Copyright ©The Histochemical Society, Inc.

Primary Antibody–Fab Fragment Complexes : A Flexible Alternative to Traditional Direct and Indirect Immunolabeling Techniques

Jeremy K. Brown, Alan D. Pemberton, Steven H. Wright and Hugh R.P. Miller

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


    Summary
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Immunolabeling with immune complexes of primary and secondary antibodies offers an attractive method for detecting and quantifying specific antigen. Primary antibodies maintain their affinity for specific antigen after labeling with Fab fragments in vitro. Incubation of these immune complexes with excess normal serum from the same species as the primary antibody prevents free Fab fragments from recognizing immunoglobulin. Effectively a hybrid between traditional direct and indirect immunolabeling techniques, this simple technique allows primary antibodies to be non-covalently labeled with a variety of reporter molecules as and when required. Using complexes containing Fab fragments that recognize both the Fc and F(ab')2 regions of IgG, we show that this approach prevents nonspecific labeling of endogenous immunoglobulin, can be used to simultaneously detect multiple antigens with primary antibodies derived from the same species, and allows the same polyclonal antibody to be used for both antigen capture and detection in ELISA. (J Histochem Cytochem 52:1219–1230, 2004)

Key Words: endogenous immunoglobulin • Fab • fluorescence • multicolor • multilabeling • flow cytometry • ELISA


    Introduction
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
SINCE ITS INCEPTION over 60 years ago (Coons et al. 1941Go,1942Go), immunocytochemistry (ICC) has made an almost unparalleled contribution to a wide variety of fields among the life sciences. The use of antibodies to detect and localize individual or multiple antigens in situ remains a powerful and widely exploited technology (Brandtzaeg 1998Go; Coleman 2000Go).

ICC was first applied to the simultaneous detection of multiple antigens within a single sample using directly conjugated primary antibodies (Bernier and Cebra 1964Go). Subsequent advances in optical and digital imaging (Schrock et al. 1996Go), in conjunction with the availability of fluorescent dyes with sufficiently distinct emission spectra (Panchuk-Voloshina et al. 1999Go), have allowed directly conjugated antibodies to be used to discriminate as many as seven different antigens in a single sample (Tsurui et al. 2000Go). However, covalent labeling of antibodies is not a routine procedure in the majority of laboratories, requires relatively large quantities of purified antibody (Mao 1999Go) 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 1999Go), have led the majority of researchers to rely on the indirect method (Coons et al. 1955Go) for their multilabeling requirements (Staines et al. 1988Go; Ferri et al. 1997Go).

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. 1997Go), 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)Go 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. 1990Go; Krenacs et al. 1991Go). This technique has been adapted to allow indirect dual labeling with primary antibodies derived from the same species (Kroeber et al. 1998Go). 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. 1994Go; Lu and Partridge 1998Go). The use of monovalent IgG Fc-specific Fab fragments avoids this issue (van der Loos and Gobel 2000Go) 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.


    Materials and Methods
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Unless stated otherwise, reagents were purchased from Fisher Scientific UK (Loughborough, UK). All experimental procedures involving laboratory animals were approved by the University of Edinburgh's Biological Services ethical review committee and were performed under license, as required by the United Kingdom's Animals (Scientific Procedures) Act 1986.

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-{alpha}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-{alpha}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. 2003Go). 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. 1999Go; Wright et al. 2002Go; Brown et al. 2003Go).

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)Go and Valnes and Brandtzaeg (1981)Go. 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 Antibody–Fab 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. 2003Go) 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 antibody–Fab fragment complexes for 60 min at 21C. After washing, wells were incubated for 30 min at 21C with 50 µl of streptavidin–biotinylated 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 streptavidin–biotinylated 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. 2003Go) standard (0.1–5 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 streptavidin–biotinylated 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 Antibody–Fab 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 antibody–Fab fragment complexes on mouse samples is represented schematically in Figure 1 .



View larger version (23K):
[in this window]
[in a new window]
 
Figure 1

Schematic representation of the procedure for generating and using primary antibody–Fab fragment complexes in the context of using primary mouse antibodies to probe mouse tissues. While the sample is blocked with 10% normal mouse serum (A), primary mouse antibody is incubated for 20 min with fluorescent anti-mouse Fab fragments (B). The resultant primary antibody–Fab fragment complexes are incubated with excess normal mouse serum for 10 min to block free Fab fragment paratopes (C), diluted to working concentration, and centrifuged before use to remove any insoluble precipitates (D). Primary mouse antibody–Fab fragment complexes are then applied to the sample (E), resulting in specific labeling in the absence of spurious crossreactions with endogenous mouse Ig (F).

 
Indirect Single-color Labeling of the Integrin {alpha}6, ß1, and ß7 Subunits
Samples were washed with staining buffer and blocked for 1 hr in staining buffer + 10% NMS. Sections were then incubated for 1 hr with 2 µg/ml of rat IgG2a anti-integrin-{alpha}6, ß1, ß7, or control rat IgG2a in staining buffer + 10% NMS. Sections were washed with PBS and incubated for 30 min with cyanine-5 (Cy5) conjugated goat anti-rat IgG monovalent Fab fragments diluted to 4 µg/ml in staining buffer + 10% NMS and centrifuged at 13,000 x g for 10 min before use to remove insoluble precipitates.

Simultaneous Detection of Integrin-{alpha}6, ß1, and ß7 Using Primary Antibody–Fab 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-{alpha}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-ß1–FITC, anti-integrin-{alpha}6–Rhodamine Red-X (RRX), control IgG2a–FITC, control IgG2a–RRX, and control IgG2a–Cy5. 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-ß7–Cy5 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-{alpha}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 antibody–Fab fragment complexes and a 10-fold excess of unlabeled anti-integrin-{alpha}6, -ß1 or -ß7.

Simultaneous Detection of Integrin-{alpha}E and -ß7 on Viable Cells Using Primary Antibody-Fab Fragment Complexes
Mouse mucosal mast cell homologues (Miller et al. 1999Go) 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-{alpha}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-{alpha}E–Cy5, control IgG2a–Cy5, and control IgG2a–FITC. 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-ß7–FITC 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. 1994Go) and Photoshop (Adobe Systems UK; Uxbridge, UK). Object-Image is a public domain software package, based on NIH Image (Rasband and Bright 1995Go), 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.


    Results
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Primary Antibody–Fab Fragment Complex Formation
Pilot studies established that primary antibodies labeled in solution with polyclonal anti-IgG (heavy and light chain-specific) Fab fragments retain their ability to recognize specific antigen (Figure 2A) . In agreement with data published by Molecular Probes for their commercial Fc-specific anti-mouse IgG1 Zenon Fab fragments (Haugland 2002Go), biotinylated anti-rat IgG (heavy and light chain-specific) Fab fragments formed immune complexes with polyclonal rat anti-mMCP-2 antibodies within minutes when mixed in a minimal volume (10 µl per µg of primary antibody). Complex formation occurred within 2–4 min (Figure 2A) and did not appear to be adversely affected by the presence of glycerol, 0.5% Tween-80, and/or concentrations of up to 0.5 M NaCl (data not shown). A 10-min incubation at 21C with ≥0.1 µl of NRtS significantly (p<0.01; unpaired Student's t-test with Welch correction) reduced the affinity of 1 µg of biotinylated goat anti-rat IgG Fab fragments for normal Rat IgG immobilized on ELISA plates (Figure 2B). Maximal inhibition of IgG binding was achieved by incubation with ≥5 µl of NRtS per µg of biotinylated anti-rat IgG Fab fragments (Figure 2B).



View larger version (14K):
[in this window]
[in a new window]
 
Figure 2

Rat anti-mMCP-2 IgG and biotinylated goat anti-rat IgG Fab fragments form immune complexes capable of recognizing mMCP-2 immobilized on ELISA plates within minutes when mixed in vitro (A). Preincubation with NRtS prevents biotinylated anti-rat Fab fragments from binding to immobilized normal rat IgG in a dose-dependent manner (B). A sandwich ELISA was developed using polyclonal rat anti-mMCP-2 Ig as the capture antibody and biotinylated primary antibody–Fab fragment complexes containing the same antibody as the detection reagent (C–F). Standard curves from three independent assays are shown (C–E; error bars represent one SEM; n=4). Supernatant from an in vitro mucosal mast cell homologue culture (diluted 1:500; n=16) was used as an unknown for the purposes of calculating intra-assay variation (%CV, inset in lower right of C–E) and inter-assay variation (%CV in parentheses in F).

 
Development of a Sandwich ELISA Incorporating Primary Antibody–Fab Fragment Complexes
An mMCP-2-specific ELISA was developed using polyclonal rat antibodies to capture mMCP-2 on the microtiter plate surface and complexes containing the same antibody, together with biotinylated anti-rat IgG Fab fragments at a ratio of 1:1, for quantitative detection of bound mMCP-2 (Figures 2C–2F). Incubation of mMCP-2-specific Fab fragments complexes with excess NRtS for 10 min at 21C prevented nonspecific labeling of the capture antibody, allowing the resolution of mMCP-2 concentrations of ≥0.1 ng/ml (Figures 2C–2F). The sensitivity and dynamic range obtained using this modified version of the assay were similar to those observed for the indirect mMCP-2 ELISA reported by Pemberton et al. (2003)Go. Intra-assay variability was low in all three assays (Figure 5C, % CV = 4.48; Figure 5D, % CV = 5.73; Figure 5E: % CV = 3.39), and inter-assay variability (Figure 2F, % CV = 1.14) was lower than that reported using sheep capture and rat detection antibodies (Pemberton et al. 2003Go).



View larger version (36K):
[in this window]
[in a new window]
 
Figure 5

Flow cytometric analysis of mouse mucosal mast cell homologues dual-labeled with Cy5- and FITC-labeled Fab fragment complexes containing primary rat IgG2a antibodies specific for integrin-{alpha}E and -ß7. Primary rat IgG2a antibodies were prelabeled with fluorophore-conjugated anti-rat IgG Fab fragments to generate the following Fab fragment complexes: anti-integrin-ß7–FITC, control IgG2a–FITC, anti-integrin-{alpha}E–Cy5, and control IgG2a–Cy5. (A) Viable cells (outlined) were identified on the basis of their forward (x-axis, FSC-H) and side scatter characteristics (y-axis, SSC-H). (B) Cells probed with Fab fragment complexes specific for both components of the integrin-{alpha}Eß7 heterodimer exhibited closely correlated FITC (x-axis, FL1-H) and Cy5 (y-axis, FL4-H) fluorescence. (C,D) Single negative controls, in which control rat IgG2a was substituted for anti-integrin-{alpha}E (C) or -ß7 (D), were used to confirm Fab fragment complex specificity.

 
Immunofluorescent Labeling of Antigens in Mouse Tissues Using Mouse Primary Antibodies
The hypothesis that Fab fragment complexes can be used to label specific antigen in samples derived from the same species as the primary antibody in the absence of spurious labeling of endogenous Ig was tested using primary mouse antibodies and frozen sections of normal mouse jejunum. Serial sections were probed with ß-catenin-specific mouse IgG1 using directly conjugated antibodies, Fab fragment complexes, or indirect ICC (Figure 3) .



View larger version (100K):
[in this window]
[in a new window]
 
Figure 3

Immunofluorescent labeling of frozen serial sections from normal mouse jejunum with mouse anti-ß-catenin IgG1. (A,B) Direct ICC. Samples were probed with (A) FITC-conjugated mouse anti-ß-catenin IgG1 or (B) FITC-conjugated control IgG1. (C,D) Fab fragment complex ICC. Samples were probed with ß-catenin-specific (C) or control mouse IgG1 (D) that had been prelabeled with FITC-conjugated goat anti-mouse IgG Fab fragments. Arrows highlight diffuse cytoplasmic staining in C. (E,F) Indirect ICC including a blocking step for endogenous mouse Ig. Samples were blocked with excess unlabeled anti-mouse IgG Fab fragments and then probed with (E) mouse anti-ß-catenin IgG1 or (F) control IgG1. (G,H) Indirect ICC. Samples were probed with (G) mouse anti-ß-catenin IgG1 or (H) control IgG1. FITC-conjugated goat anti-mouse Fab fragments were used to visualize mouse IgG labeling in both indirect procedures (E–H). Bars = 25 µm.

 
Consistent with its association with the intracellular domain of E-cadherin (Ozawa et al. 1990Go), ß-catenin-specific labeling was most evident on the lateral surfaces (zonula adherens) of epithelial cells (Figures 3A, 3C, 3E, and 3G). There was also evidence for diffuse cytoplasmic labeling in this cell type, typically concentrated towards the basement membrane (Figure 3C, arrows) but no conclusive evidence for ß-catenin expression within the nuclei of basal crypt enterocytes, as has been described in normal murine colon epithelium (van de Wetering et al. 2002Go). Although specific ß-catenin labeling was most intense in indirectly labeled sections (excitation and exposure conditions: 0.6 numerical aperture objective; 0.05 mW excitation at 488 nm; 35 frames accumulated in fast photon counting mode), there was also considerable nonspecific labeling of endogenous Ig (Figures 3E–3H). Although this was reduced in sections that had been incubated with excess unlabeled goat anti-mouse Fab fragments before application of the primary antibodies, it was not completely abolished, and plasma cells within the lamina propria commonly fluoresced more intensely than the ß-catenin primary antibody (Figures 3E–3F). Sections labeled with directly conjugated antibodies exhibited considerably less intense specific fluorescence than those labeled using the indirect approach (excitation and exposure conditions: 0.6 numerical aperture objective; 0.15 mW excitation at 488 nm; 170 frames accumulated in fast photon counting mode) but were not compromised by spurious labeling of endogenous Ig (Figures 3A and 3B). Spurious labeling of endogenous Ig was virtually abolished in sections probed with ß-catenin-specific and control Fab fragment complexes (Figures 3C and 3D). Sections stained with ß-catenin-specific Fab fragment complexes (Figure 3C) also exhibited substantially brighter labeling (excitation and exposure conditions: 0.6 numerical aperture objective; 0.15 mW excitation at 488 nm; 70 frames accumulated in fast photon counting mode) than those probed with directly conjugated antibody (Figure 3A). These results were successfully reproduced using samples from three other 129 mice and at least two different BALB/c mice. Specific labeling of mouse samples in the absence of crossreactivity with endogenous Ig was also obtained with Fab fragment complexes containing several other primary mouse monoclonals (data not shown).

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 4A–4C) 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-{alpha}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.



View larger version (53K):
[in this window]
[in a new window]
 
Figure 4

Single-color indirect immunofluorescent labeling of frozen sections from normal mouse jejunum (A–D). Sections were probed with anti-integrin-ß1 (A), -ß7 (B, arrows highlight intraepithelial leukocytes), -{alpha}6 (C, arrow highlights epithelial basement membrane; arrowhead highlights endothelium), or isotype-matched control (D) antibodies. Specific labeling was visualized using Cy5-conjugated secondary antibodies. Triple-color immunofluorescent labeling of frozen sections from normal mouse jejunum with Fab fragment complexes containing primary antibodies derived from the same host species (E–L). Primary rat IgG2a antibodies were prelabeled with fluorophore-conjugated anti-rat IgG Fab fragments to generate the following Fab fragment complexes: anti-integrin-ß1–FITC or control IgG2a–FITC (pseudocolored red); anti-integrin-ß7–Cy5 or control IgG2a–Cy5 (pseudocolored green); and anti-integrin-{alpha}6–RRX or control IgG2a–RRX (pseudocolored blue). Images of murine jejunum labeled with all three integrin-specific Fab fragment complexes (E,F). (G–I) High-resolution images from sections probed with various combinations of anti-integrin and control IgG2a–Fab fragment complexes. (G) Anti-integrin-ß1 (red), control (blue), and control (green). (H) Control (red), anti-integrin-ß7 (green), and control (blue). (I) Control (red), control (green), and anti-integrin-{alpha}6 (blue). (J–l) High-resolution images of jejunal epithelium probed with all three anti-integrin–Fab fragment complexes plus a 10-fold excess of unlabeled rat IgG2a anti-integrin-ß1 (J), -integrin-ß7 (K), or -integrin-{alpha}6 (L). Identical CLSM settings were used to acquire images from samples probed with anti-integrin antibodies and their respective controls. Bars = 10 µm.

 
After stabilization with excess serum from the same species as the primary antibody, Fab fragment complexes containing primary antibodies derived from the same species can be mixed and used in multilabeling studies (Figures 4E–4L). Images of frozen sections of normal mouse jejunum that were triple-labeled with Fab fragment complexes containing primary rat IgG2a monoclonal antibodies specific for integrins-ß1 (pseudocolored red), -ß7 (pseudocolored green), and integrin-{alpha}6 (pseudocolored blue) are shown in Figures 4E and 4F. The staining morphology obtained using anti-integrin–Fab fragment complexes (Figures 4E–4L) was virtually identical to that observed with single-color indirect ICC (Figures 4A–4C). Although there was a reduction in signal intensity, fully saturated images were readily acquired at relatively low-level excitation (0.05–0.5 mW) and photon multiplier tube sensitivity (40 frames accumulated in fast photon counting mode using a 1.4 numerical aperture x63 objective). There was little evidence for crossreaction between Fab fragment complexes in triple-labeled samples (Figure 4E and 4F). Single positive controls, in which control rat IgG2a–Fab fragment complexes were substituted for two of the anti-integrin antibody–Fab fragment complexes, were used to confirm staining fidelity. Nonspecific background staining was low in all cases and there was negligible evidence for Fab fragment transfer from control antibodies to anti-integrin antibodies (Figures 4G–4I). Fab fragment complex stability was also examined using competitive controls, where tissue sections were probed with all three anti-integrin–Fab fragment complexes in the presence of a 10-fold excess of unlabeled integrin-ß1 (Figure 4J), -ß7 (Figure 4K), or -{alpha}6 (Figure 4L) specific antibodies. Excess unlabeled primary antibodies almost completely inhibited Fab fragment complex labeling in all three cases (Figures 4J–4L). These results were successfully reproduced using samples from several different 129 and BALB/c mice. They were also largely unaffected by changing the primary antibody/fluorophore combinations, although the signal obtained with Cy5-labeled anti-integrin-{alpha}6 complexes was commonly so intense that it resulted in substantial bleed-through into the RRX channel.

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. 2002Go), identified on the basis of their forward and side scatter characteristics (Figure 5A). The majority of cells incubated with integrin-{alpha}E- and integrin-ß7-specific Fab fragment complexes exhibited bright FITC and Cy5 fluorescence (Figure 5B). Consistent with the expression of integrins-{alpha}E and 7 as a heterodimer (Kilshaw 1999Go), 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-{alpha}E (Figure 5C) or integrin-ß7-specific (Figure 5D) primary antibodies were substituted with control rat IgG2a.


    Discussion
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Various methods involving the sequential application and elution (Nakane 1968Go) or denaturation (Wang and Larsson 1985Go; Tornehave et al. 2000Go) of antibodies have been developed for indirect multilabeling using primary antibodies derived from the same species. However, their application is limited to tissues and antigens that are resistant to damage by the relatively harsh conditions involved. The use of monovalent IgG Fab fragments to block endogenous Ig was first introduced for the screening of human antibodies on human tissue sections (Nielsen et al. 1987Go). Monovalent Fab fragments, as opposed to divalent IgG or F (ab')2 fragments, allow endogenous Ig epitopes to be sterically blocked while eliminating the possibility of crosslinking them with subsequent primary antibody steps. Complete blocking of endogenous Ig with Fab fragments can be difficult to achieve (Nielsen et al. 1987Go). Nevertheless, this approach has been adapted for dual-labeling studies using primary antibodies derived from the same species (Lewis-Carl et al. 1993Go; Negoescu et al. 1994Go; Donat et al. 1999Go). All three methods, although subtly different, rely on indirect labeling of the first antigen followed by (Lewis-Carl et al. 1993Go) or incorporating (Negoescu et al. 1994Go; Donat et al. 1999Go) the use of Fab fragments to fully saturate the first primary antibody and block its recognition by the secondary antibody used in the indirect labeling of the second antigen. Brouns et al. (2002)Go have recently employed this approach, in combination with tyramide signal amplification (Bobrow et al. 1989Go; Shindler and Roth 1996Go), to successfully delineate three different antigens using polyclonal rabbit primary antibodies.

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)Go 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. 1990Go) 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 2000Go; Martin et al. 2003Go), 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 antibody–Fab 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 1:2–1:4 (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. 2003Go), 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 2C–2F).

Primary antibody–Fab 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 antibody–Fab 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.


    Acknowledgments
 
Supported by the Wellcome Trust (grant #060312).

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-{alpha}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.


    Footnotes
 
Received for publication November 12, 2003; accepted April 21, 2004


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

Bernier GM, Cebra JJ (1964) Polypeptide chains of human gamma-globulin: cellular localization by fluorescent antibody. Science 144:1590–1591[Medline]

Bobrow MN, Harris TD, Shaughnessy KJ, Litt GJ (1989) Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J Immunol Methods 125:279–285[CrossRef][Medline]

Brandtzaeg P (1998) The increasing power of immunohistochemistry and immunocytochemistry. J Immunol Methods 216:49–67[CrossRef][Medline]

Brouns I, Van-Nassauw L, Van-Genechten J, Majewski M, Scheuermann DW, Timmermans JP, Adriaensen D (2002) Triple immunofluorescence staining with antibodies raised in the same species to study the complex innervation pattern of intrapulmonary chemoreceptors. J Histochem Cytochem 50:575–582[Abstract/Free Full Text]

Brown JK, Knight PA, Wright SH, Thornton EM, Miller HRP (2003) Constitutive secretion of the granule chymase mouse mast cell protease-1 and the chemokine, CCL2, by mucosal mast cell homologues. Clin Exp Allergy 33:132–146[CrossRef][Medline]

Coleman R (2000) The impact of histochemistry: a historical perspective. Acta Histochem 102:5–14[Medline]

Coons AH, Creech H, Jones R (1941) Immunological properties of an antibody containing a fluorescent group. Proc Soc Exp Biol Med 47:200–202

Coons AH, Creech H, Jones R, Berliner E (1942) The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J Immunol 45:159–170

Coons AH, Leduc EH, Connolly JM (1955) Studies on antibody production. I. A method for the histochemical demonstration of specific antibody and its application to a study of the hyperimmune rabbit. J Exp Med 102:49–60[Abstract/Free Full Text]

Donat ME, Wong K, Staines WA, Krantis A (1999) Heme oxygenase immunoreactive neurons in the rat intestine and their relationship to nitrergic neurons. J Autonom Nerv Syst 77:4–12[CrossRef][Medline]

Ferri GL, Gaudio RM, Castello IF, Berger P, Giro G (1997) Quadruple immunofluorescence: a direct visualization method. J Histochem Cytochem 45:155–158[Abstract/Free Full Text]

Haugland RP (2002) Zenon technology—versatile reagents for immunolabeling. In Gregory J, ed. Handbook of Fluorescent Probes and Research Products. Eugene, OR, Molecular Probes, 197–204

Hierck BP, Iperen LV, Gittenberger-de-Groot AC, Poelmann RE (1994) Modified indirect immunodetection allows study of murine tissue with mouse monoclonal antibodies. J Histochem Cytochem 42:1499–1502[Abstract/Free Full Text]

Kilshaw PJ (1999) Alpha E beta 7. Mol Pathol 52:203–207[Abstract]

Kingston D, Pearson JR (1981) The use of the peroxidase reaction to obliterate staining of eosinophils by fluorescein-labelled conjugates. J Immunol Methods 44:191–198[CrossRef][Medline]

Krenacs T, Uda H, Tanaka S (1991) One-step double immunolabeling of mouse interdigitating reticular cells: simultaneous application of pre-formed complexes of monoclonal rat antibody M1–8 with horseradish peroxidase-linked anti-rat immunoglobulins and of monoclonal mouse anti-Ia antibody with alkaline phosphatase-coupled anti-mouse immunoglobulins. J Histochem Cytochem 39:1719–1723[Abstract]

Kroeber S, Schomerus C, Korf HW (1998) A specific and sensitive double-immunofluorescence method for the demonstration of S-antigen and serotonin in trout and rat pinealocytes by means of primary antibodies from the same donor species. Histochem Cell Biol 109:309–317[CrossRef][Medline]

Lewis-Carl SA, Gillete-Ferguson I, Ferguson DG (1993) An indirect immunofluorescence procedure for staining the same cryosection with two mouse monoclonal primary antibodies. J Histochem Cytochem 41:1273–1278[Abstract/Free Full Text]

Lu QL, Partridge TA (1998) A new blocking method for application of murine monoclonal antibody to mouse tissue sections. J Histochem Cytochem 46:977–984[Abstract/Free Full Text]

Mao S-Y (1999) Conjugation of fluorochromes to antibodies. In Javois L, ed. Immunocytochemical Methods and Protocols. Totowa, NJ, Humana Press, 35–38

Martin K, Hart C, Liu J, Leung WY, Patton WF (2003) Simultaneous trichromatic fluorescence detection of proteins on Western blots using an amine-reactive dye in combination with alkaline phosphatase- and horseradish peroxidase-antibody conjugates. Proteomics 3:1215–1227[CrossRef][Medline]

Miller HRP, Wright SH, Knight PA, Thornton EM (1999) A novel function for transforming growth factor-beta1: upregulation of the expression and the IgE-independent extracellular release of a mucosal mast cell granule-specific beta-chymase, mouse mast cell protease-1. Blood 93:3473–3486[Abstract/Free Full Text]

Nakane PK (1968) Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study on pituitary glands of the rat. J Histochem Cytochem 16:557–560[Medline]

Negoescu A, Labat-Moleur F, Lorimier P, Lamarcq L, Guillermet C, Chambaz E, Brambilla E (1994) F(ab) secondary antibodies: a general method for double immunolabeling with primary antisera from the same species. Efficiency control by chemiluminescence. J Histochem Cytochem 42:433–437[Abstract/Free Full Text]

Nielsen B, Borup-Christensen P, Erb K, Jensenius JC, Husby S (1987) A method for the blocking of endogenous immunoglobulin on frozen tissue sections in the screening of human hybridoma antibody in culture supernatants. Hybridoma 6:103–109[Medline]

Ozawa M, Ringwald M, Kemler R (1990) Uvomorulin-catenin complex formation is regulated by a specific domain in the cytoplasmic region of the cell adhesion molecule. Proc Natl Acad Sci USA 87:4246–4250[Abstract]

Panchuk-Voloshina N, Haugland RP, Bishop-Stewart J, Bhalgat MK, Millard PJ, Mao F, Leung WY (1999) Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates. J Histochem Cytochem 47:1179–1188[Abstract/Free Full Text]

Pemberton AD, Brown JK, Wright SH, Knight PA, McPhee ML, McEuen AR, Forse PA, Miller HRP (2003) Purification and characterization of mouse mast cell proteinase-2 and the differential expression and release of mouse mast cell proteinase-1 and -2 in vivo. Clin Exp Allergy 33:1005–1012.[CrossRef][Medline]

Rasband WS, Bright DS (1995) NIH Image: a public domain image processing program for the Macintosh. Microbeam Anal 4:137–149

Schrock E, Du-Manoir S, Veldman T, Schoell B, Wienberg J, Ferguson-Smith MA, Ning Y, et al. (1996) Multicolor spectral karyotyping of human chromosomes. Science 273:494–497[Abstract]

Shindler KS, Roth KA (1996) Double immunofluorescent staining using two unconjugated primary antisera raised in the same species. J Histochem Cytochem 44:1331–1335[Abstract/Free Full Text]

Staines WA, Meister B, Melander T, Nagy JI, Hökfelt T (1988) Three-color immunofluorescence histochemistry allowing triple labeling within a single section. J Histochem Cytochem 36:145–151[Abstract]

Tornehave D, Hougaard DM, Larsson L (2000) Microwaving for double indirect immunofluorescence with primary antibodies from the same species and for staining of mouse tissues with mouse monoclonal antibodies. Histochem Cell Biol 113:19–23[CrossRef][Medline]

Tsurui H, Nishimura H, Hattori S, Hirose S, Okumura K, Shirai T (2000) Seven-color fluorescence imaging of tissue samples based on Fourier spectroscopy and singular value decomposition. J Histochem Cytochem 48:653–662[Abstract/Free Full Text]

Tuson JR, Pascoe EW, Jacob DA (1990) A novel immunohistochemical technique for demonstration of specific binding of human monoclonal antibodies to human cryostat tissue sections. J Histochem Cytochem 38:923–926[Abstract]

Valnes K, Brandtzaeg P (1981) Selective inhibition of nonspecific eosinophil staining or identification of eosinophilic granulocytes by paired counterstaining in immunofluorescence studies. J Histochem Cytochem 29:595–600[Abstract]

van der Loos CM, Gobel H (2000) The animal research kit (ARK) can be used in a multistep double staining method for human specimens. J Histochem Cytochem 48:1431–1437[Abstract/Free Full Text]

van de Wetering M, Sancho E, Verweij C, de Lau W, Oving I, Hurlstone A, et al. (2002) The beta-catenin/TCF-4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 111:241–250[Medline]

Vischer NOE, Huls PG, Woldringh CL (1994) Object-Image: an interactive image analysis program using structured point collection. Binary 6:160–166

Wang BL, Larsson LI (1985) Simultaneous demonstration of multiple antigens by indirect immunofluorescence or immunogold staining. Novel light and electron microscopical double and triple staining method employing primary antibodies from the same species. Histochemistry 83:47–56[Medline]

Wright SH, Brown J, Knight PA, Thornton EM, Kilshaw PJ, Miller HRP (2002) Transforming growth factor-beta1 mediates coexpression of the integrin subunit alphaE and the chymase mouse mast cell protease-1 during the early differentiation of bone marrow-derived mucosal mast cell homologues. Clin Exp Allergy 32:315–324[CrossRef][Medline]





This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Brown, J. K.
Articles by Miller, H. R.P.
Articles citing this Article
PubMed
PubMed Citation
Articles by Brown, J. K.
Articles by Miller, H. R.P.


Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]