BRIEF REPORT |
Correspondence to: Palle Serup, Hagedorn Research Institute, Niels Steensensvej 6, DK-2820 Gentofte, Denmark. E-mail: pas@novonordisk.com
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Summary |
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Six distinct fibroblast growth factors (FGFs) have been detected in pancreatic islets by immunohistochemistry (IHC) using commercially available antisera. We show here that these antisera are useful for Western blotting but that only two are suited for IHC. By Western blotting, these antisera detect recombinant FGFs. Detection can be eliminated by preabsorption with immunizing peptide but not with irrelevant peptide. By IHC we find specific labeling of islets with anti-FGF1 and anti-FGF2 antisera. Labeling can be abolished by preabsorption with the immunizing peptides. In contrast, prominent staining of islets by anti-FGF4, -FGF5, -FGF7, and -FGF10 antisera is unspecific because the staining cannot be competed by preabsorption with the immunizing peptides.
(J Histochem Cytochem 51:397400, 2003)
Key Words: FGF, FGFR, growth factor, islet, ß-cell, diabetes, autocrine, pancreas
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Introduction |
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FIBROBLAST GROWTH FACTORS (FGFs) are secreted proteins encoded by a multigene family containing at least 22 members. FGFs regulate many aspects of cellular behavior including stimulation of proliferation, instructive signaling during embryonic development, and chemoattraction as well as chemorepulsion (
We first tested whether the antisera could detect recombinant FGFs in Western blotting experiments and, if so, whether the signal could be competed out by preabsorption with the cognate immunizing peptide. We used goat anti-FGF1 (cat RDI-FGF1Cabg), goat anti-FGF2 (cat RDI-FGFBCbg), goat anti-FGF4 (cat RDI-FGF4Cabg), goat anti-FGF5 (cat RDI-MFGF5Cabg), goat anti-FGF7 (cat RDI-FGF7Cabg), goat anti-FGF10 (cat RDI-FGF10Cabg), rabbit anti-human FGFR1 (flg) (cat RDI-FGFR1 abr), mouse anti-human FGFR2 (bek) (cat RDI-FGFR2abm), rabbit anti-human FGFR3 (cat RDI-FGFR3abr), and rabbit anti-human FGFR4 (cat RDI-FGFR4abr). All FGF and FGFR antibodies and corresponding peptides were purchased from Research Diagnostics Inc. (Flanders, NJ). Preabsorption of antisera was performed by incubating with immunizing peptide at a fivefold excess (w/w) in PBS supplemented with 0.25% BSA and 0.3% Triton X-100 for 13 hr at room temperature (RT) on a rocking platform. Western blotting was performed using precast 15% SDS-polyacrylamide gels (Bio-Rad; Hercules, CA). Samples containing 50 ng recombinant human FGF protein (R&D Systems; Minneapolis, MN) were denatured for 5 min at 90C in a 20-µl volume in 1 x formamide gel loading buffer [62.5 mM Tris-HCl (pH6.8), 13% sucrose, 2% SDS, and 0.005% bromphenol blue]. After electrophoresis proteins were transferred to a Hybond nitrocellulose membrane and washed three times for 5 min in TBST [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween-20]. Thereafter the membrane was blocked in TBST with 2% skimmed milk and 2% BSA for 1 hr. Goat anti-FGF antibodies were used at a dilution of 1:200 in blocking buffer as suggested by the manufacturer. Membranes were incubated overnight at 4C, followed by three washes in TBST for 5 min and 1 hr of blocking. Secondary HRP-conjugated antibody was used at a dilution of 1:2000 in blocking buffer and was incubated with the membrane for 1 hr at RT. The membranes were washed three times in TBST for 5 min and again twice for 5 minutes in TBS (TBST without Tween-20). The blots were developed using an ECL+ Western blotting detection system (Amersham Pharmacia Biotech; Poole, UK). As shown in Fig 1, all the tested anti-FGF antisera recognized the relevant recombinant FGF and the signal could be blocked by preabsorption with the immunizing peptide but not with an irrelevant peptide. Therefore, all the tested antisera are capable of specific detection of the cognate antigen in Western blotting experiments. We also conclude that the immunizing peptides are able to block the antigen recognition sites of the antibodies.
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We next tested whether the same antisera could detect a specific signal by IHC staining of sections of adult mouse pancreas. Pancreatic tissue from adult mice was fixed in 4% PFA, sucrose-protected, and 4-µm frozen sections were cut on a cryostat. FGF and FGFR antibodies were tested at a range of dilutions and were used at dilutions of 1:20 (anti-FGFs) or 1:200 (anti-FGFRs). Guinea pig -insulin (diluted 1:500) and mouse
-glucagon (diluted 1:50) were both from Novo Nordisk (Bagsværd, Denmark). Cy2- or Texas Red-conjugated secondary antibodies raised in donkey were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). Preincubation of antisera with immunizing peptide was performed as described for Western blotting, except that a 20-fold excess (w/w) of peptide was used. Before addition of antibody, the sections were rinsed three times in PBS and blocked in 10% donkey serum (Jackson ImmunoResearch). Antibodies diluted in PBS supplemented with 0.25% BSA and 0.3% Triton X-100 were added to each slide and incubated overnight. Before addition of secondary antibody, the sections were washed in PBS three times for 5 min and incubated with secondary antibody for 45 min, followed by three 5-min washes in PBS. Before pictures were taken the sections were mounted in fluorescence mounting media (Kierkegaard; Gaithersburg, MD). Negative controls included stainings without primary antibody and were blank, as were stainings incubated with immunizing peptide without primary antibody. As shown in Fig 2, all six antisera labeled the islets of Langerhans but only the signal obtained with the anti-FGF1 and anti-FGF2 antisera could be competed out by preabsorption with the immunizing peptides despite the fact an amount fourfold higher than what was able to eliminate binding in Western blotting experiments was used. Therefore, whether FGF4, FGF5, FGF7, and FGF10 are truly expressed in islet ß-cells cannot be determined by use of these antisera for IHC staining. We therefore conducted RT-PCR analysis of FGF expression in RNA prepared from isolated islets and used RNA isolated from E12 whole embryos and NIH3T3 cells as positive control. cDNA synthesis and RT-PCR analysis were prepared as described in
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We also assessed the expression of FGF receptors by RT-PCR on RNA from isolated islets. As shown in Fig 3, we could detect expression of FGFR1b, FGFR1c, FGFR2b, FGFR2c, FGFR3b, and FGFR4. Again, we cannot conclude which of the many cell types present in the islet preparations give rise to these bands. We attempted to determine if islet ß-cells expressed any of the FGF receptors by IHC, but, as shown in Fig 2, even though islet ß-cells were labeled by antisera against FGFR1, FGFR3, FGFR4, and endothelial cells were labeled by FGFR2 antiserum, we could not compete the labeling with the immunizing peptides. Therefore, we cannot conclude with any certainty from these data that a particular cell type in the pancreas expresses a given FGF receptor.
Our results could have implications for the autocrine FGF signaling loop among ß-cells proposed by
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Acknowledgments |
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Supported by funds from the Juvenile Diabetes Research Foundation and the Danish National Research Academy.
We thank Ragna Jørgensen and Helle V. Petersen for expert technical assistance.
Received for publication November 7, 2002; accepted November 13, 2002.
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Literature Cited |
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Bhushan A, Itoh N, Kato S, Thiery JP, Czernchow P, Bellusci S, Scharfmann R (2001) Fgf10 is essential for maintaining the proliferative capacity of epithelial progenitor cells during early pancreatic organogenesis. Development 128:5109-5117
Hart A, Baeza N, Apelqvist A, Edlund H (2000) Attenuation of FGF signalling in mouse b-cells leads to diabetes. Nature 408:864-868[Medline]
Jensen J, Heller RS, FunderNielsen T, Pedersen EE, Lindsell C, Weinmaster G, Madsen OD et al. (2000) Independent development of pancreatic - and ß-cells from neurogenin3-expressing precursors. Diabetes 49:163-176[Abstract]
Jensen J, Serup P, Karlsen C, FunderNielsen T, Madsen OD (1996) mRNA Profiling of rat islet tumors reveals Nkx6.1 as a ß-cell-specific homeodomain transcription factor. J Biol Chem 271:18749-18758
Miller DL, Ortega S, Bashayan O, Basch R, Basilico C (2000) Compensation by fibroblast growth factor 1 (FGF1) does not account for the mild phenotypic defects observed in FGF2 null mice. Mol Cell Biol 20:2260-2268
Ornitz DM, Itoh N (2001) Fibroblast growth factors. Genome Biol 2:3005. 13005.12
Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM et al. (2000) Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230-238
Yang X, Dormann D, Munsterberg A, Weijer C (2002) Cell movement patterns during gastrulation in the chick are controlled by positive and negative chemotaxis mediated by FGF4 and FGF8. Dev Cell 3:425-437[Medline]