Primary AntibodyFab Fragment Complexes : A Flexible Alternative to Traditional Direct and Indirect Immunolabeling Techniques
Department of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Veterinary Centre, Midlothian, United Kingdom
Correspondence to: Jeremy K. Brown, Dept. of Veterinary Clinical Studies, University of Edinburgh, Easter Bush Veterinary Centre, Midlothian EH25 9RG, UK. E-mail: Jeremy.brown2{at}ed.ac.uk
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Summary |
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Key Words: endogenous immunoglobulin Fab fluorescence multicolor multilabeling flow cytometry ELISA
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Introduction |
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ICC was first applied to the simultaneous detection of multiple antigens within a single sample using directly conjugated primary antibodies (Bernier and Cebra 1964). Subsequent advances in optical and digital imaging (Schrock et al. 1996
), in conjunction with the availability of fluorescent dyes with sufficiently distinct emission spectra (Panchuk-Voloshina et al. 1999
), have allowed directly conjugated antibodies to be used to discriminate as many as seven different antigens in a single sample (Tsurui et al. 2000
). However, covalent labeling of antibodies is not a routine procedure in the majority of laboratories, requires relatively large quantities of purified antibody (Mao 1999
) and, to maintain flexibility for multilabeling experiments, it is often necessary to have a choice of several different fluorophore conjugates of each primary and control antibody. These factors, together with the widely held perception that direct ICC is relatively insensitive (Mao 1999
), have led the majority of researchers to rely on the indirect method (Coons et al. 1955
) for their multilabeling requirements (Staines et al. 1988
; Ferri et al. 1997
).
The use of labeled secondary anti-immunoglobulin (Ig) antibodies in the indirect method introduces the possibility of spurious staining resulting from crossreactions with endogenous Ig and between different primary and secondary antibody combinations. Although indirect ICC has been used to detect up to four antigens in the same sample using primary antibodies derived from different species and species-specific secondary antibodies (Ferri et al. 1997), it is often necessary to use primary antibodies derived from the same host species in multilabeling studies. The challenge imposed by this situation, i.e., the need to differentiate two or more primary antibodies that have virtually identical antigenic properties, is essentially the same as that faced when attempts are made to localize antigens using primary antibodies derived from the same species as the sample material.
Tuson et al. (1990) first described the use of preformed immune complexes of primary and secondary antibodies to prevent nonspecific labeling of endogenous Ig when probing human tissues with human monoclonal antibodies. Effectively a hybrid of indirect and direct ICC, this technique involves non-covalent labeling of the primary antibody with a covalently labeled secondary antibody in vitro. After absorption of unbound secondary antibody paratopes with excess serum from the same species as the primary antibody, the resultant complexes retain the antigen specificity of the primary antibody but do not crossreact with endogenous Ig when applied to tissue samples homologous to the primary antibody species (Tuson et al. 1990
; Krenacs et al. 1991
). This technique has been adapted to allow indirect dual labeling with primary antibodies derived from the same species (Kroeber et al. 1998
). However, the use of divalent secondary antibodies in these protocols may lead to the formation of crosslinked complexes, and the ratio of primary to secondary antibodies must be carefully optimized (Hierck et al. 1994
; Lu and Partridge 1998
). The use of monovalent IgG Fc-specific Fab fragments avoids this issue (van der Loos and Gobel 2000
) and has been developed commercially by DakoCytomation (ARK; DakoCytomation, Ely, UK) and Molecular Probes (Zenon; Eugene, OR).
Here we describe a method for the in vitro non-covalent labeling of primary antibodies with commercial monovalent Fab fragments that recognize both the Fc and F(ab')2 regions of IgG (Jackson ImmunoResearch Laboratories; West Grove, PA). The resultant immune complexes do not crossreact with endogenous Ig, can be used to simultaneously detect multiple antigens with primary antibodies derived from the same species, and allow the same polyclonal antibody to be used for both antigen capture and detection in ELISA.
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Materials and Methods |
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Antibodies
Normal sera were heat-inactivated at 59C for 2 hr before use. Purified and fluorescein isothiocyanate (FITC)-conjugated monoclonal mouse IgG1 anti-ß-catenin (clone 14), purified and FITC-conjugated control mouse IgG1 (clone MOPC-21), purified monoclonal rat IgG2a anti-mouse integrin-ß1 (clone KM16), rat IgG2a anti-human integrin-6 (clone GoH3), and control rat IgG2a (clone R35-95) were purchased from BD Biosciences (Cowley, UK). Rat IgG2a anti-integrin-ß7 (clone M293) hybridoma supernatant and purified rat IgG2a anti-integrin-
E (clone M290) were provided by Dr. Peter Kilshaw (Babraham Institute; Babraham, Cambridge, UK). Polyclonal rat and sheep anti-mouse mast cell protease-2 (mMCP-2) antibodies were prepared as described previously (Pemberton et al. 2003
). Secondary goat anti-mouse IgG and anti-rat IgG monovalent Fab fragments (Jackson ImmunoResearch Laboratories) were purchased from Stratech Scientific (Soham, UK).
Generation of Mouse Mucosal Mast Cell Homologues
Bone marrow cells prepared from 12-week-old male BALB/c mice were cultured for 21 days ex vivo in the presence of recombinant human TGF-ß1 (1 ng/ml; Sigma-Aldrich, Poole, UK), recombinant mouse IL-3 (1 ng/ml; R & D Systems, Abingdon, UK), recombinant mouse IL-9 (5 ng/ml; R & D Systems), and recombinant mouse SCF (50 ng/ml; PeproTech EC, London, UK) as described previously (Miller et al. 1999; Wright et al. 2002
; Brown et al. 2003
).
Tissue Samples
Freshly dissected jejunum samples from normal 129 and BALB/c mice (B and K Universal; Hull, UK) were embedded in OCT compound (BDH Laboratory Supplies; Poole, UK) and snap-frozen in isopentane cooled on dry ice. Cryostat sections (10 µm) were mounted on Snow Coat X-tra charged slides (Surgipath Europe; Peterborough, UK), air-dried for 10 min, and stored at 70C.
Fixation and 3,3'-Diaminobenzidine Pretreatment of Tissue Sections
Sections were thawed for 10 min at 21C, fixed in absolute methanol for 10 min at 20C, and air-dried for a further 10 min under forced air. Sections were washed and rehydrated in PBS containing 0.5% Tween-80 (Sigma-Aldrich). Eosinophil auto-fluorescence was quenched using a modified version of the procedures described by Kingston and Pearson (1981) and Valnes and Brandtzaeg (1981)
. Non-eosinophil-derived endogenous peroxidase activity was quenched for 30 min with 1% H2O2 in PBS + 0.5% Tween-80. Sections were then washed with PBS and incubated for 3 min with DAB (3,3'-diaminobenzidine) substrate (Vector Laboratories; Peterborough, UK). Sections were washed thoroughly with PBS before proceeding with ICC.
Primary AntibodyFab Fragment Complex Formation Kinetics
Immulon-4 96-well microtiter plates (Thermo Shandon) were coated overnight at 4C with 50 µl/well of polyclonal sheep anti-mMCP-2 (1 µg/ml in pH 9.6 carbonate buffer). Wells were washed (six times with 0.15 M NaCl + 0.05% Tween-20) and alternate columns incubated for 1 hr at 21C with 50 µl of purified mMCP-2 (Pemberton et al. 2003) diluted to 5 ng/ml in ELISA buffer (pH 7.4 PBS + 0.05% Tween-20). The remaining columns were incubated with ELISA buffer alone to provide blanks for each time point. Biotinylated mMCP-2-specific Fab fragment complexes were generated as follows. Twelve µg of polyclonal rat anti-mMCP-2 were mixed with 12 µg of biotinylated goat anti-rat IgG Fab fragments in 120 µl of pH 7.4 PBS (1:1 ratio determined through titration). Complex formation was stopped by diluting 20-µl aliquots with 980 µl of ELISA buffer + 5% heat-inactivated normal rat serum (NRtS: Sigma-Aldrich) at 15 sec, 30 sec, 45 sec, 60 sec, 120 sec, and 240 sec time points and the resultant mixture allowed to stand for
10 min at 21C. Wells were washed and incubated with 50 µl of primary antibodyFab fragment complexes for 60 min at 21C. After washing, wells were incubated for 30 min at 21C with 50 µl of streptavidinbiotinylated horseradish peroxidase complex (Amersham Biosciences; Little Chalfont, UK) diluted 1:2000 in PBS + 0.05%Tween-20. Wells were washed and incubated at 21C for 15 min with 50 µl of TMB substrate (Insight Biotechnology; Wembley, UK). The reaction was stopped with 0.18 M sulfuric acid and the absorbance (450 nm) of each well measured using a microplate reader (Model 550; Bio-Rad Laboratories, Hemel Hempstead, UK).
Blocking of IgG-specific Fab Fragment Paratopes with Normal Serum
Immulon-4 96-well microtiter plates were coated overnight at 4C with 50 µl/well of normal rat IgG (0.1 µg/ml in pH 9.6 carbonate buffer). Wells were washed (six times with 0.15 M NaCl + 0.05% Tween-20) and incubated for 1 hr at 21C with 50 µl of goat anti-rat IgG Fab fragments (1 µg/ml in ELISA buffer) that had been preincubated for 10 min at 21C with various amounts of normal rat serum (0, 0.1, 1, 2, 4, 5, 10, 20, 40, or 80 µl of NRtS per µg of Fab fragments). Plates were then incubated sequentially with streptavidinbiotinylated horseradish peroxidase complex, TMB substrate, and 0.18 M sulfuric acid before being read as described above.
An mMCP-2 Sandwich ELISA Incorporating Fab Fragment Complexes
Immulon-4 96-well microtiter plates were coated overnight at 4C with 50 µl/well of polyclonal rat anti-mMCP-2 (3 µg/ml in pH 9.6 carbonate buffer). Wells were washed (six times with 0.15 M NaCl containing 0.05% Tween-20) and incubated for 1 hr at 21C with 50 µl of purified mMCP-2 (Pemberton et al. 2003) standard (0.15 ng/ml) or samples, diluted appropriately in pH 7.4 PBS + 0.05% Tween-20 + 5% NRtS. Biotinylated mMCP-2-specific Fab fragment complexes were generated as follows. For each 96-well plate, 10 µg of polyclonal rat anti-mMCP-2 was incubated at 21C with 10 µg of biotinylated goat anti-rat IgG Fab fragments in pH 7.4 PBS (1:1 ratio in 100 µl final volume). After 20 min, 4900 µl of PBS + 0.05% Tween-20 + 5% NRtS was added and the resultant mixture allowed to stand for 10 min at 21C. Wells were washed and incubated for 1 hr at 21C with 50 µl of Fab fragment complexes. Plates were then incubated sequentially with streptavidinbiotinylated horseradish peroxidase complex, TMB substrate, and 0.18 M sulfuric acid before being read as described above. Three independent assays were performed and coefficient of variation (CV) values for intra- (n=16) and inter-assay (n=3) variability were determined using supernatants from mouse mucosal mast cell homologue cultures (day 21) diluted 1:500 in ELISA buffer + 5% NRtS as unknown samples.
Immunocytochemistry
Standard Conditions and Reagents
Unless stated otherwise, ICC procedures were performed at 21C under humidified conditions in a Sequenza immunostaining center (Thermo Shandon; Runcorn, UK). Antibodies and blocking sera were diluted in pH 7.4 PBS containing 0.5 M NaCl and 0.5% Tween-80 (staining buffer). After ICC labeling, samples were washed in PBS and mounted with 1.5 (0.17 mm thickness) glass coverslips (BDH Laboratory Supplies) using Mowiol (pH 8.5) mounting medium (EMD Biosciences; San Diego, CA).
Direct Single-color Labeling of ß-Catenin
Tissue sections were washed with staining buffer and blocked for 1 hr in staining buffer containing 10% heat-inactivated normal mouse serum (NMS; Sigma-Aldrich). Sections were then incubated for 1 hr with FITC-conjugated mouse IgG1 anti-ß-catenin or FITC-conjugated control mouse IgG1 diluted to 5 µg/ml in staining buffer + 10% NMS and centrifuged at 13,000 x g for 10 min before use to remove insoluble precipitates.
Indirect Single-color Labeling of ß-Catenin
Tissue sections were washed with staining buffer and blocked for 1 hr in staining buffer containing 10% heat-inactivated normal goat serum (NGS; Sigma-Aldrich) with or without 50 µg/ml of unlabeled goat anti-mouse Fab fragments. Sections were then washed in PBS and incubated for 1 hr with 5 µg/ml of purified mouse IgG1 anti-ß-catenin or control mouse IgG1 in staining buffer + 10% NGS. Sections were washed with PBS and incubated for 30 min with FITC-conjugated goat anti-mouse IgG monovalent Fab fragments diluted to 4 µg/ml in staining buffer + 10% NGS and centrifuged at 13,000 x g for 10 min before use to remove insoluble precipitates.
Single-color Labeling of ß-Catenin Using Primary AntibodyFab Fragment Complexes
Tissue sections were washed with staining buffer and blocked for 1 hr with staining buffer containing 10% NMS. During this time, ß-cateninspecific and control mouse IgG1 primary antibodies were incubated for 20 min at 21C with FITC-conjugated mouse IgG specific Fab fragments at a ratio of 1:2 (optimized by titration) in a small volume, typically 10 µl/1 µg of primary antibody, of staining buffer in a microcentrifuge tube. The resultant complexes were diluted in staining buffer + 10% NMS to give the equivalent concentration of 5 µg/ml of primary mouse IgG1 and incubated for 10 min at 21C to block unbound Fab fragment paratopes. Complexes were then centrifuged at 13,000 x g for 10 min, to remove insoluble precipitates, and incubated with tissue sections for 1 hr. The procedure for generating and using primary mouse antibodyFab fragment complexes on mouse samples is represented schematically in Figure 1 .
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Simultaneous Detection of Integrin-6, ß1, and ß7 Using Primary AntibodyFab Fragment Complexes
Tissue sections were washed with staining buffer and blocked for 1 hr with staining buffer containing 10% NRtS. During this time, integrin-specific and control rat IgG2a primary antibodies were incubated for 20 min at 21C with fluorophore- conjugated rat IgG-specific Fab fragments in a small volume, typically 10 µl/1 µg of primary antibody, of staining buffer in a microcentrifuge tube. Rat anti-integrin-ß1, anti-integrin-6, and control IgG2a were labeled, separately in solution, with fluorophore-conjugated goat anti-rat IgG Fab fragments at a ratio of 1:2 to yield the following complexes: anti-integrin-ß1FITC, anti-integrin-
6Rhodamine Red-X (RRX), control IgG2aFITC, control IgG2aRRX, and control IgG2aCy5. Rat IgG2a anti-integrin-ß7 hybridoma supernatant was combined with Cy5-conjugated goat anti-rat IgG Fab fragments at a ratio of 8 µl/1 µg to yield anti-integrin-ß7Cy5 Fab fragment complexes. Fab fragment complexes were subsequently diluted to three times their working concentration in staining buffer + 10% NRtS and incubated for 10 min at 21C to block unbound Fab fragment paratopes. Complexes were then mixed 1:1:1 in various combinations to give final primary antibody concentrations equivalent to 5 µg/ml of anti-integrin-ß1/control IgG2a, 2 µg/ml of anti-integrin-
6/control IgG2a, and a 1:40 dilution of anti-integrin-ß7 hybridoma supernatant/2 µg/ml control IgG2a. Complexes were centrifuged at 13,000 x g for 10 min to remove insoluble precipitates, and incubated with tissue sections for 1 hr. For competitive controls, samples were simultaneously incubated with all three integrin-specific primary antibodyFab fragment complexes and a 10-fold excess of unlabeled anti-integrin-
6, -ß1 or -ß7.
Simultaneous Detection of Integrin-E and -ß7 on Viable Cells Using Primary Antibody-Fab Fragment Complexes
Mouse mucosal mast cell homologues (Miller et al. 1999) were divided into aliquots of 5 x 105 cells, washed in two changes of PBS, resuspended in 50 µl of PBS containing 10% NRtS, and incubated on ice for 40 min. Rat anti-integrin-
E and control IgG2a were labeled, separately in solution, with FITC- or Cy5-conjugated Fab fragments at a ratio of 1:4 to yield the following complexes: anti-integrin-
ECy5, control IgG2aCy5, and control IgG2aFITC. Rat IgG2a anti-integrin-ß7 hybridoma supernatant was combined with Cy5-conjugated goat anti-rat IgG Fab fragments at a ratio of 8 µl/1 µg to yield anti-integrin-ß7FITC Fab fragment complexes. After 20 min, complexes were diluted in PBS + 10% NRtS to give final concentrations equivalent to 10 µg/ml of primary antibody or a 1:20 dilution of M293 hybridoma supernatant and incubated for 10 min at 21C. After centrifugation at 13,000 x g for 10 min to remove insoluble precipitates, 50 µl of complexes were added in various combinations to each aliquot of 5 x 105 cells. Cells were incubated on ice for 30 min, washed in PBS, and fixed for 5 min in PBS + 2% formaldehyde. Quantitative analysis was performed on 10,000 cells/sample using a flow cytometer (FACSCalibur; Becton Dickinson, Richmond, CA) with linear amplification for forward/side scatter and logarithmic amplification for FITC and Cy5 fluorescence.
Microscopy and Imaging
Fluorescent images were acquired using an MRC-600 confocal laser scanning microscope (CLSM: Bio-Rad Laboratories) mounted on an Axiovert 100 inverted microscope equipped with Plan-Apochromat objective lenses (Carl Zeiss; Oberkochen, Germany). Fluorophores were excited and imaged sequentially using the 488 nm (FITC), 568-nm (RRX), and 647-nm (Cy5) lines from a 15 mW Kr/Ar laser (Bio-Rad Laboratories). Images were prepared for publication using Object-Image (Vischer et al. 1994) and Photoshop (Adobe Systems UK; Uxbridge, UK). Object-Image is a public domain software package, based on NIH Image (Rasband and Bright 1995
), developed by Norbert Vischer (University of Amsterdam; Amsterdam, The Netherlands) and is freely available via the Internet at http://simon.bio.uva.nl/object-image.html.
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Results |
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Triple Immunofluorescent Labeling with Primary Antibodies Derived from the Same Species
Three different rat monoclonal IgG2a anti-integrin antibodies that produce distinct staining patterns when applied individually to frozen sections of normal mouse jejunum (Figures 4A4C)
were selected for use in this proof of function study. Rat anti-integrin-ß1 (Figure 4A) predominantly labeled smooth muscle and endothelial cells within the lamina propria. Rat anti-integrin-ß7 labeling (Figure 4B) was restricted to leukocytes, the majority of which were located within the epithelium (Figure 4B, arrow). Rat anti-integrin-6 labeling (Figure 4C) was predominant along the basement membrane (Figure 4C, arrow) but was also present on the basolateral surfaces of epithelial cells and on endothelial cells in the lamina propria (Figure 4C, arrowhead). Sections probed with isotype-matched control rat IgG2a (Figure 4D) confirmed staining specificity.
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Dual Fluorescent Labeling of Viable Cells for Analysis by Flow Cytometry
The potential for multilabeling with Fab fragment complexes containing primary antibodies derived from the same species was also investigated in the context of flow cytometry (Figure 5) . Analysis was restricted to viable mucosal mast cell homologues (Wright et al. 2002), identified on the basis of their forward and side scatter characteristics (Figure 5A). The majority of cells incubated with integrin-
E- and integrin-ß7-specific Fab fragment complexes exhibited bright FITC and Cy5 fluorescence (Figure 5B). Consistent with the expression of integrins-
E and -ß7 as a heterodimer (Kilshaw 1999
), there was a strong correlation between FITC and Cy5 signal intensity in dual-labeled samples (Figure 5B). Staining specificity was confirmed using single negative controls in which integrin-
E (Figure 5C) or integrin-ß7-specific (Figure 5D) primary antibodies were substituted with control rat IgG2a.
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Discussion |
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A major drawback to sequential staining methods is that they become increasingly time-consuming as the desired number of labels increases, to the extent that the triple labeling procedure described by Brouns et al. (2002) requires 3 days to perform. Although this can be regarded as a fairly trivial point in the context of labeling fixed samples, prolonged staining times can severely reduce the viability of live samples, precluding the use of most sequential labeling procedures in applications such as flow cytometry. The use of preformed immune complexes (Tuson et al. 1990
) offers an alternative approach to sequential labeling and has the potential to substantially reduce the amount of time required for multilabeling studies using primary antibodies raised in the same host species.
Here we describe a method by which primary antibodies can be non-covalently labeled in vitro with a reporter molecule using monovalent Fab fragments that recognize both the Fc and F(ab')2 regions of IgG (Figure 1). Like primary antibodies labeled with Fc-specific Fab fragments (van der Loos and Gobel 2000; Martin et al. 2003
), such as the ARK (DakoCytomation) and Zenon (Molecular Probes) reagents, these immune complexes retain the antigen specificity of the original primary antibody and can be used in a variety of applications that would normally require covalently conjugated primary antibodies. The monovalent nature of Fab fragments prevents the formation of large crosslinked complexes and, once stabilized with excess serum from the same species as the primary antibody, Fab fragment complexes can be used to label specific antigen in tissues homologous to the primary antibody without resulting in spurious labeling of endogenous Ig (Figure 3). Furthermore, multiple Fab fragment complexes containing the same species and isotype of primary antibodies can be combined and used to simultaneously label multiple antigens in the same sample in a single incubation, in the absence of erroneous crossreactions between different primary and secondary antibody pairs (Figures 4 and 5). Although lower than that obtained through conventional indirect ICC (Figure 4), the signal-to-noise ratio achieved with primary antibodyFab fragment complexes typically exceeds that normally achievable using covalently conjugated primary antibodies (Figure 3). Moreover, the ability to label as much, or as little, primary antibody with a choice of different reporter molecules as and when required provides a similar level of flexibility as traditional indirect ICC.
The ratio of primary antibodies to Fab fragments required for the formation of complexes that produce optimal immunolabeling of specific antigen does not appear to vary dramatically with primary antibody specificity or species. Primary antibody to Fab fragment ratios of 1:21:4 (weight for weight, based on concentration data supplied by manufacturers) typically produced optimal results with primary mouse and rat monoclonal antibodies, and over-labeling, to the extent that their affinity for specific antigen is significantly compromised, was not a significant problem. Preliminary results suggest that similar primary antibodies to Fab fragment ratios produce optimal immunolabeling with Fab fragment complexes containing primary rabbit or goat polyclonal antibodies (data not shown). Primary antibody to Fab fragment ratios can be reduced to 1:1 for the purposes of ELISA quantification of antigen without compromising the sensitivity or dynamic range of the assay. This effectively reduces the requirements for ELISA development to a single species of polyclonal antibody and protein standard. Indeed, the original mMCP-2 sandwich ELISA (Pemberton et al. 2003), which requires two different species of polyclonal anti-mMCP-2 antibodies, offers no discernible advantage over the modified version of the assay described here (Figures 2C2F).
Primary antibodyFab fragment complexes provide an attractive alternative to covalently conjugated primary antibodies. They can be generated rapidly, as and when required, using inexpensive reagents that are readily available from a variety of commercial sources. Although they do not offer the same level of signal amplification as traditional indirect ICC, they do not crossreact with other immunoglobulins and significantly reduce the amount of time required for simultaneous labeling of multiple antigens. We envisage that primary antibodyFab fragment complexes, used either alone or in conjunction with indirect and direct ICC, will greatly facilitate the full exploitation of the vast array of fluorescent markers now available and the spectral resolution offered by modern microscopic and flow cytometric equipment. They also have the potential to greatly facilitate sandwich ELISA development.
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Acknowledgments |
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We thank Dr Mike Wilkinson (GlaxoSmithKline) for the donation of the BIO-RAD MRC600 confocal microscope and Dr Peter Kilshaw (Babraham Institute) for providing the anti-integrin-E and -ß7 monoclonal antibodies. We are also grateful to Judith A. Pate and Elisabeth M. Thornton for technical assistance and to Mara Rocchi (Moredun Research Institute; Penicuik, Midlothian, UK) for assistance with the flow cytometry analysis.
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Footnotes |
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