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Contents lists available at SciVerse ScienceDirect

Acta Histochemica

jou rna l h o mepage: www.elsev ier .de /ac th is

ignal amplification in immunohistochemistry: Loose-jointed deformableeteropolymeric HRP conjugates vs. linear polymer backbone HRP conjugates

gor Buchwalow ∗, Werner Boecker, Eduard Wolf, Vera Samoilova, Markus Tiemannnstitute for Hematopathology, Fangdieckstr. 75a, 22547 Hamburg, Germany

r t i c l e i n f o

rticle history:eceived 23 October 2012eceived in revised form6 December 2012ccepted 17 December 2012vailable online xxx

a b s t r a c t

Improvements in reagents and protocols for immunohistochemistry have led to increased sensitivityof detection systems. A significant level of signal amplification was achieved by the chain-polymerconjugate technology utilizing enzyme-labeled inert “backbone” molecule of dextran (Dako). However,the relatively large size of the dextran molecule in aqueous phase appears to create spatial hindrancecompromising the penetrative ability of the detection reagent. Novel AmpliStainTM detection systems(SDT GmbH, Baesweiler, Germany) seem to overcome these constraints offering a more compact anddeformable conjugate design that facilitates agile penetration through the narrowest diffusion pathways

eywords:mmunohistochemistryntibodiesignal amplificationetection systemsuclear antigensifferential diagnosis

in tissue sections. Here, we compared the level of signal amplification achievable with AmpliStainTM-HRP(SDT) and EnVisionTM+-HRP (Dako). Our results show that the AmpliStainTM-HRP systems allow higherdilutions of primary antibodies in both immunohistochemistry and ELISA. Compared with EnVisionTM+,anti-mouse AmpliStainTM enables at least three times more sensitive detection of mouse antibodies,whereas anti-rabbit AmpliStainTM is ten times more sensitive than anti-rabbit EnVisionTM+.

ntroduction

The peroxidase-labeled antibody (Ab) method, introduced in968, was the first practical application of antibodies (Abs) toaraffin-embedded tissues for most clinical and research studiesNakane, 1968). However, conventional secondary Ab(IgG)-HRPonjugates are basically limited to not more than 3 molecules ofRP per single IgG molecule, because of the functional bindingapacity of Ab coupled with HRP using regular periodate, benzo-uinone or thiol-maleimide conjugation chemistries (Niemeyer,004; Jameson and Wong, 2009). This molar ratio of conjugation

s sufficient in routine immunostaining, but may lack the sensi-ivity required for detecting targets present in low picogram toemtogram amounts without the use of additional steps to amplifyhe signal (Harlow and Lane, 1999; Buchwalow and Boecker, 2010).

During the past few decades, improvements in the reagents androtocols used for immunohistochemistry have led to increasedensitivity of detection systems. A several-fold higher antigenetectability than those achieved in enzyme–anti-enzyme immuneomplex techniques (PAP and APAAP) (Sternberger et al., 1970) or

Please cite this article in press as: Buchwalow I, et al. Signal amplification inHRP conjugates vs. linear polymer backbone HRP conjugates. Acta Histoch

n standard avidin/streptavidin–biotin-complex (ABC) and LabeledtreptAvidin-Biotin (LSAB) protocols can be gained with the chain

∗ Corresponding author.E-mail address: buchwalow@pathologie-hh.de (I. Buchwalow).

065-1281/$ – see front matter © 2013 Elsevier GmbH. All rights reserved.ttp://dx.doi.org/10.1016/j.acthis.2012.12.008

© 2013 Elsevier GmbH. All rights reserved.

polymer-conjugate technology developed in the last decade of theformer century (Bisgaard et al., 1993; Van der Loos et al., 1996).

The chain polymer-conjugate technology utilizes an inert poly-mer molecule of dextran or synthetic peptide (poly-l-lysine) inthe capacity of a backbone carrying both multiple enzymes andAbs. Thus, for instance, up to 40 molecules of HRP and approxi-mately 11 molecules of Ab can be attached to one 500 kDa dextranmolecule (manufacturer’s data; http://www.dako.com/). Dextranbackbone-based polymer conjugates of Dako (Glostrup, Denmark)known as EnVisionTM+ reagents demonstrated highly improveddetection sensitivity, efficiency and reliability compared to conven-tional secondary Ab conjugates or to enzyme–anti-enzyme and ABCtechniques (Sabattini et al., 1998). The staining costs with the useof EnVisionTM+ could reportedly be reduced by about 95% (Bellinget al., 1999).

However, the larger volume that such a hydrated backbonemolecule as dextran occupies in aqueous environment appears tocreate spatial hindrance, thus reducing the penetrative ability ofthe detection reagent. Consequently, sensitivities of the polymer-enhanced system vary greatly for different antigens, especially withmuch lower sensitivity observed in staining of hidden nuclear anti-gens (for review see: Shi et al., 1999 and citations therein).

The ever-increasing demands from immunohistology for more

immunohistochemistry: Loose-jointed deformable heteropolymericemica (2013), http://dx.doi.org/10.1016/j.acthis.2012.12.008

compact and flexible conjugates with better penetrative abilityhave recently led to the AmpliStainTM 1-Step heteropolymericdetection systems manufactured by Stereospecific DetectionTechnologies (SDT GmbH, Baesweiler, Germany). These systems

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Table 1Primary antibodies used in this study.

Antibodies Source Dilutiona Tissuesb

� Smooth Muscle Actin (mouse Ab) Dako 1/50 1, 3, 4, 5� Smooth Muscle Actin (rabbit Ab) AbCam 1/200 1, 3, 4, 5Cytokeratins 5 (rabbit Ab) Medac 1/100 4, 5, 9, 11Cytokeratin 7 (mouse Ab) Dako 1/200 4, 5, 10, 11Cytokeratin 10 (mouse Ab) Dako 1/200 5, 9Cytokeratins 8/18/19 (mouse Ab) Immunotech 1/100 4, 5, 6, 7, 11Estrogen receptors (mouse Ab) Dako 1/200 4, 5Estrogen receptors (rabbit Ab) Thermo 1/200 4, 5Ki67 (rabbit Ab) Thermo 1/100 2, 4, 5, 8, 12, 13MIB1 (mouse Ab) Dako 1/50 2, 4, 5, 8

a Starting dilution for series of further dilutions of twofold increments.b Tissue samples immunostained in this study: human kidney (1), human tonsil

(2), human muscle tissue (3) human mammary gland (4), human breast tumors (5),htr

ccDcehtldm(egAu

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uman salivary gland (6), human salivary gland tumors (7), human gastrointestinalissue (8), human skin (9), human pancreas (10), cow and pig mammary gland (11),at and mouse spleen (12), and rat and mouse gut (13).

omprise StrongZymeTM anti-mouse, anti-rabbit and anti-goatonjugates made of specially engineered decarboxylated Highensity Nude HRPTM di-, tri- tetra- and pentamers covalentlyoupled to the affinity purified secondary Abs using the propri-tary SnakeLinkerTM technology. Decarboxylation decreases theydrodynamic radius of the enzymatic label making it effec-ively smaller in size. Application of the flexible extendable springinkers yields a loose-jointed compact and flexible conjugateesign. Moreover, StrongZymeTM design has a very strong enzy-atic signal-generating moiety. According to the manufacturer

http://www.sdt-reagents.de/), these factors make conjugates bothxceptionally active in detection and good at penetration. Theoal of the present study was to compare the sensitivities of thempliStainTM 1-Step detection systems of SDT GmbH with the pop-lar marketed EnVisionTM+ systems manufactured by Dako.

aterials and methods

issue probes

Human tissue probes were from Institute of Pathology of theniversity of Münster and Institute for Hematopathology, Ham-urg. All cases were from adult patients (22–69 years of age). Thistudy was carried out in compliance with the Helsinki Declaration,pproved by an internal review board (Internal Ethics Committeeor Hematopathological and Pathological Scientific Studies, Ham-urg) and done in accordance with the ethical standards of theegional committee on human studies (DAkkS, Deutsche Akkred-tierungsstelle, Berlin). Tissue probes from experimental animals

ere used from earlier experiments performed in accordance withhe Helsinki Declaration and The Guiding Principles in the Care andse of Animals.

mmunohistochemistry

Tissue probes were fixed in buffered 4% formaldehyde and rou-inely embedded in paraffin. Four-micrometer-thick paraffin tissueections were deparaffinized with xylene and graded ethanol. Afterre-treatment for antigen retrieval by heating in 10 mM sodiumitrate buffer, pH 6.0, at 95 ◦C for 30 min in a domestic vegetableteamer (Buchwalow and Boecker, 2010), tissue sections weremmunoreacted with primary Abs (Table 1) applied for 1 h atoom temperature in several dilutions of twofold increments (1:50,

Please cite this article in press as: Buchwalow I, et al. Signal amplification inHRP conjugates vs. linear polymer backbone HRP conjugates. Acta Histoch

:100, 1:200, 1:400, 1:800, 1:1600). PBS was used for all dilutionsnd washings. Blocking the endogenous Fc receptors prior to incu-ation with primary Abs was omitted, because we have recentlyeported that endogenous Fc receptors in routinely fixed cell and

PRESSemica xxx (2013) xxx– xxx

tissue probes do not retain their ability to bind Fc fragments of Abs(Buchwalow et al., 2011).

After immunoreacting with primary Abs, tissue sections weretreated for 10 min with methanol containing 0.6% H2O2 to quenchendogenous peroxidase. Bound primary Abs were detected withEnVisionTM+ Horse Radish Peroxidase (HRP) Systems (Dako Cor-poration, Hamburg, Germany) or with an AmpliStainTM HRPconjugates (SDT GmbH, Baesweiler, Germany) 30 min at room tem-perature. HRP labeling was visualized using a NovaRed substrate kit(Vector Laboratories, Burlingame, CA, USA). Immunostaining withheteropolymeric AmpliStainTM and dextran backbone EnVisionTM+was performed on two immediately adjacent sections for each Abdilution. Chromogene development was monitored under micro-scopic control and stopped simultaneously for the entire series ofthe primary Ab dilutions.

The exclusion of either the primary or the secondary Ab fromthe immunohistochemical reaction, substitution of primary Abswith non-specific mouse or rabbit IgG at the same concentrationsresulted in lack of immunostaining.

ELISA tests

High Binding EIA ImmunoPlate strips coated with tetanus toxoid(rows R1–R4) and F1 antigen (rows M1–M4) were used as spe-cific immunosorbents. Rabbit anti-tetanus hyperimmune serum(R1–R4) and culture fluid containing anti-F1mouse monoclonal IgGAbs (M1–M4) were applied in several dilutions of twofold incre-ments to the wells of the ImmunoStrips in quadruplicates andincubated 30 min at room temperature on the orbital shaker at700 rpm. After washing with Tween–PBS, bound rabbit and mouseIgGs were detected in 30-min incubation with AmpliStainTM-HRP (anti-rabbit, rows R3–R4, and anti-mouse, rows M3–M4) andwith EnVisionTM-HRP (anti-rabbit, rows R1–R2, and anti-mouse,rows M1–M2). For the comparison in ELISA, both ready-to-use IHC staining conjugates were diluted 1:100 in UniversalIHC Diluent/Blocker/Stabilizer (#UDBS of SDT GmbH, Baesweiler,Germany). After incubation with secondary Ab conjugates stripswere washed 5 times and reactions were visualized using #esTMBsubstrate (SDT) (Fig. 4). Substrate color development was ter-minated with stop reagent (#SER from SDT). After termination,OD at 450 nm was measured vs. 620 nm, as reference, in ELISA-spectrophotometer Anthos HT-2 (Fig. 4a and b).

Image acquisition

Immunostained sections were examined on a Zeiss Axio ImagerZ1 microscope. Microscopic images were captured using AxioCamdigital microscope cameras and AxioVision image processing (CarlZeiss Vision, Oberkochen, Germany). Images shown are represen-tative of two independent experiments which gave similar results.The images were acquired at 96 DPI and submitted with the finalrevision of the manuscript at 300 DPI.

Results

In order to compare EnVisionTM+ and AmpliStainTM 1-Stepdetection systems, we performed immunostaining of 15 tissuespecimens with 10 mouse monoclonal and rabbit monoclonal andpolyclonal primary Abs (Table 1). For paraffin embedding, tis-sue probes were routinely fixed with 4% formaldehyde in PBS,pH 7.4. PBS was used for all washes and dilutions. Paraffin tis-

immunohistochemistry: Loose-jointed deformable heteropolymericemica (2013), http://dx.doi.org/10.1016/j.acthis.2012.12.008

sue sections (4 �m thick) were deparaffinized with xylene andgraded ethanol, while antigen retrieval was achieved by heat-ing the sections in 10 mM sodium citrate buffer, pH 6.0, at 95 ◦Cfor 30 min in a domestic vegetable steamer. Immunostaining was

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Fig. 1. Immunohistochemical staining of human skin for cytokeratin 10 (mouse IgG Ab concentration 100 �g/ml). Primary Abs were applied at dilutions 1:200 (a and e),1 ted usk

pitsDawCc

:400 (b and f), 1:800 (c and g) and 1:1600 (d and h). Bound primary Abs were detecit. Nuclei were counterstained with Ehrlich’s hematoxylin.

erformed according to a standard protocol routinely used formmunohistopathology (Buchwalow and Boecker, 2010). Paraffinissue sections were immunostained with primary Abs in series ofeveral dilutions of twofold increments under identical conditions.etection of bound primary Abs with AmpliStainTM-HRP conjugate

Please cite this article in press as: Buchwalow I, et al. Signal amplification inHRP conjugates vs. linear polymer backbone HRP conjugates. Acta Histoch

nd EnVisionTM+ backbone polymer conjugate was performed pair-ise on two immediately adjacent sections for each Ab dilution.hromogene development after application of the polymeric HRPonjugates was monitored under microscopic control until optimal

ing AmpliStainTM (a–d) or EnVisionTM+ (e–f) and visualized with NovaRed substrate

staining and stopped simultaneously for the entire series of primaryAb dilutions. Dilutions ranged from 1:50 to 1:1600.

Immunostaining results obtained using a panel of 10 antibodiesrevealed that the sensitivity of the AmpliStainTM 1-Step detectionsystems was greater than that of EnVisionTM+. The detection effi-

immunohistochemistry: Loose-jointed deformable heteropolymericemica (2013), http://dx.doi.org/10.1016/j.acthis.2012.12.008

ciency of the AmpliStainTM 1-Step detection reagents was superior,as exemplified by immunohistochemical staining of human skinfor cytokeratin 10 (Fig. 1a–f), human tonsil for Ki67 (Fig. 2a–f) andhuman breast carcinoma for estrogen receptors (ER) (Fig. 3a–f).

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F centr1 ed usik

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AamsoQ

ig. 2. Immunohistochemical staining of human tonsil for Ki67 (rabbit IgG Ab con:200 (b and f), 1:400 (c and g) and 1:800 (d and h). Bound primary Abs were detectit. Nuclei were counterstained with Ehrlich’s hematoxylin.

otably, the superiority of AmpliStainTM detection systems wasspecially pronounced in detection of nuclear antigens, such as Ki67nd ER (Figs. 2 and 3).

In order to quantify the immunostaining efficiency ofmpliStainTM and EnVisionTM+, we run ELISA tests for bothnti-rabbit IgG (rows R1, R2, R3 and R4 in Fig. 4) and anti-

Please cite this article in press as: Buchwalow I, et al. Signal amplification inHRP conjugates vs. linear polymer backbone HRP conjugates. Acta Histoch

ouse IgG (rows M1, M2, M3 and M4 in Fig. 4) specificystems. ELISA tests demonstrated a markedly higher efficiencyf AmpliStainTM detection systems compared with EnVisionTM+.uantitative ELISA data (Fig. 4b and c) showed that, compared

ation approx. 1000 �g/ml). Primary Abs were applied at dilutions 1:100 (a and e),ng AmpliStainTM (a–d) or EnVisionTM+ (e–f) and visualized with NovaRed substrate

with EnVisionTM+-HRP, anti-mouse AmpliStainTM-HRP enables atleast three times more sensitive detection of mouse Abs, whereasanti-rabbit AmpliStainTM is on the average ten times stronger thanEnVisionTM+ goat-anti-rabbit.

Discussion

immunohistochemistry: Loose-jointed deformable heteropolymericemica (2013), http://dx.doi.org/10.1016/j.acthis.2012.12.008

Compared with EnVisionTM+, better immunostaining resultsachieved with AmpliStainTM are apparently due to two main fac-tors: a strong enzymatic signal-generating moiety and a higher

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Fig. 3. Immunohistochemical staining of human breast carcinoma for estrogen receptors (mouse IgG Ab concentration 395 �g/ml). Primary Abs were applied at dilutions1 ry AbsN

pcaesbsbw

:200 (a and e), 1:400 (b and f), 1:800 (c and g) and 1:1600 (d and h). Bound primaovaRed substrate kit. Nuclei were counterstained with Ehrlich’s hematoxylin.

enetrating ability of the compact and flexible StrongZymeTM

onjugates. Backbone EnVisionTM polymer conjugates are usu-lly depicted as linear (Fig. 5a), but in practice they comprisessentially rigid globular structures. Intact carrier polymers pere may indeed be predominantly linear and flexible, but covalent

Please cite this article in press as: Buchwalow I, et al. Signal amplification inHRP conjugates vs. linear polymer backbone HRP conjugates. Acta Histoch

inding of enzymes and Abs to dextran polymer via divinyl-ulfone chemistry (EnVisionTM+ of Dako) obviously takes awayoth flexibility and its relative linearity. The same likely occursith poly-l-lysine after coupling with enzymes and Fab’ antibody

were detected using AmpliStainTM (a–d) or EnVisionTM+ (e–f) and visualized with

fragments using Mal-SH chemistry (N-Histofine® of Nichirei Bio;http://www.nichirei.co.jp/bio/english/). The size of the backbonepolymer is not only a factor restricting penetration into the tis-sue section, but also a measure of the bulky substance presentin the conjugate structure that has no other but solely carrier

immunohistochemistry: Loose-jointed deformable heteropolymericemica (2013), http://dx.doi.org/10.1016/j.acthis.2012.12.008

function. This appears to create spatial hindrance, thus compromis-ing the penetrative ability of the conjugates of complex backbonepolymer design. Moreover, because such conjugates increase dra-matically in size, the enzyme density per unit surface may not

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F TM e (#ASs . Indir( ith Am

b1

obo(peiiMgsmjmdesa

ig. 4. ELISA tests. (a) Side by side comparison of AmpliStain 1-Step anti-mousystems with EnVisionTM+ anti-mouse (rows M1–M2) and anti-rabbit (rows R1–R2)b and c) Quantitative assay of specific mouse and rabbit IgG Abs using detection w

e increased to the degree that would be desired (Shi et al.,999).

Novel re-adjustable heteropolymeric AmpliStainTM conjugatesf SDT offer compact and at the same time light conjugate designased on the smaller HRP oligomers loosely coupled to the sec-ndary anti-mouse or anti-rabbit Abs using expandable linkersFig. 5b). Proprietary SnakeLinkerTM chemistry yields runny, meta-lastic conjugates that can change their shape conforming to thenvironment (Fig. 5c). Deformable structure of the conjugate facil-tates agile penetration through the narrowest diffusion pathwaysn the tissue sections when targeting hidden nuclear markers.

oreover, AmpliStainTM conjugates bear very powerful signal-enerating charge composed of multiple dissipated over its surfacemaller HRP molecules. In the AmpliStainTM conjugate, one Abolecule binds 12–24 HRP molecules. In the EnVisionTM+ con-

ugate, its “backbone” molecule of dextran bears Ab and HRPolecules in a ratio amounting approx. to 1:4 (manufacturer’s

Please cite this article in press as: Buchwalow I, et al. Signal amplification inHRP conjugates vs. linear polymer backbone HRP conjugates. Acta Histoch

ata). As a result, combination of the above properties (higher pen-trative ability and stronger signal-generating charge) enables veryensitive detection of poorly expressed low-abundance and hiddenntigens.

-M1-HRP – rows M3–M4) and anti-rabbit (#AS-R1-HRP – rows R3–R4) detectionect sandwich ELISA test systems, 15-min TMB substrate reaction (before stopping).pliStainTM and EnVisionTM+ HRP conjugates in ELISA.

In our study, the stronger enzymatic signal generation ofAmpliStainTM conjugates is amply evident from immunohisto-chemical stainings (Figs. 1–3) and particularly from quantitativeELISA readout data (Fig. 4). Moreover, because of their higherpenetrative ability, AmpliStainTM conjugates have proved to beespecially effective in detecting nuclear antigens, such as ER andKi67 (Figs. 2 and 3).

Immunohistochemistry for nuclear hormone (estrogen andprogesterone) receptors (ER and PR) is indispensable in histopatho-logical assessment of human breast and uterine tumors (Bonfittoand Andrade, 2003; Boecker, 2006; Dabbs, 2010). The clinicalresponsiveness to anti-hormonal treatments correlates with theexpression of the ER and PR (Masood, 1994). Enhanced nuclearstaining for ER or PR means that estrogen or progesterone is caus-ing the tumor to grow, and that the cancer should respond wellto hormone suppression treatments. Therefore, the score of ER-and PR-positive cells in human neoplasms is not only of diagnostic

immunohistochemistry: Loose-jointed deformable heteropolymericemica (2013), http://dx.doi.org/10.1016/j.acthis.2012.12.008

importance, but also has independent prognostic value (Masood,1994; Molino et al., 1997b; Bonfitto and Andrade, 2003), and thehigher sensitivity of the AmpliStainTM HRP system could contributeto the diagnostic and prognostic precision.

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Fig. 5. (a) Linear polymer backbone HRP conjugates (EnVisionTM+ System) developed by Dako (adapted from the manufacturer’s catalog). (b and c) AmpliStainTM design ofp lly gloh ningsg r diffu

md1tcldTsSiat(

dAs(ptth

seAHptel

rinciple. (b) Intact, “relaxed” conjugate in aqueous phase has a loose-jointed, basicaydrodynamic radius when diffusing through nuclear pores or through smaller opelobular to ellipsoidal. Extension of SnakeLinkersTM further contributes to the faste

Similarly, other nuclear proteins, such as cell proliferationarker Ki-67, have potential applications in determining clinical

iagnostic decisions for patients with malignant tumors (Gerdes,990; Gerdes et al., 1991; Dabbs, 2010). Thus, it has been shownhat high proliferative activity is a poor prognostic factor in breastarcinoma (Molino et al., 1997a; Nakagomi et al., 1998). However,ower sensitivity of detection systems may reveal inconsistent gra-ation of Ki-67 expression in histological and cytological samples.hus, Ki-67 expression in human neoplasms has been reported byeveral groups sometimes with conflicting results (Gerdes, 1990;aiz et al., 2002; Colozza et al., 2005; Song et al., 2011). Therefore,ncreasing sensitivity of detection systems, especially for nuclearntigens, will help to improve the diagnostic accuracy in differen-ial diagnosis between malignant and benign lesions in neoplasiaGerdes, 1990; Dabbs, 2010).

As a more sensitive method, the AmpliStainTM allows higherilutions of primary Abs. Higher dilutions of primary Abs withmpliStainTM HRP system enable achieving more reliable results,ince higher Ab dilutions prevent unwanted background stainingBuchwalow et al., 2011). Based on the German market pricing,rice per test with the AmpliStainTM system is considerably lowerhan that with the EnVisionTM+. Moreover, the aggregate cost perest with AmpliStainTM can be further reduced thanks to the muchigher dilution of primary Abs.

To summarize, AmpliStainTM detection systems are more sen-itive than existent backbone polymer detection systems, as.g. EnVisionTM+. The stronger enzymatic signal generation ofmpliStainTM conjugates results from the increased molar ratio ofRP molecules to Abs with more HRP molecules being available

Please cite this article in press as: Buchwalow I, et al. Signal amplification inHRP conjugates vs. linear polymer backbone HRP conjugates. Acta Histoch

er binding event to react with the substrate. The higher pene-rative ability of the compact and flexible AmpliStainTM conjugatesnables more sensitive and reliable detection of hidden nuclear andow abundance antigens.

bular form. (c) At penetration, the conjugate can anisotropically reduce its effective in cellular membranes occurring after aldehyde fixation. Its form can change fromsion.

Acknowledgments

We thank colleagues from our Institute for sharing reagents andtissue samples and Dr. Elena Gromakowski for performing the ELISAassays.

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