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PERSPECTIVE
Multiple Immunoenzyme Staining: Methods and Visualizationsfor the Observation With Spectral Imaging
Chris M. van der Loos
Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands
SUMMARY Several staining concepts and color combinations exist to perform successfuldouble immunoenzyme staining on human tissue specimens. Most of these concepts arebased on differences between both primary antibodies: animal species, mouse Ig isotype orIgG subclasses, conjugates, or concentrations. Traditionally, double immunoenzyme staininghas used chromogens selected to provide maximum color contrast when observed with theunaided eye. Unfortunately, visually good color combinations always include at least onediffuse chromogen, because of the paucity of appropriate chromogen colors. This situationis drastically changed with the use of spectral imaging, where multicolor microscopy can beunmixed in individual images based on their spectral characteristics. Spectral unmixing canbe performed even up to quadruple immunoenzyme staining. This work contains practicalsuggestions for immunoenzyme double staining procedures for some frequently encoun-tered primary antibody combinations: rabbit–mouse, goat–mouse, mouse–mouse, andrabbit–rabbit. The suggested protocols are all suitable for a classical red-brown color com-bination plus blue nuclear counterstain that is composed of peroxidase activity (diamino-benzidine tetrahydrochloride), alkaline phosphatase activity (Liquid Permanent Red), andhematoxylin, respectively. Although the red and brown chromogens do not contrast verywell visually, they both show a crisp localization and can be perfectly unmixed by spectralimaging. (J Histochem Cytochem 56:313–328, 2008)
KEY WORDS
immuno-double staining
immuno-quadruple staining
chromogens
spectral imaging
unmixing
THROUGHOUT LIFE SCIENCES, tissue specimens are oftenselectively stained using single immunohistochemical(IHC) techniques to visualize one particular antigenin the tissue by a colored enzymatic reaction product.To study the relationship between two antigens, mul-tiple antigens can also be localized using differentlycolored reaction products. Unfortunately, the multiplestaining techniques are often believed to be restrictedto researchers with “golden hands” because these tech-niques are often strongly tailored to a particular aim,can include the preparation of special reagents, and areprone to spurious mixed-staining. This work containsdouble immunoenzyme staining protocols for humantissue specimens that can be performed with commer-cially available reagents. These generic protocols can be
easily adapted to many different applications and anti-body combinations. Attention will be paid to the mostfrequently encountered primary antibody double stain-ing combinations: mouse–rabbit, mouse–goat, mouse–mouse, and rabbit–rabbit. The problems related tocombining two mouse antibodies that potentially maylead to unwanted cross-reactions will be discussed here.
Traditionally, when using the unaided eye or any typeofRGBcamera for observationof double staining results,a good visual contrast between the two enzymatic reac-tion products plays an essential role. If two antigens arelocalized at the same cellular structure (5colocalization),a mixed-color is present that needs to have a good con-trast with the two basic colors. To accomplish this, oneneeds to compromise with the chromogen selection re-garding the sensitivity/efficiency and microscopic resolu-tion of the colored reaction products.
Spectral imaging with the ability to unmix multi-plexed images is only marginally applied for fluores-cence microscopy (Tsurui et al. 2000; Siboni et al.2001; Greenbaum et al. 2002;Weier et al. 2005). Papersdescribing spectral imaging of bright field tissue samples
Correspondence to: Chris M. van der Loos, PhD, Departmentof Pathology, Academic Medical Center, M2-230 Meibergdreef 9,NL-1105 AZ Amsterdam, The Netherlands. E-mail: [email protected]
Received for publication November 19, 2007; accepted Novem-ber 26, 2007 [DOI: 10.1369/jhc.2007.950170].
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C The Histochemical Society, Inc. 0022-1554/07/$3.30 313
Volume 56(4): 313–328, 2008
Journal of Histochemistry & Cytochemistry
http://www.jhc.org
are rare (Ornberg et al. 1999; Ornberg 2001; Levensonand Mansfield 2006). On introduction of spectral im-aging of bright field microscopical specimens, the wholeconcept of contrasting basic colors and mixed color isdrastically changed. Based on the spectral characteristicsof the reaction products, spectral imaging offers thegreat advantage of unmixing the double staining pat-terns into two single staining images, and visual contrast
is no longer a requirement (de Boer et al. 2007a,b;Hoozemans et al. 2007; Scheper et al. 2007).
Traditional Chromogen Combinations forDouble Staining
When observing double staining results with the unaidedeye, the chromogen combination is of essential impor-tance because visual contrast is the key requirement.Especially when the observation of colocalization is themain target, thereneeds tobeanoptimal contrastbetweenthe two basic colors and the mixed component. Duringthe history of IHC, many different chromogen combina-tions for double staining have been proposed (van derLoos 1999), but only two have proven to be suitable forthe direct visual observation of both the individualchromogens and a mixed color at sites of colocalization:red–blue (with a brown–purple intermediate color) andturquoise–red (with a blue–purple intermediate color).
The red–blue color combination is composed of alka-line phosphatase (AP) activity in blue using Fast BlueBB/Napthhol-AS-MX-phosphate and horseradish per-
Table 1 Primary antibodies used in this study
Antibody Species Clone Vendor
Foxp3 Mouse IgG1 Clone 236A/E7 Abcam, Cambridge, UKCD3 Rabbit Clone SP7 Thermo/LabVisionCD20 Mouse IgG1 Clone L26 Thermo/LabVisionCD34 Mouse IgG1 Clone QBend10 Thermo/LabVisionCD8 Mouse IgG1 Clone C8/144B DakoGranzyme B-7 Mouse IgG2a Clone GrB-7 DakoCD25 Mouse IgG1 Clone ACT-1 DakoCD68 Mouse IgG3 Clone PG-M1 DakoCD3, FITC Mouse IgG1 Clone SK7 BD Biosciences,
San Jose, CA
Table 2 Secondary steps, other immunoreagents, and chromogens used in this study
Reagent Dilution Vendor Code
Polymer anti-mouse/HRP Undiluted Thermo/LabVision TL-017-ADJ-MPolymer anti-mouse/AP Undiluted Thermo/LabVision TL-017-AFJ-MPolymer anti-rabbit/HRP Undiluted Thermo/LabVision TL-017-ADJ-RPolymer anti-rabbit/AP Undiluted Thermo/LabVision TL-017-AFJ-RDonkey anti-goat IgG/APa 1:50 Jacksonc 705-055-147Donkey anti-goat IgG/HRPa 1:100 Jackson 705-035-147Donkey anti-goat IgG/biotina 1:200 Jackson 705-065-147Rabbit anti-goat Ig 1:5000 Dako Z0454Normal goat serum 1:10 Dako X0907Normal mouse serum 1:10 Dako X0910Rabbit anti-FITC 1:1000 Serotec/Biogenesisd 4510-7804Streptavidin/AP 1:100 Dako D0396Streptavidin/HRP 1:400 Dako P0397Goat anti-mouse IgG1/APb 1:50 Southern Bioteche 1070-04Goat anti-mouse IgG1/HRPb 1:100 Southern Biotech 1070-05Goat anti-mouse IgG1/biotinb 1:100 Southern Biotech 1070-08Goat anti-mouse Ig/AP 1:50 Dako D0486Goat anti-mouse Ig/HRP 1:100 Dako P0447Goat anti-rabbit Ig/AP 1:50 Dako D0487Goat anti-rabbit Ig/HRP 1:100 Dako P0448Endogenous biotin blocking system Dako X0590Animal Research Kit (ARK) Dako K3954Tyramide signal amplification kit (CSA II) Dako K1497Diaminobenzidine tetrahydrochloride enhanced kit (DAB1) Dako K3468Liquid Permanent Red kit Dako K0640Vector Blue Vector Labs SK-4300Vector VIP Vector Labs SK-4600Mayers hematoxylin 1:10 Dako S3309VectaMount Vector Labs H-5000
aSimilar dilutions for AP, HRP, and biotin donkey anti-mouse IgG conjugates.bSimilar dilutions for AP, HRP, and biotin conjugates of anti-mouse IgG2a, IgG2b, and IgG3.cJackson ImmunoResearch (West Grove, PA).dAbD Serotec/Biogenesis (Oxford, UK).eSouthern Biotech Associates (Birmingham, AL).HRP, horseradish peroxidase; AP, alkaline phosphatase.
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oxidase (HRP) activity in red with 3-amino-9-ethyl-carbazole (Boorsma 1984; van der Loos 1999). Bothreaction products dissolve in organic mounting media,and therefore, aqueous mounting is needed here. Alter-natively, two commercially available chromogens fromVector Laboratories (Burlingame, CA) can be applied:Vector Blue and Vector NovaRed (van der Loos 2005).Aqueous mounting for Vector Blue and NovaRed isnot possible (NovaRed disappears over time!), but or-ganic mounting after complete drying of the specimens
using an alcohol- and xylene-free mountant (Vecta-Mount) yields excellent microscopical results withboth cryostat and paraffin tissue sections. A counter-stain in combination with the red–blue color combina-tion needs to be tested per antibody combination. Themost obvious choice is methyl green (0.1% in acetatebuffer, pH 5.5), yielding weakly green nuclei. However,methyl green appears to bind to the red reaction prod-uct, shifting its color to brown. As such, this lowersthe color contrast, hampering the observation of co-
Figure 1 Mouse–rabbit double staining.
Figure 2 Goat–mouse indirect double staining.
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localization by mixed colors. In some instances, a weakeosin counterstain (dip in eosin solution for 30 sec,rinse in running tap water, and check microscopicallyuntil an optimal result is obtained) or weak NuclearRed counterstain may suffice and give a faint image of
unstained tissue elements. The problem in the red–bluecombination is that blue AP reaction products (FastBlue, Vector Blue) are relatively insensitive and ratherdiffusely localized compared with the peroxidase re-action product. Visualizing AP activity with nitro blue
Figure 3 Goat–mouse multistep double staining.
Figure 4 Mouse–mouse sequential double staining.
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tetrazolium/5-bromo-4-chloro-3-indolyl-phosphateyields a dark blue purple–colored reaction product thatdoes not allow the observation of a mixed color.
The turquoise–red color combination is composedof b-galactosidase (b-GAL) activity in turquoise andAP activity in red (van der Loos et al. 1993). b-GALactivity is visualized using 5-bromo-4-chloro-3-indolylb-galactoside (X-gal) with ferro-ferri iron cyanide salts(Bondi et al. 1982). This reaction product is very sta-ble and does not dissolve in alcohols or other organicliquids. AP activity can be visualized in red usingFast Red TR, Fast Red Violet LB/Naphthol-AS-MX-phosphate (Boorsma 1984; van der Loos 1999), orother commercially available red AP visualizationmethods. Sensitive and elegantly in use (one can preparethe exact amount of reagent needed!) is Liquid Perma-nent Red (LPR) from Dako (Carpinteria, CA), whichalso allows organic mounting after skipping the dehy-dration in alcohols (dry specimens at hot plate and cover-slip organically). A weak blue hematoxylin counterstaincontrasts fairly well with the basic colors but is nearlyidentical in color with the blue–purple intermediate colorat sites of colocalization. The problem in the turquoise–red combination is b-GAL activity being certainly farless sensitive/efficient and more diffusely localized com-pared with HRP reaction products or AP activity in red.
Alternatively, a red–green combination can be ob-tained from green HRP activity visualized with3,3¶,5,5¶-tetramethylbenzidine (TMB)/dioctyl sodiumsulphosuccinate (Buckel and Zehelein 1981) and AP
activity in red (van der Loos et al. 1988). The turquoise/green reaction product is pretty accurate and verysensitive/efficient; however, it is not very stable afteraqueous mounting (Speel et al. 1994). Because of theextreme sensitivity/efficiency of these chromogens, acareful titration of primary and secondary reagents isstrongly recommended. Generally, TMB-based visual-ization of HRP activity, including the commerciallyavailable chromogens TrueBlue (Kirkegaard and Perry,Gaithersburg, MD) and Vector TMB are considered“difficult” and do not seem to work well with everyantibody in each situation. A scientific explanationfor these inconsistent and variable results is lackingthus far.
As we saw here, both red–blue and turquoise–redcolor combinations have the drawback of containingone diffusely localized and rather insensitive/inefficientchromogen: Fast Blue BB, Vector Blue in the red–bluecombination, and X-gal in the turquoise–red combina-tion. This drawback makes it necessary to re-titrateprimary antibodies for double staining situations andmakes it impossible to perform successful double stain-ing with antibodies that show obscure and weak stain-ing by nature or staining of tiny cellular constituents.
The red–brown color combination is composed ofHRPactivitywith diaminobenzidine tetrahydrochloride(DAB) in brown and AP activity with Dako’s LPR inred. This chromogen combination ensures two sensitive/efficient enzymatic visualization procedures both witha crisp microscopical appearance. Nuclear counterstain
Figure 5 Mouse–mouse double staining, different IgG subclasses.
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with hematoxylin is optional. For observation with theunaided eye, this color combination has been appliedfor decades bymany investigators (Nakane 1968;Malikand Daymon 1982) and is used in several commerciallyavailable double staining kit systems. The red–browncolor combination combined with visual observationis only useful in showing two different cell populationsor constituents without any overlap. Visualization ofcolocalization, however, is not possible with this colorcombination because a visually distinct red–brownmixed color is lacking (van der Loos 1999).
Color Combinations for Spectral Imaging
Because of the ability of spectral imaging system toseparate chromogens that are visually nearly indis-tinguishable, the visual color of a reaction product isno longer important. Therefore, chromogens can beselected now for multiple staining that are sensitive/efficient, have a crisp localization pattern, and that canbe mounted organically, best fitting with paraffin tis-sue sections rather than for their visual assessmentproperties. The double staining protocols in this workare based on the brown–red color combination: HRPactivity in brown by an enhanced version of DAB(DAB1; Dako) and AP activity in red by LPR (Dako)with a nuclear counterstain in blue using hematoxylin.To show the power of spectral unmixing, it is shownhere that even quadruple IHC can be unmixed into
four individual images. Quadruple IHC is performedsequentially combining two double staining procedureswith a heat-induced epitope retrieval (HIER) step in be-tween for removing all immunoreagents from the firststaining sequence (Lan et al. 1995). In the second stain-ing sequence, AP activity in blue with Vector Blue andHRP activity in purple with Vector VIP (Vector) areused. Although the visual contrast after multiple stain-ing is rather poor and colocalization by a mixed color iseasily missed with the unaided eye, these enzymatic re-action products serve as an exquisite basis for analysisof multiple staining by spectral imaging.
Materials and Methods
Tissue samples (tonsil, rejected kidney transplant)were routinely fixed with buffered 4% formalin for atleast 48 hr and routinely processed to paraffin blocks.Five-mm sections were cut and mounted on coatedslides and dried overnight at 37C. Slides were dewaxedin xylene and hydrated using graded alcohols to tapwater. Endogenous peroxidase activity in formalin-fixed, paraffin-embedded (FFPE) sections was blockedwith 0.3% peroxide in methanol (20 min, room tem-perature). Ensuring a well-preserved tissue morphologyduring HIER, this step was performed in the PreTreat-ment Module (PTModule; Thermo Scientific/Lab-Vision, Fremont, CA) (Gray et al. 2006) for 20 min
Figure 6 Mouse–mouse double staining, combining high-sensitive tyramide amplification and low-sensitive indirect staining.
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at 98C with a cool-down to 65C. For optimizing thestaining results of all antibodies involved in double stain-ing, four HIER solutions were tested (Shi et al. 1997):citrate pH6.0, EDTApH8.0, Tris-HCl1 EDTApH9.0,and Tris-HCl pH 10.0 (Thermo Scientific/LabVision).After washing with running tap water, a non-serumproteinblockwasapplied for15minat roomtemperature(Ultra V Block; Thermo Scientific/LabVision).
Fresh tonsil tissue was snap-frozen in liquid nitrogenand stored at 280C. Five-mm cryostat tissue sectionswere dried overnight at room temperature, fixed withcold acetone (10 min), and air dried (2 min). Endog-enous peroxidase activity in cryostat tissue sections wasblocked with Tris-HCl–buffered saline (TBS) 1 0.3%peroxide and 0.1% sodium azide (10 min, room tem-perature) (Li et al. 1987). After washing with TBS, anon-serum protein block was applied for 15 min atroom temperature (Ultra V Block; Thermo Scientific/LabVision). Primary antibodies used in this study arelisted in Table 1. All other immunoreagents fitting intothe general protocols in Figures 1–8 are listed in Table 2.
Antibodies and conjugates were diluted in TBS 1
1% BSA (Sigma, St. Louis, MO), and TBS was used aswashing buffer for all further steps (three times for3 min). From here on, a double staining protocol in-cluding enzymatic visualization was performed asdescribed under Double Staining Protocols and sche-
matically represented in Figures 1–8. Double stainedspecimens were counterstained using 1:10 diluted hema-toxylin (in tap water) ensuring a moderate, but essen-tially transparent nuclear staining. Specimens were driedon a hot plate (50C) and organically (without alcohol orxylene) coverslipped with VectaMount (Vector).
Specimens were observed with a Leica BM5000microscope (Leica Microsystems; Wetzlar, Germany)with a Nuance VIS-FL Multispectral Imaging System(Cambridge Research Instrumentation; Woburn, MA)connected using a standard 13 c-mount. Spectra wereacquired from 460 to 660 nm at 10-nm intervals.Nuance software version 2.4 was used for analysis.
Double Staining Protocols
The protocol for a double staining procedure is highlydependent on the primary antibody combination withrespect to animal species, Ig isotype, mouse IgG subclass,or direct labeling. Usually investigators perform singlestaining first and then select the best antibody combi-nations for double staining. This strategy means that adouble staining protocol needs to be composed of pri-maries thathavebeenoptimized for single stainingbefore.Consequently, a double staining procedure has to be de-signed based on the characteristics of the primary anti-bodies. The following section discusses the advantages,disadvantages, and problems with regularly encountered
Figure 7 Mouse–mouse/FITC indirect-direct multistep double staining.
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primary combinations of mouse–rabbit, mouse–goat,mouse–mouse, and rabbit–rabbit.
Mouse–Rabbit Combination
The mouse–rabbit primary antibody combination isthe easiest double staining procedure to perform tech-nically. A cocktail of both primary antibodies and thenboth secondary antibodies followed by the subsequentvisualization of AP activity and HRP activity makes ita short and user-friendly procedure. In the fall of 2007,the MultiVision kit system for staining a rabbit–mouseprimary antibody combination including red and bluechromogens was introduced by Thermo Scientific/LabVision. Many double staining combinations canbe composed of any mouse monoclonal antibody com-bined with either a classical rabbit polyclonal antibodyor one of the recently introduced commercially avail-able rabbit monoclonal antibodies (Rossi et al. 2005).A biotin-free detection system is applied using singlespecies anti-mouse and anti-rabbit polymers attachedwith either HRP or AP enzymes (Sabattini et al. 1998).Because all anti-mouse, anti-rabbit polymers contain asecondary antibody of goat origin, there is no inter-species cross-reaction problem. Figure 1 shows a gen-eral protocol and schematic diagram. The step numbers
in the schematic diagram refer to the step number inthe protocol.
Mouse–Goat Combination
Basically, the combination of mouse and goat primaryantibodies is similar to the mouse–rabbit situation: twoprimaries of different species. When no problems withendogenous biotin are expected and the higher stainingsensitivity/efficiency of polymers is not needed, one mayuse traditional conjugates raised in donkey as second-ary antibodies against mouse or goat directly labeledwith AP, HRP, or biotin (Jackson; West Grove, PA)(Figure 2). Care should be taken to block endogenousbiotin when using biotin-conjugated antibodies (Woodand Warnke 1981). However, blocking of endogenousbiotin in some tissues (liver, kidney, spleen) after HIERusing Tris-EDTA pH 9.0 may be unsuccessful (Vybergand Nielsen 1998). A special problem is encounteredwhen a biotin-free polymer detection system is neededfor more sensitivity/efficiency. Because all anti-mousepolymers contain an antibody raised in goat, cross-reaction with the anti-goat detection system is very ob-vious.Alternatively,amultistepapproachwithablockingstep in between can be applied successfully (Figure 3).In this multistep protocol, HRP activity can be developed
Figure 8 Mouse–mouse/Animal Research Kit (ARK)-biotin indirect-direct multistep double staining after ARK-biotin labeling.
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after step 3 (option 1) or as a last chromogen step(option 2). Option 2 is preferred, but this may lead to aweak or even negative HRP staining result.
To date, there are anti-goat polymers available,but it is undisclosed what species the anti-goat anti-body is raised in (Invitrogen/Zymed; Carlsbad, CAand BioCare; Concord, CA). Without the proper knowl-edge of the host, these polymers may cross-react un-expectedly with other species involved in the stainingsequence and are therefore not useful for this type ofdouble staining.
Mouse–Mouse or Rabbit–Rabbit Combinations
Because many antibodies are of murine monoclonalorigin, it is obvious that a mouse–mouse combination
is regularly needed. Several successful double stainingprocedures have been published dealing with the mainproblem of cross-reaction between the two anti-mousedetection systems.
1. Sequential double staining combining two unlabeledmouse or rabbit antibodies in two separate detec-tion sequences (Figure 4). To prevent cross-reactionbetween the two staining sequences, investigatorsinitially removed the immunoreagents from the firststaining sequence by low pH buffers. This stepleaves the colored reaction product intact (Nakane1968). Several methods have been suggested for thisremoval (extreme pH buffers, high salt, electric cur-rent), but none of them was successful in removinghigh-affinity primary antibodies (Tramu et al. 1978).
Figures 9–10
Figure 9 Spectra tab from the Nuance software (version 2.4) showing the individual spectra of Liquid Permanent Red (LPR)-strong (red), LPR-weak (magenta), diaminobenzidine tetrahydrochloride (DAB)-strong (green), DAB-weak (yellow), and hematoxylin (blue) from 460 to 660 nm.Note the distinct difference between the spectra of DAB-strong reaction product and DAB-weak, whereas there is minimal difference betweenLPR-strong and LPR-weak.
Figure 10 Spectra tab from the Nuance software (version 2.4) showing the individual spectra of LPR (red), DAB (green), Vector Blue (yellow),and Vector VIP (blue) from 460 to 660 nm.
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Subsequently, it was shown that cross-reaction be-tween reagents used in the first and second stainingsequence is prevented by using DAB as chromogenafter the fist staining sequence. It is claimed thatthe brown DAB reaction product is the only knownchromogen that effectively covers immunoreagentsfrom the first staining sequence and therefore pre-vents cross-reactions (Sternberger and Joseph 1979).However, in case both antigens are in close proxim-ity, the effective sheltering by the DAB reactionproduct may also shelter the second antigen as well(Valnes and Brandtzaeg 1984). Titration of the firstprimary antibody has proven to be an important keyfactor for the successful performance of sequentialdouble staining. A too high concentration of the firstprimary antibody may result in incomplete shelter-ing by DAB reaction product, leading to unwantedcross-reactions with the second staining sequence(van der Loos, unpublished data). Generally, se-quential double staining is applied for the identifica-tion of two different cell types or cell constituents. Itis not recommended for the observation of colocali-zation by mixed colors. Control experiments are in-dispensable for the safe performance of this type ofdouble staining.
2. Sequential double staining with a HIER step in be-tween the two staining sequences is described by Lanet al. (1995). This heating step has been proven tobe effective even for the removal of high-affinityprimary antibodies and can be combined with anytype of chromogen that survives the heating step.This enables chromogen combinations of one’s ownchoice and may circumvent the use of DAB.
3. Indirect/indirect protocol based on two mousemonoclonal primary antibodies of different Ig type(IgG–IgM) or different IgG subclass (IgG1, IgG2a,IgG2b, IgG3; Figure 5) (Tidman et al. 1981).Because nearly 90% of all mouse monoclonal anti-bodies are of IgG1 subclass, this indirect/indirectdouble staining protocol can be only rarely applied.High-quality mouse IgG isotype–specific second
step reagents labeled with different enzymes, fluoro-chromes, or biotin can be found at Southern BiotechAssociates (Birmingham, AL). As with indirect/indirect double staining using antibodies of differentanimal species, this type of double staining can alsobe performed by applying a cocktail of two primaryantibodies and a cocktail of two secondary anti-bodies. To date, no polymers are available for thistype of detection.
4. Performance of a first unlabeled primary antibodyat a very high dilution followed by the highly sen-sitive/efficient tyramide amplification and a secondunlabeled primary antibody visualized by a rela-tively low-sensitive/efficient IHC detection protocol.The high dilution of the first primary prohibits cross-reaction with the second detection system. If anycomponent of the second detection system attachesto components of the first detection system, it re-mains below detection level and will not visible.This method was first described with fluorochromes(Brouns et al. 2002) and recently with enzymaticmarkers (van der Loos 2006) (Figure 6). Obviously,control experiments are needed for the safe in-terpretation of results obtained with this type ofdouble staining.
5. Indirect/direct multistep protocol combining an un-labeledmouseprimaryantibodywith a secondmouseprimary antibody that is directly conjugated (fluoro-chrome, hapten, biotin, or enzyme). The multistepprocedure starts with the unlabeled primary anti-body, for example, detected with an enzyme-labeledanti-mouse polymer. Next, a normal mouse serumblocking step is applied to saturate all anti-mousebinding sites. After the directly conjugated secondmouse primary antibody, subsequent detection isperformed with an (indirect) enzyme labeled anti-fluorochrome or hapten or enzyme-labeled strepta-vidin (Figure 7) (van der Loos et al. 1989; van derLoos 1999). As such, directly labeled primary anti-bodies are commercially available mainly for fluo-rescent cell sorter systempurposes, andmanyof those
’
Figures 11–12
Figure 11 Microscopic detail of a hyperplastic tonsil (human) showing double immunoenzyme staining with CD3 (rabbit monoclonal SP7) andFoxp3 (mouse clone 236A/E7) antibodies on a formalin-fixed, paraffin-embedded (FFPE) tissue section. Indirect/indirect double stainingprotocol is based on primary antibodies of different animal species (Figure 1). (A) RGB image of the original FFPE tissue section showing CD3in brown (DAB1), Foxp3 in red (LPR), and nuclear counterstain in blue (hematoxylin). After unmixing with spectral imaging, a compositefluorescent-like image in pseudo-colors clearly shows the presence of Foxp-3 (red)-positive nuclei in the CD3 (green) T cells (detail in insertboxed area in B). (C–E) Individual layers after unmixing; CD3 (C), Foxp-3 (D), and hematoxylin (E). Bar 5 0.1 mm.
Figure 12 Microscopic detail of a rejected kidney transplant (human) showing double immunoenzyme staining with CD8 (mouse clone C8/144, IgG1) and Granzyme B-7 (mouse clone GrB-7, IgG2a) antibodies on an FFPE tissue section. Indirect/indirect double staining protocol isbased onmouse IgG subclass difference (Figure 5). (A) RGB image of the original FFPE tissue section showing CD8 in brown (DAB1), GrB-7 in red(LPR), and nuclear counterstain in blue (hematoxylin). After unmixing with spectral imaging, a composite fluorescent-like image in pseudo-colors clearly shows the colocalization of GrB-7–positive granules (red) with the CD8 (green) T cells (arrows), identifying these cells showingcolocalization as cytotoxic T cells (B). GrB-7 single-positive cells are natural killer cells. (C–E) Individual layers after unmixing; CD8 (C), GrB-7 (D),and hematoxylin (E). Bar 5 0.1 mm.
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fluorochrome conjugates may also work in IHC.Applicability of this multistep double staining proce-dure has been valuable in several publications fromour group (Naruko et al. 1996; de Boer et al. 1997;Hosono et al. 2003).
6. Whenever no directly conjugated mouse primaryantibody is available, one may adopt the AnimalResearch Kit (ARK) in vitro labeling system. TheARK provides a biotinylated anti-mouse IgG Fab-fragment (biotinylation reagent) that is in vitro mixedwith the primary antibody (15 min, room tempera-ture). After blocking the unbound biotinylationreagent with normal mouse IgG (5 min, room tem-perature), a ready-to-use biotinylated primary anti-body can be applied in the multistep indirect/directprotocol as described above (van der Loos and Gobel2000) (Figure 8). Because the ARK contains an anti-mouse Fab fragment, this option is not available forprimaries raised in other species.
Expanding to Triple or Quadruple IHC
Any combination of suitable double staining com-binations described above can be applied to set upquadruple IHC. A first staining sequence ends withdevelopment of HRP activity with DAB1 in brown andAP activity with LPR in red. To remove all immuno-reagents involved with the first staining sequence,the specimens were subjected to a second HIER treat-ment (Lan et al. 1995) using a buffer of choice best fit-ting the second pair of primary antibodies. Obviously,this second HIER treatment only fits with FFPE sec-tions and not with acetone-fixed cryostat tissue sec-tions. A HIER treatment of 10 min at 98C was foundto be sufficient to remove all antibodies. RemainingAP activity does not survive the second HIER treat-ment, and remaining HRP activity was blocked with
3% peroxide in TBS. The second double stainingprocedure involved the staining of AP activity in blue(Vector Blue) and HRP activity in purple (Vector VIP),in that order. For triple IHC staining, only VectorVIP is applied. Control experiments to monitor po-tential cross-reaction between the two double stainingprocedures consisted of a complete quadruple IHCstaining procedure but omitting the second pair of pri-mary antibodies.
Results
After double staining, the brown and red reactionproducts from HRP (DAB1) and AP (LPR) activitiesdid not contrast visually. Colocalization by mixed coloris exceptionally difficult to recognize. Control experi-ments replacing one or both primary antibodies by non-immune Ig of the same species, Ig isotype, IgG subclass,and concentration yielded consistent negative results.These “half double staining” specimens served as a con-trol sample for obtaining the individual spectra by theNuance spectral imaging system. The individual spectraof DAB1, LPR, and hematoxylin from 460 to 660 nmacquired from these single stained samples and usedas a spectral library are depicted in Figures 9 and 10.After loading this spectral library, the Nuance softwareis used to unmix spectral data cubes acquired fromdouble stained specimens into three individual mono-chrome grayscale images, each of which shows thedistribution and abundance of one of the chromogens.A pseudo-colored composite image is depicted in a sim-ulated fluorescence mode for optimal visual contrast.As such, this unmixing was performed with three anti-body combinations plus a nuclear counterstain as de-picted in Figures 11–13.
Expanding double IHC staining into quadruplestaining can be performed using LPR and DAB1 inthe first staining sequence, followed by a second HIER
’
Figures 13–14
Figure 13 Microscopic detail of hyperplastic tonsil (human) showing a double immunoenzyme staining with CD25, interleukin-2 receptor(mouse clone ACT-1), and FITC-conjugated CD3 (mouse monoclonal SK7) on an acetone-fixed cryostat tissue section. Indirect/direct multistepdouble staining protocol is based on one unlabeled primary and one FITC-conjugated primary antibody (Figure 7). (A) RGB image of theoriginal tissue section showing T cells with CD3 in brown (DAB1) and CD25 in red (LPR). After unmixing with spectral imaging, a compositefluorescent-like image in pseudo-colors clearly shows the presence of activated T cells marked by CD3–CD25 colocalization (red and greenmerged to yellow). Resting T cells remain single stained (green), and a macrophage subpopulation remains single stained (red). (C) CD3 T cellsand (D) CD25 show the individual layers after unmixing. (E) Resulting image of a Boolean “and–operation” with C and D images. Note that thisimage shows exclusively CD3–CD25 colocalization similar to the yellow cells in B. Bar 5 0.1 mm.
Figure 14 Microscopic detail of hyperplastic tonsil (human) showing a quadruple immunoenzyme staining with CD3 (rabbit monoclonal SP7),CD20 (mouse clone L26), CD68 (mouse clone PG-M1, IgG3), and CD34 (mouse clone QBend10, IgG1) antibodies on an FFPE tissue section. First,an indirect/indirect double staining protocol is performed based on primary antibodies of different animal species (Figure 1) with CD3 andCD20. Next, the section is subjected to a second heat-induced epitope retrieval procedure to remove immunoreagents from the first doublestaining procedure. Subsequently, a second indirect/indirect double staining protocol is performed based on mouse IgG subclass difference(Figure 5) with CD68 and CD34. (A) RGB image of the original tissue section showing T cells with CD3 in brown (DAB1), B cells with CD20 in red(LPR), macrophages in blue (Vector Blue), and endothelium in purple (Vector VIP). Note the moderate staining intensity of the usedchromogens. After unmixing with spectral imaging, a composite fluorescent-like image in pseudo-colors clearly shows the presence of T cells(green), B cells (red), macrophages (yellow), and endothelium (blue) (B). (C–F) Individual layers after unmixing. Bar 5 0.1 mm.
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step. Both LPR and DAB reaction products survive theheating step completely unchanged. After completingthe quadruple staining with Vector Blue and Vector VIPchromogens, the four reaction products can be spec-
trally unmixed using the spectral library depicted inFigure 10. From Figure 14, it is clear that quadruplestaining with CD3, CD20, CD68, and CD34 exclu-sively shows T cells, B cells, macrophages, and endo-
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Multiple Immunoenzyme Staining and Spectral Imaging 325
thelial cells, respectively. Spectral imaging does notreveal any overlap between these four antibodies as isnot expected for those four different cell types in ahyperplastic tonsil. Controls involving all steps of thequadruple IHC procedure with omission of the secondpair of primary antibodies did not yield any stainingwith Vector Blue and Vector VIP. As such, this indicatesno remaining AP or HRP activity and no cross-reactivitybetween the two double staining procedures.
Discussion
As expected, the brown–red color combination com-posed of the crisp reaction products fromHRP (DAB1)and AP (LPR) activity plus hematoxylin nuclear coun-terstain is not useful for the observationof colocalizationusing the unaided eye. However, this color combina-tion is a good basis for successful double stainingusing spectral imaging because the visual color contrastis no longer important. Based on the different spectralcharacteristics of both chromogens and the nuclearcounterstain, it is possible to spectrally unmix the orig-inal observation into three different images, each dis-playing the distribution and abundance of the individualchromogen. The software facilitates the visualization ofthese different images by creating a composite imagein different layers that can be shut on and off inde-pendently. In addition, this composite image can bedisplayed in a simulated fluorescence mode, which canaid visual assessment. This seems to be helpful for study-ing colocalization. In this context, it is also possible toshow colocalization using either the Multiple MarkerMolecular plug-in within the Nuance 2.4 software orimport two individual images after unmixing into ad-vanced imaging software (such as Image-Pro Plus,version 5.0; Media Cybernetics, Bethesda, MD) andperform a thresholded Boolean “AND-operation.” Theresulting new image (Figure 13E) exclusively displaysthose pixels that show colocalization, whereas singlestaining is unseen here (de Boer et al. 2007a).
The success of unmixing the DAB and LPR reactionproducts is dependent on the staining intensities of thecolored reaction products. An absolute prerequisite forsuccessful spectral imaging is a relative transparency (orlow optical density) of the colored reaction products.This is especially true for DAB, whose reaction productsuffers from the problem that it clogs at high stainingintensities, yielding a dark brown deposit. This darkbrown reaction product has different spectral charac-teristics compared with the transparent yellow–brownreaction product. For example, dark DAB deposits willbe “missed” after unmixing using a spectrum createdfrom a moderate yellow–brown DAB deposit. More-over, spectra from any dark chromogen deposit tendto be similar and will therefore “bleed” into other chro-mogen layers after spectral unmixing.
The brown DAB reaction product is not a trueabsorber of light, but a scatterer of light, and has a verybroad, featureless spectrum (Figure 9). This means thatDAB does not follow the Beer-Lambert law, whichdescribes the linear relationship between the concen-tration of a compound and its absorbance, or opticaldensity. As a consequence, darkly stained DAB has adifferent spectral shape than lightly stained DAB. Inaddition, this type of spectrum is sometimes hard todiscriminate from other spectra of light-scatteringmaterials using the Nuance software. For example,the spectrum of DAB is similar to that of melanin, andthe two can hardly be unmixed (van der Loos,unpublished data). In contrast, the LPR reactionproduct is a true absorber of light and follows theBeer-Lambert law.
Having different spectra at different staining inten-sities of the chromogen also creates a potential problemwith quantification of the immunostaining results. Inthis respect, the DAB chromogen is less suitable forquantification than, for example, LPR, which showsvery similar spectra at high and low staining intensities(Figure 9), as is expected from a compound that followsthe Beer-Lambert law.
The concerns about the DAB reaction productmentioned above certainly do not exclude DAB froma double staining application unmixed by spectralimaging. Its crisp reaction product is ideal for a goodmicroscopic resolution, and dark brown staining in-tensities can be simple avoided by diluting the primaryantibody of interest. Furthermore, it is observed thatother HRP chromogens (3-amino-9-ethylcarbazole,Vector NovaRed) share similar problems as seenwith the DAB reaction product (van der Loos, unpub-lished data).
In this study, three different chromogens (DAB, LPR,hematoxylin) could simply be unmixed by spectralimaging. This therefore raises the question of whetherspectral unmixing would work for combinations offour or five colors. Because the enzymatic reaction prod-ucts are visualized one by one, layering of more chro-mogens may lead to a generalized “darkening” of thespecimen. As such, this does not fit with the conceptdescribed above that spectral imaging works ideallywith transparent chromogens. However, surprisingly,the first tests with quadruple IHC, composed of LPRand DAB1 (in the first sequence) and Vector Blue andVector VIP (in the second sequence) showed that spec-tral unmixing is feasible when moderate staining inten-sities of the individual chromogens have been applied.However, the set-up of triple and quadruple IHC stain-ing protocols inherits many technical problems (van derLoos 1999). Most promising seems a sequential multi-staining technique in which the immunoreagents fromthe first double staining procedure are removed by aHIER step (Lan et al. 1995; Van den Brink et al. 2000)
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and continue with either single staining (for triple IHC)or another double staining procedure (for quadrupleIHC). Figure 14 shows that, although antibodies fromthe same species are involved in the first and seconddouble staining sequence, no overlapping staining pat-terns were found because of cross-reactivity.
In conclusion, spectral imaging is a true asset for theanalysis of immunoenzyme multiple staining specimens.Whenever chromogens are applied ensuring moderatestaining intensities, multiple staining can be perfectly un-mixed in individual TIF images. These individual imagescan be subjected to standard imaging procedures and co-localization can be exclusively visualized using a Boolean“AND” operation. Good multiple staining methods, upto fourdifferentmarkers, in concertwith spectral imaginganalysis, opens the way to understand the more complexrelationships in all kinds of cellular processes.
Acknowledgments
The author thanks Alton D. Floyd, PhD (Edwardsburg,MI), and Jim R. Mansfield (CRi, Woburn, MA) for helpfulsuggestions and critical reading of this manuscript.
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