immunohistochemical studies on intestinal TGF-{alpha} used paraffin sections from fixed tissues and mostly used a monoclonal antibody raised against the carboxyl terminal 17 amino-acid peptide of human TGF-{alpha}. Whereas some studies showed either cytoplasmic or supranuclear staining in both surface epithelium and crypt base epithelium in the large intestine (Perez-Tomas et al. 1993 ), others revealed staining mainly in the surface and upper crypt epithelium (Thomas et al. 1992 ; Christensen and Poulsen 1996 ). In the small intestine, Bernard et al. 1991 and Christensen and Poulsen 1996 found distribution of TGF-{alpha} immunoreactivity along the villi with a tendency for increased expression at the villous tip, whereas Thomas et al. 1992 and Alison et al. 1993 saw more intense staining in the lower two thirds of the villi. Whether the data from most of these immunohistochemical studies with paraffin sections truly represent the TGF-{alpha} distribution in the untreated and nonprocessed intestine is not certain, because tissue fixation and processing might modify the antigenicity of TGF-{alpha} peptide. Furthermore, although some of these studies demonstrated that the TGF-{alpha} staining can be blocked by incubating the antibody with an excess amount of recombinant TGF-{alpha} peptide, the possibility remains that the antibody was detecting a similar sequence in an alternative tissue antigen to TGF-{alpha}. Controversy also exists over the exact site of TGF-{alpha} mRNA synthesis. Whereas the in situ hybridization results of Babyatsky et al. 1996 revealed localization of TGF-{alpha} mRNA mainly in the villous epithelium in the small intestine and in the differentiated surface epithelium in the large intestine, those of Dvorak et al. 1994 and of Campbell et al. 1996 showed the mRNA localization in the undifferentiated crypt cells in the small intestine.

This study was designed to determine if the controversy in TGF-{alpha} peptide localization in the mucosa of the normal colon could be explained by tissue fixation/processing or by the different antibodies used. We first examined staining patterns in frozen and paraffin sections from human colon by using different primary antibodies, then investigated staining specificity by using various negative controls including colon tissues from TGF-{alpha} knockout mice and by performing Western immunoblot analysis. In situ hybridization was also performed to see if TGF-{alpha} mRNA and peptide have a similar localization in the colon.


  Materials and Methods
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Colon Tissues from Human and TGF-{alpha} Wild-type or Knockout Mice
With approval from the Human Ethics Committee of the Women's and Children's Hospital (Adelaide, South Australia), surgically resected colon tissues were freshly collected. Specimens of normal macroscopic appearance were either OCT-embedded and snap-frozen or were fixed in 10% buf-fered formalin for 24 hr or in Methacarn fixative for 2 hr, followed by alcohol dehydration and paraffin-embedding. Some colon tissues were also freshly frozen at -80C to be used in total protein extraction for Western blotting study.

Colonies of TGF-{alpha} wild-type and knockout mice were bred from breeding pairs (Mann et al. 1993 ) provided by Dr. Ashley R Dunn (Ludwig Institute of Cancer Research; Melbourne, Australia). The homozygous TGF-{alpha} gene knockout resulted in a complete lack of TGF-{alpha} peptide synthesis in vivo, as demonstrated previously (Mann et al. 1993 ). The lack of TGF-{alpha} synthesis does not affect reproduction and general health of the mice apart from inducing waviness of the fur and curliness of the whiskers (Mann et al. 1993 ), which provides a marker for easy identification from the wild types. Male mice 9 weeks old were sacrificed by a CO2 overdose. Colon specimens were collected as for the human surgical samples. These procedures were undertaken with approval of the Animal Ethics Committee of the Women's and Children's Hospital.

Immunohistochemical Analyses of TGF-{alpha}
To compare any potential difference in the localization of TGF-{alpha} immunoreactivity between paraffin and frozen sections, immunohistochemical detection of TGF-{alpha} was performed on both 8-µm cryosections and 3-µm paraffin sections mounted on gelatin-coated glass slides. To block nonspecific binding sites, the paraffin and frozen sections were treated for 90 min at room temperature (RT) with Tris-buffered saline (TBS, pH 7.4) containing 1% bovine serum albumin (BSA) and 5% normal rabbit serum (for sections to be incubated subsequently with secondary antibody raised in rabbit) or pig serum (for sections to be incubated with a secondary antibody raised in pig). On some occasions, antigen retrieval procedures were carried out with paraffin sections before the above blocking step as a means to enhance TGF-{alpha} staining. Sections were either treated with 0.1% trypsin in PBS, pH 7.4, for 5 min at RT, incubated with 0.05% saponin for 30 min, or heated in just boiled Antigen Retrieval AR-10 solution (BioGenex; San Ramon, CA). After blocking, three anti-TGF-{alpha} antibodies were used in this study, including a mouse monoclonal IgG (GF10) raised against human TGF-{alpha} C-terminal peptide 34–50 (Oncogene Science; Cambridge, MA) at 1:100 dilution, a polyclonal sheep serum raised against rat TGF-{alpha} recombinant peptide (a gift from Dr. R. Coffey; Vanderbilt University, Nashville, TN) at 1:1000 dilution, or a rabbit polyclonal IgG made against human recombinant TGF-{alpha} (PeproTech; Rocky Hill, NJ) at 1:200 dilution in TBS containing 1% BSA. Sections were incubated overnight with one of the above antibodies at 4C, followed by washing twice in TBS containing 0.01% Tween-20 for 10 min each and once in TBS for 5 min. Then sections were respectively incubated with a rabbit anti-mouse biotinylated IgG (Dako; Carpinteria, CA), a rabbit anti-goat biotinylated IgG (Dako), or a swine anti-rabbit biotinylated IgG (Dako). After washing as above, the sections were detected with Cy3–streptavidin (Zymed Laboratories; San Francisco, CA). Immunoperoxidase detection was also used on some occasions instead of the Cy3 fluorescent detection. For this, sections were incubated for 20 min in cold 0.3% H2O2 in methanol to quench any endogenous peroxidase activity before blocking with normal serum. After the secondary antibody step, sections were treated with avidin and biotinylated horseradish peroxidase reagents (Dako) and developed with diaminobenzidine tetrahydrochloride (DAB) substrate (Sigma; St Louis, MO) with hematoxylin counterstaining.

For verification of staining, apart from using colon sections from TGF-{alpha} knockout mice in comparison with wild-type animals, other negative controls included preabsorption for 2 hr at RT of the anti-human TGF-{alpha} monoclonal antibody in the presence of an excess amount of human TGF-{alpha} (40 µg/ml) (GroPep Pty; Adelaide, Australia), incubation of the anti-rat TGF-{alpha} primary antibody in the presence of rat TGF-{alpha} (20 µg/ml) (Peninsula Laboratories; Belmont, CA), and replacement of the primary antibody by a normal mouse isotypic monoclonal IgG (Dako), a normal sheep serum, or a normal rabbit IgG (Dako) at the same dilution as the corresponding primary antibody. The sheep anti-rat TGF-{alpha} was demonstrated by us (not shown) and by a previous study (Beauchamp et al. 1989 ) using radioimmunoassays to have no crossreaction with mouse or human epidermal growth factor (EGF), a homologue of TGF-{alpha}. The two anti-human TGF-{alpha} antibodies have also been shown by suppliers (PeproTech and Oncogene Science) to have no crossreactions with human EGF.

TGF-{alpha} mRNA In Situ Hybridization
To examine whether the TGF-{alpha} mRNA is localized similarly to the protein, a nonradioactive in situ hybridization was carried out. A plasmid pGEM7(+Z) containing a 270-BP human TGF-{alpha} cDNA was used to generate sense and antisense digoxigenin (DIG)-labeled RNA probes as described (Miettinen and Heikinheimo 1992 ) using a DIG RNA labeling kit (Boehringer Manheim; Manheim, Germany). Following a protocol modified from a previous study (Wang et al. 1996 ), in situ hybridization was performed on 4-µm sections of fomalin-fixed, paraffin-embedded human colon tissue mounted on 3-aminopropyltriethoxysilane-coated glass slides. Dewaxed and hydrated sections were treated with 0.2 N HCl for 20 min at RT, and permeabilized with 75 µg/ml proteinase K (Sigma) for 20 min at 37C. After postfixation for 5 min with 2% paraformaldehyde in 10 mM PBS, pH 7.5, sections were neutralized for 10 min with 0.2% glycine in PBS at RT. After washing and dehydration, 25 µl hybridization mix (Wang et al. 1996 ) containing 0.5 µg/ml sense or antisense probe was added to each section, covered with a glass coverslip, and incubated in a humidified chamber for 18 hr at 50C. Posthybridization washes were undertaken at RT as follows, with gentle shaking: once in 2 x SSC solution (0.3 M NaCl and 30 mM Na citrate, pH 7.0) for 5 min to remove coverslips; once in 2 x SSC containing 100 µg/ml RNase A (Sigma) at 37C for 30 min to destroy unbound probes; twice in 2 x SSC for 15 min; and once in 1 x SSC for 10 min. After blocking of nonspecific binding sites with 10% normal sheep serum for 30 min at RT, the sections were incubated for 30 min at 37C with an alkaline phosphatase-coupled sheep anti-DIG IgG (1:450) (Boehringer Mannheim) and developed by incubating in NBT/X-phosphate substrate for 18 hr in the dark (Boehringer Mannheim). Finally, sections were lightly counterstained with methyl green and mounted in glycerol–gel mounting medium (Dako). In this study we found it unnecessary to include a step of blocking the endogenous alkaline phosphatase activity because the activity is minimal in the formalin-fixed and paraffin-embedded specimens, consistent with a previous finding (Bromley et al. 1994 ).

TGF-{alpha} Western Blotting
To further demonstrate specificities of the anti-TGF-{alpha} antibodies, Western blotting was performed with protein samples isolated from frozen colon tissues using the acid–ethanol extraction method (Beauchamp et al. 1989 ) or from fixed tissues using Laemmli's lysis buffer as described (Lalani et al. 1995 ). First, the protein concentration of the protein extract was quantitated using Bradford reagent (Sigma) with BSA (Sigma) as a standard. Equal amounts of protein (200 µg) from each sample, 250 ng of human recombinant TGF-{alpha}, and 2 µg of a broad range of biotinylated protein molecular weight markers (Bio-Rad; Hercules, CA) were treated with a reducing sample buffer, separated on a 12.5% SDS-PAGE mini-gel, and electroblotted onto a 0.2-µm nitrocellulose filter. The filters were probed with the rabbit anti-human recombinant TGF-{alpha} IgG at 1:1000 dilution or with the sheep anti-rat recombinant TGF-{alpha} serum at 1:5000 dilution, detected with a swine anti-rabbit or a rabbit anti-sheep biotinylated IgG as for the immunohistochemical analysis. Some filters were also probed with the mouse monoclonal anti-human TGF-{alpha} C-terminal IgG and a biotinylated rabbit anti-mouse IgG. Immunoreactivity was detected by avidin–biotinylated horseradish peroxidase reagents, and developed as enzyme chemoluminescence signal using the ECL Western blotting system (Amersham; Poole, UK).

On some occasions, before Western analysis with the anti-TGF-{alpha} monoclonal antibody, immunoprecipitation was performed to enrich any TGF-{alpha}-like immunoreactive material in protein extracts from frozen colon tissues. The protein extracts (1 mg each) were first incubated for 1 hr with protein G–Sepharose beads (Pharmacia Biotech; Uppsala, Sweden) to remove IgG in the homogenates. Then the supernatants were incubated with the monoclonal antibody (1:100) for 2 hr, followed by protein G immunobeads for a further 2 hr at RT to precipitate the monoclonal antibody bound with TGF-{alpha}-like molecule(s). After brief centrifugation, the beads were washed with TBS and heated for 10 min at 100C with 40 µl reducing SDS gel loading buffer to elute the precipitated TGF-{alpha}-immunoreactive material and the monoclonal antibody. The supernatants were then loaded for electrophoresis and Western analysis.


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Staining in Human Frozen or Paraffin Colon Sections with Different Antibodies
In this study we aimed to systematically examine the localization of TGF-{alpha} immunoreactivity in the human colon using three different antibodies in both frozen and fixed/paraffin-embedded sections and to verify the staining with various negative controls. We conducted these comparisons with colon sections derived from a normal region of a surgical specimen. On frozen sections, the three antibodies, the rabbit anti-human recombinant TGF-{alpha} IgG (Figure 1A), the sheep anti-rat TGF-{alpha} serum (not shown), and the mouse monoclonal anti-human TGF-{alpha} C-terminal peptide IgG (not shown) gave a similar, uniform cytoplasmic staining on the surface epithelium and on the epithelium along the entire crypt. The negative controls all consistently gave negative staining. These controls included preabsorption of the anti-human TGF-{alpha} antibodies with an excess amount of human TGF-{alpha} (Figure 1B) and substitution of the primary antibodies with a normal rabbit IgG, a normal sheep serum, or with an isotypic normal mouse IgG at the same dilution as the respective primary antibody (not shown).



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Figure 1. TGF-{alpha} immunoreactivity and mRNA detection in human transverse colon. (A) Immunofluorescent staining with the rabbit polyclonal anti-human TGF-{alpha} on a frozen section. (B) Staining on a frozen section with the rabbit antibody preincubated with human TGF-{alpha} (40 µg/ml) for 2 hr. (C) A paraffin section stained with the monoclonal antibody raised against human TGF-{alpha} C-terminal peptide. (D) A paraffin section stained with the rabbit polyclonal antibody. (E) A paraffin section stained as for D but with the trypsin antigen retrieval procedure. (F) A negative control for (E) with normal rabbit IgG in place of the primary antibody. (G) In situ hybridization detection of TGF-{alpha} mRNA in a paraffin section. (H) In situ hybridization control with a sense probe. Bars: A,B,G,H = 200 µm; CF = 100 µm.

On paraffin sections, however, different staining patterns were obtained compared with those in frozen sections. The monoclonal antibody against the human TGF-{alpha} C-terminal peptide still gave intense staining, which was localized predominantly on the surface epithelium and the upper quarter of crypt epithelium, plus some nuclear staining at the base of crypts (Figure 1C). This staining appeared specific because it could be abolished by preincubating the antibody with an excess amount of human TGF-{alpha} (not shown), as had been reported in various previous studies using this antibody on paraffin sections. On the other hand, both the rabbit anti-human TGF-{alpha} IgG (Figure 1D) and the sheep anti-rat TGF-{alpha} serum (not shown) gave faint staining, localized mainly on the surface and the upper crypt epithelium. Staining on paraffin sections was not affected by the type of fixative because there were no differences in staining between specimens from the same resected human colon tissue fixed in parallel either by formalin or by Methacarn (Figure 1D, showing a section from a formalin-fixed specimen). In view of these differences between the polyclonal and monoclonal antibodies, and between frozen and fixed tissue sections, we introduced several modifications to the immunostaining method in an attempt to enhance the detection in paraffin-embedded sections. Three different kinds of antigen retrieval protocols were tried. Whereas the retrieval steps with either saponin or heat treatment in AR-10 Antigen Retrieval solution (BioGenex) had little effect in enhancing TGF-{alpha} staining with the polyclonal antibodies (not shown), the antigen retrieval procedure with trypsin considerably improved staining on paraffin sections (Figure 1E vs 1D), which was localized uniformly on the surface and crypt epithelium, a pattern also seen in frozen sections (Figure 1A). The enhanced staining with the trypsin antigen retrieval step appeared to be specific because low background staining was obtained when the primary antibody was replaced by a normal rabbit IgG at the same dilution (Figure 1F). By using the immunoperoxidase staining method with DAB as a color substrate, similar results for all three antibodies were obtained as with the immunofluorescence staining (not shown).

In Situ Hybridization Detection of TGF-{alpha} mRNA
To examine whether TGF-{alpha} mRNA was distributed similarly to its peptide in the colon mucosa, in situ hybridization was performed on formalin-fixed, paraffin-embedded tissue sections. TGF-{alpha} mRNA was found mainly on the crypt epithelium and on the surface epithelium in colon mucosa (Figure 1G), consistent with the distribution pattern of TGF-{alpha} immunoreactivity. Minimal background labeling only was seen in negative controls with the sense probe (Figure 1H) or in sections pretreated with RNase A (not shown), indicating that the staining reflected hybridization between tissue TGF-{alpha} mRNA and the antisense probe.

TGF-{alpha} Immunostaining Specificity Test with Knockout Mice
By comparing staining patterns in colon sections from TGF-{alpha} wild-type and knockout mice, we further attempted to examine the staining specificities of various antibodies. In frozen sections from wild-type mice, TGF-{alpha} immunoreactivity, as detected by the sheep anti-rat TGF-{alpha} serum, was localized on the epithelial cells in surface epithelium and to a slightly lower extent along the entire crypt epithelium, with no immunoreaction in the cells within the lamina propria (Figure 2A and Figure 2B), a pattern similar to that with a frozen section of human colon. Incubation of the antiserum in the presence of rat TGF-{alpha} peptide (20 µg/ml) abolished most of the staining in all crypt regions (Figure 2C). In paraffin sections of wild-type mouse colon (fixed by formalin or Methacarn), however, no or only minimal staining was seen (not shown). Replacement of the antiserum with a normal sheep serum at the same dilution gave only minimal background staining in the wild-type animals (not shown). In the frozen sections from TGF-{alpha} knockout mice, no staining on the crypt epithelial regions was detected by this anti-rat TGF-{alpha} antiserum (Figure 2D). These results indicate that the TGF-{alpha} staining using this polyclonal antiserum in the colon was specific and that tissue fixation and processing destroyed most of the antigenicity of TGF-{alpha} peptide that could be recognized by this antiserum, as similarly observed with paraffin sections of fixed human colon tissues. Similar results were obtained with the rabbit anti-human recombinant TGF-{alpha} IgG in staining TGF-{alpha} wild-type and knockout mice (not shown). These results indicate that polyclonal antibodies raised against either rat or human recombinant TGF-{alpha} peptide generated specific TGF-{alpha} staining in the colon.



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Figure 2. TGF-{alpha} immunofluorescent staining in colon sections from TGF-{alpha} wild-type or knockout mice. (A) High-power view of a frozen colon section from a wild-type mouse stained with the sheep anti-rat TGF-{alpha} antiserum. (B) Low-power view of the same section as in A. (C) Frozen section from a wild-type mouse stained in the presence of recombinant rat TGF-{alpha} at 20 µg/ml. (D) Frozen section from a TGF-{alpha} knockout mouse stained with the same antibody as for A. (E) Paraffin section from a wild-type mouse stained with the mouse anti-human TGF-{alpha} C-terminal peptide monoclonal antibody. (F) Paraffin section from a knockout mouse stained with the monoclonal antibody. Bars: A,E,F = 80 µm; BD = 200 µm.

To test the specificity of the mouse monoclonal anti-human TGF-{alpha} C-terminal IgG (GF10) in immunostaining, we did not include frozen colon sections from wild-type and knockout mice because of the limitations of the detection system used. An anti-mouse secondary antibody would recognize the bound primary antibody (IgG) raised in a mouse as well as the IgG intrinsically present in the frozen sections of the mouse colon. However, in paraffin sections, most of the antigenicity of IgG intrinsically present in tissue had been destroyed during tissue fixation and processing, rendering it unrecognizable by the secondary anti-mouse IgG used in our detection system. When an isotypic monoclonal normal mouse IgG was used instead of the anti-TGF-{alpha} peptide IgG, there was no staining on colon paraffin sections from wild-type mice (not shown), indicating that this detection system could be used to visualize only the IgG molecules that bound to tissue TGF-{alpha}-like material but not the IgG molecules intrinsically present in tissue sections. As shown in Figure 2, this antibody gave a strong staining in paraffin sections from wild-type mice (Figure 2E), with a pattern similar to that found in human colon paraffin sections. This monoclonal antibody also gave some intense staining in colon mucosa from knockout mice (Figure 2F), localized cytoplasmically in some cells in the surface and upper crypts and in the nuclei of some cells of lower crypts. This suggests that the antibody may have crossreacted with some antigens in addition to mouse TGF-{alpha}.

Western Immunoblotting for Antibody Specificity Analysis
To further confirm the specificities of these antibodies, Western blot analyses were conducted with samples of total protein extracted from colon specimens of human and TGF-{alpha} wild-type or knockout mice and with recombinant human TGF-{alpha} peptide as a standard. When the polyclonal anti-human or rat recombinant TGF-{alpha} antibodies were used to probe the blots (Figure 3A, showing representative results with the rabbit anti-human recombinant TGF-{alpha} IgG), TGF-{alpha} immunoreactivity was shown as a 6-kD band with the recombinant peptide (Figure 3A, Lane 2), and three bands with human colon protein of predominantly 6 kD, and around 15–18 and 25–28 kD in molecular weight (Figure 3A, lane 3). The two larger molecular species probably represent precursor peptides of TGF-{alpha} which were also shown in some previous studies (Hansard et al. 1997 ; Hoffmann et al. 1997 ; Tureaud et al. 1997 ; Wang et al. 1997 ). Using this antibody, no TGF-{alpha} immunoreactivity was found in colon protein samples from knockout mice (Figure 3A, Lane 4), but one band of around 25–28 kD was revealed in the sample from wild-type mice (Figure 3A, Lane 5). These results further indicate that the polyclonal antibodies raised from recombinant TGF-{alpha} are TGF-{alpha}-specific.



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Figure 3. TGF-{alpha} Western blotting analysis using different antibodies on colon protein extracts from frozen or fixed colon specimens of human and TGF-{alpha} wild-type or knockout mice. (A) A Western blot probed with the rabbit anti-human TGF-{alpha} polyclonal IgG, showing molecular markers (Lane 1), 250 ng recombinant human TGF-{alpha} peptide (Lane 2), 200 µg protein extract from frozen colon tissues of human (Lane 3), TGF-{alpha} knockout mouse (Lane 4), and wild-type mouse (Lane 5). (B) A Western blot probed with the mouse anti-human TGF-{alpha} C-terminal peptide monoclonal antibody, illustrating immunoreactivity of 250 ng recombinant human TGF-{alpha} peptide (Lane 2); immunoprecipitates using the same antibody from 1 mg of protein extracts from frozen colon tissues of TGF-{alpha} wild-type mouse (Lane 3), knockout mouse (Lane 4), and human (Lane 5); protein extract from formalin-fixed colon from a wild-type mouse (Lane 6) or a knockout mouse (Lane 7); protein extract from human colon specimens either formalin-fixed (Lane 8) or Methacarn-fixed (Lane 9); and 500 ng normal mouse monoclonal IgG2a control as detected by the anti-mouse secondary antibody (Lane 10).

Similarly, the monoclonal antibody against human TGF-{alpha} C-terminal peptide was used in probing Western blots containing protein samples prepared from frozen or fixed/paraffin-embedded colon tissues of human, TGF-{alpha} wild-type, and TGF-{alpha} knockout mice. As expected, this antibody recognized the recombinant human TGF-{alpha} peptide as a 6-kD band (Figure 3B, Lane 2). To detect any TGF-{alpha}-like immunoreactive material in colon extracts, the protein extracts were first immunoprecipitated by the monoclonal antibody and the eluted antibody and TGF-{alpha}-like material were subjected to Western analysis using the same antibody (Figure 3B, Lanes 3–5). No TGF-{alpha}-like bands were detected in colon samples from wild-type (Figure 3B, Lane 3) or knockout mice (Figure 3B, Lane 4) except for the presence of the antibody itself that was used in the immunoprecipitation. In human colon extract (Figure 3B, Lane 5), apart from the presence of the immunoglobulin IgG2a light chain (around 30 kD) and heavy chain (around 50 kD), which correspond to the bands of the normal mouse IgG2a control (Figure 3B, Lane 10), there was a band of around 25–28 kD. This 25–28-kD band, also seen in some previous studies (Hansard et al. 1997 ; Hoffmann et al. 1997 ; Wang et al. 1997 ), could be assumed to be a TGF-{alpha} precursor. After human colon was fixed in either formalin (Figure 3B, Lane 8) or in Methacarn (Figure 3B, Lane 9) and processed for paraffin embedding, the immunoreactive material in the protein extracts by this monoclonal antibody appeared to be around 31 and 66 kD in size, the identities of which remain to be determined. However, in colon protein extracts prepared from formalin-fixed, paraffin-embedded colon specimens from TGF-{alpha} wild-type (Figure 3B, Lane 6) and knockout (Figure 3B, Lane 7) mice, this antibody detected a major band of around 35 kD in both mice, indicating that this monoclonal antibody detected some non-TGF-{alpha} material in fixed mouse colon, which also confirms the positive staining result by this antibody in the fixed colon from the knockout mice (Figure 2F).


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In an attempt to explain the controversy of the distribution of TGF-{alpha} immunoreactivity in the colon mucosa, this study compared the immunostaining patterns in both frozen and paraffin sections with three different kinds of anti-TGF-{alpha} antibodies and used TGF-{alpha} knockout mice as a negative control to confirm staining specificity. We have demonstrated that three sources of antibodies (anti-rat TGF-{alpha} polyclonal, anti-human TGF-{alpha} polyclonal, and anti-human TGF-{alpha} C-terminal peptide monoclonal) gave similar staining patterns in frozen human colon sections, with intense cytoplasmic staining on the surface and along the entire crypt epithelium, consistent with the findings of Hoffmann et al. 1997 on frozen rat colon sections with an anti-rat TGF-{alpha} polyclonal antibody. Using negative controls, including antibody preabsorption with human or rat recombinant TGF-{alpha} peptide and colon tissues from TGF-{alpha} knockout mice, as well as by Western blotting, we have demonstrated the specificity of TGF-{alpha} colon staining using the polyclonal anti-human or rat recombinant TGF-{alpha} antibodies. By in situ hybridization, we have shown that TGF-{alpha} mRNA had a distribution similar to its immunoreactivity in the colon.

In paraffin sections, the two polyclonal antibodies gave minimal staining, which could be enhanced to a certain extent by trypsin pretreatment of the sections. This indicates that tissue fixation and/or tissue processing for paraffin embedding, such as dehydration or heat treatment, could destroy or alter TGF-{alpha} antigenicity. Therefore, TGF-{alpha} staining with paraffin sections does not accurately represent the distribution of TGF-{alpha} immunoreactivity.

In contrast to the polyclonal anti-TGF-{alpha} antibodies, the monoclonal anti-human TGF-{alpha} C-terminal peptide antibody still gave intense staining in paraffin-embedded human colon samples, but with a different pattern from that in frozen sections. Whereas the polyclonal antibodies gave more uniform staining on the surface and along the entire crypt epithelium in frozen sections, the monoclonal antibody mainly stained the surface and upper crypt epithelium, as well as some nuclear staining in the lower crypt epithelial cells in paraffin sections.

The staining by this monoclonal antibody on paraffin sections appeared to be specific because it could be blocked by excess human TGF-{alpha} recombinant peptide. However, this abolition of staining by excess TGF-{alpha} peptide does not exclude the possibility of crossreactivity with other molecules apart from TGF-{alpha} in tissue sections. In this study we showed that the staining pattern of the monoclonal antibody on paraffin sections was also present in the TGF-{alpha} knockout mice and in the wild-type mice, suggesting a lack of specificity in the mouse colon. The monoclonal antibody has been shown by the supplier to have no crossreactions with human and mouse epidermal growth factor (EGF) and some other proteins, such as BSA, but there is no information on its crossreactivity with other members of the EGF family that are also expressed in the colon, such as amphiregulin and betacellulin (Barnard et al. 1995 ). It is noteworthy that in one earlier study (Code et al. 1987 ), an antiserum made against the carboxyl terminus of mature TGF-{alpha}, the same antigen as for the monoclonal antibody used in the present study, had a significant level of crossreaction to the synenkephalin in the brain. Using Western blotting, we have shown that this monoclonal antibody recognized a protein of around 35 kD present in fixed colon of both wild-type and knockout mice. The identity of this antigen remains to be determined. While the current study was in completion, Aoyama et al. 1997 reported that this anti-human TGF-{alpha} monoclonal antibody was indeed not rodent TGF-{alpha}-specific, crossreacting with proteins from mouse kidney and uterus, probably including carbonic anhydrase II with a molecular weight of around 30 kD. Taken together, these data suggest that the monoclonal anti-human TGF-{alpha} C-terminal peptide is not reliable for colon TGF-{alpha} localization studies, at least in tissues from rodents. Therefore, the data in the previous studies on TGF-{alpha} immunoreactivity distribution on paraffin sections of rodent tissues using this monoclonal antibody may be questionable. A recent report has demonstrated that, using this monoclonal antibody, the type of chemical fixative could dramatically affect the localization of TGF-{alpha}-like immunoreactivity in the rat colon (Hardman and Cameron 1998 ), further supporting the unsuitability of this antibody for immunohistochemical studies in rodent tissues.

In summary, this study has shown that frozen sections, rather than paraffin sections, should be used for an accurate representation of TGF-{alpha} distribution in the colon tissues. Using specimens from TGF-{alpha} knockout mice and other negative controls, we have confirmed the specificity of the two polyclonal antibodies raised against rat or human recombinant TGF-{alpha} peptide in both colon TGF-{alpha} immunostaining and Western blotting. We have also demonstrated a nonspecificity in immunostaining on paraffin sections of fixed rodent colon tissues using the monoclonal antibody raised against the C-terminal peptide of human TGF-{alpha}, the most commonly used antibody in TGF-{alpha} immunohistochemistry.


  Acknowledgments

Supported in part by grants from the Australian National Health and Medical Research Council (to CJX and LCR), Channel 7 Children's Medical Research Foundation of South Australia (to LCR and CJX), J.H. & J.D. Gunn Medical Research Foundation (to LCR), and the Australian Rotary Health Research Fund (to LCR).

We wish to thank Dr A. Dunn (Lugwid Institute for Cancer Research; Melbourne, Australia) for providing breeding pairs of TGF-{alpha} wild-type and knockout mice, Prof R. Coffey (Vanderbilt University, Nashville, TN) for providing the sheep anti-rat TGF-{alpha} antiserum, Prof D. Lee (University of North Carolina; Chapel Hill, NC) for valuable advice, and Dr P. J. Miettinen (Department of Pathology, Children's Hospital; University of Helsinki, Finland) for her generous gift of the human TGF-{alpha} ribovector.

Received for publication December 23, 1998; accepted March 9, 1999.


  Literature Cited
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

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