Ultrasmall immunogold particles: Important probes for immunocytochemistry

Ultrasmall Immunogold Particles: ImportantProbes for ImmunocytochemistryJOHN M. ROBINSON,1* TOSHIHIRO TAKIZAWA,2 DALE D. VANDRE,1 AND RICHARD W. BURRY1

1Department of Cell Biology, Neurobiology, and Anatomy, Ohio State University, Columbus, Ohio2Department of Anatomy, Jichi Medical School, Tochigi, Japan

KEY WORDS immunogold; immunocytochemistry; Nanogold; FluoroNanogold; correlative mi-croscopy; microtubules; neutrophils

ABSTRACT In this article, we review the immunocytochemical literature with respect to acomparison between conventional colloidal gold and ultrasmall gold particles as immunoprobes. Wediscuss the relative advantages and disadvantages of each of these types of particles forimmunocytochemical applications. We present results from our own laboratories, in which wecompared these immunoprobes in selected experimental situations. In addition, we discuss our workon the use of a fluorescently labeled ultrasmall immunoprobe for correlative microscopy. Microsc.Res. Tech. 42:13–23, 1998. r 1998 Wiley-Liss, Inc.

INTRODUCTIONThe fields of immunocytochemistry and immunohisto-

chemistry comprise a large body of methods and tech-nologies whose purpose is to obtain spatial and tempo-ral information about biological samples with a highdegree of chemical specification. This chemical specific-ity is derived from the innate specificity of the antigen–antibody interaction. When coupled with the highdegree of topological resolution of optical and electronmicroscopes, it provides unique information that can-not be readily gained with biochemical, immunochemi-cal, or morphological methods alone. The application ofthese methodologies has been extremely important incell and developmental biology, as well as in immunopa-thology.

The major differences in immunocytochemical meth-ods are: 1) type of sample preparation (i.e., preembed-ding or postembedding) and 2) type of detection system(i.e., chromogenic, fluorochromes, or particulate). Thedemonstration of antibody binding to its antigen re-quires a reporter system. That is, antibodies must belabeled in some manner for detection of binding to anappropriate target antigen. Indeed, immunocytochem-istry as a field can be understood, to a large extent, bytracing the history of the development of these reportersystems. The primary reporter systems currently em-ployed in immunocytochemistry are fluorochromes, en-zymes (chromogenic and fluorochrome detection), andparticulate probes.

In situ immunolabeling has an interesting history.Immunocytochemistry was introduced by Coons andassociates (1942), who developed fluorochrome-labeledprimary antibodies (i.e., direct-labeling). Fluorochrome-labeled secondary antibodies (i.e., indirect-labeling)were subsequently introduced (Weller and Coons, 1954).Enzyme-labeled antibody techniques were developed inorder to amplify the immunocytochemical signal bytaking advantage of the catalytic property of horserad-ish peroxidase, which was used as the enzyme label inthese studies (Nakane and Pierce, 1966). Subsequently,bridge techniques were developed for further amplifica-

tion in the enzyme-labeled technique (Mason et al.,1969; Sternberger and Cullis, 1969; Sternberger et al.,1970). The use of the iron-containing protein ferritin asan electron-dense marker represents the introductionof ‘‘particulate probes’’ and electron microscopy to immu-nocytochemistry (Singer, 1959). This was followed bythe introduction of colloidal gold as an electron-denseimmunoprobe (Faulk and Taylor, 1971). Subsequently,gold-labeled secondary antibodies were developed (Ro-mano et al., 1974). Methods for the indirect detection ofantigen–antibody binding were further bolstered withthe advent of staphylococcal protein A-gold probes(Romano and Romano, 1977; Roth et al., 1978). Each ofthese labeling systems (i.e., fluorochromes, enzymes,and particulate probes) has its set of appropriate appli-cations and is in use today.

Independent of the labeling system employed, thereare certain conditions that should be met before success-ful immunocytochemistry can be achieved. These in-clude: 1) preservation of immunoreactivity; 2) retentionof antigens in the proper location; 3) retention ofmorphological detail (especially at the ultrastructurallevel); 4) retention of equal accessibility of antibodies toantigen molecules at different locations within thespecimen; and 5) ability to carry out multiple labeling ofdifferent antigens in the same sample (Opins et al.,1994). The extent to which these conditions are metdepends on the biological specimen and antigen(s)under investigation. For example, the immunoreactiv-ity of some antigens is quite resistant to chemicalfixation, while others are very sensitive. This may wellbe the limiting factor in any immunocytochemicalexperiment. If immunoreactivity of the antigen(s) ofinterest is diminished by fixation, then morphologicaldetail and perhaps even retention of antigens in theproper place may be compromised by having to resort to

*Correspondence to: John M. Robinson, Department of Cell Biology, Neurobiol-ogy, and Anatomy, Ohio State University, 4072 Graves Hall, 333 W. 10th Ave.,Columbus, OH 43210.

Received 20 November 1997; Accepted 12 December 1997

MICROSCOPY RESEARCH AND TECHNIQUE 42:13–23 (1998)

r 1998 WILEY-LISS, INC.

incomplete fixation conditions. Likewise, some samplefixation and embedment protocols may inhibit theaccessibility of the antibody to the target antigen.Therefore, one must seek satisfactory compromises tomeet these sometimes conflicting requirements for sam-ple preservation and antigen recognition.

A primary consideration in all immunocytochemicalprocedures is that of proper control experiments. Thisis important so that one can prevent and identifyspurious immunolocalization so that only the genuineantigen distribution is revealed. The range of controlexperiments that can be applied to samples includes: 1)omission of the primary antibody, 2) substitution of theprimary antibody, 3) absorption of the primary anti-body with the target antigen, 4) removal of the antigen,and 5) use of positive controls (for review, see Stirling,1993).

PREEMBEDDING VS. POSTEMBEDDINGIMMUNOCYTOCHEMISTRY

Simply put, preembedding immunocytochemistry re-fers to the situation where antibodies are applied to thesample prior to any kind of embedment or sectioning.Postembedding immunocytochemistry refers to the situ-ation where the sample is embedded and sectioned;antibodies are applied to the sections.

Preembedding immunocytochemistry at the ultra-structural level has often relied on the use of enzyme-labeled antibodies. Localization of antigen–antibodybinding can be detected by the precipitation of achromogen at the site of enzyme activity. The chromo-gen must have inherent electron density or the capacityto be converted to an electron-dense product to beuseful for ultrastructural studies. Horseradish peroxi-dase-labeled antibodies with diaminobenzidine as thechromogen have been of primary importance in thisregard. An advantage of this approach is that theenzyme-labeled antibodies penetrate into cells follow-ing relatively mild detergent permeabilization condi-tions. In addition, the sensitivity of detection of anti-gens is high since the catalytic activity of the enzymecan generate large amounts of chromogen precipitate. Adisadvantage associated with this methodology relatesto the possibility of reaction product drift (Courtnoy etal., 1983). When this occurs, precision of localizationdiminishes.

Postembedding immunocytochemistry uses particu-late probes that give a high level of resolution to thelabeled structures. Colloidal gold has been the probe ofchoice in most postembedding procedures at the ultra-structural level. The disadvantage of this approach,when compared to enzyme-linked antibodies, is a lossin sensitivity. Ultrastructural-level postembedding im-munocytochemistry can be carried out on ultrathincryosections of sucrose-embedded material and plastic-embedded samples. An advantage of these approachesis that intracellular antigens can be detected withoutthe need for detergent permeabilization.

COLLOIDAL GOLD ASIMMUNOCYTOCHEMICAL PROBES

The use of colloidal gold has revolutionized immuno-cytochemistry, particularly at the ultrastructural level.The fact that these probes are discrete particles thatare extremely electron-dense contributes to their wide

acceptance in immunocytochemistry. Colloidal gold par-ticles are unstable in the presence of electrolytes;however, a number of different macromolecular specieswill bind to colloidal gold particles, causing them tobecome more hydrophylic and stable in the presence ofelectrolytes. The ability to bind various macromol-ecules to colloidal gold by means of noncovalent electro-static adsorption offers the opportunity to create differ-ent affinity probes, including immunoprobes (e.g.,Geoghegan and Ackerman, 1977). Another importantattribute of colloidal gold is that particles can readily begenerated in the laboratory in various sizes (e.g., Frens,1973). The introduction of methods for isolating uni-formly sized colloidal gold particles has greatly facili-tated their use for multiple-labeling immunocytochem-istry (Slot and Geuze, 1981, 1985; Wang et al., 1985).Colloidal gold immunoprobes have been applied to thelocalization of cell surface components in preembeddingimmunocytochemistry and to intracellular molecules inpostembedding procedures (see above). It should also berecognized that colloidal gold immunolabeling has beenhighly effective in the analysis of the cell surface byscanning electron microscopy (e.g., Erlandsen et al.,1993; for recent review, see Hermann et al., 1996). Amore detailed treatment of colloidal gold chemistry andlabeling procedures is beyond the scope of this article.However, these topics have been reviewed recently(e.g., Albrecht et al., 1992; Horisberger, 1992; Ben-dayan, 1995; Roth, 1996).

One of the most tantalizing possibilities presented bycolloidal gold as an immunoprobe has been that ofquantitative immunocytochemistry. The fact that theseprobes are discrete particles permits their quantifica-tion by particle counting. Thus, the labeling densityover a particular structure or region of a cell can bedetermined. However, whether truly quantitative data(i.e., determination of the number of antigen moleculesin a given cell volume) can be obtained using theseparticulate probes has been the subject of debate.

Few studies have specifically addressed the issue ofdirectly comparing the density of colloidal gold immuno-labeling to the concentration of a specific antigen, asdetermined by an independent method(s) (e.g., enzymeassay, immunochemical assay). Where such studieshave been done it was determined that there was not aone-to-one relationship between colloidal gold particlesand antigens (e.g., Griffiths and Hoppeler, 1986; Howellet al., 1987; Slot et al., 1989; Dulhunty et al., 1993). Instudies in which the immunolabeling was carried outon ultrathin cryosections, the labeling efficiency wastypically 10–15% or less (Hortsch et al., 1985; Griffithsand Hoppeler, 1986; Howell et al., 1987; Dulhunty etal., 1993). Immunolabeling of ultrathin frozen sectionslikely offers the most sensitive conditions for postembed-ding immunocytochemistry. Thus, labeling efficiencies,with colloidal gold immunoprobes below 20% mayrepresent the best results yet obtained. On the otherhand, correlation between immunogold labeling andaminopeptidase activity has been observed in prepara-tions of membrane vesicles (Hansen et al., 1992).However, in this case the absolute concentration ofantigen was not determined.

The major reasons for the relative inefficiency oflabeling with colloidal gold immunoprobes are 1) thedegree of penetration of antibody and gold complex into

14 J.M. ROBINSON ET AL.

the sample and 2) steric hindrance effects (e.g., Griffithsand Hoppeler, 1986; Howell et al., 1987; Slot et al.,1989; Horisberger, 1992; Takizawa and Robinson, 1994).The penetration of colloidal gold into ultrathin cryosec-tions may be complicated further, since it has beenreported that the ability of the antibody and goldcomplex to penetrate into a section can vary amongsubcellular compartments in the same cell (Griffithsand Hoppeler, 1986). Opins and colleagues (1994) com-pared various preparative procedures and plastic em-bedments in an attempt to enhance labeling efficiencyby equalizing the accessibility of antigens in the variouscellular matrices.

Another important consideration for quantitativecolloidal gold immunocytochemistry is the recognitionthat labeling efficiency varies with particle size. That is,use of smaller gold particles leads to greater labelingefficiency (i.e., 5 nm . 10 nm . 15 nm). This ruleappears to hold under most, if not all, labeling condi-tions. Literature related to this topic is summarized inTable 1. Thus, even semiquantitative analysis is bestachieved with the smallest particles suitable for a givenapplication. Another important problem related to thiseffect is that of double or triple labeling with colloidalgold when particles of different sizes are used to detecttwo or three antigens in the same sample. In such casesthe larger gold particles would be less reflective of theantigen concentration than would the smaller particlesemployed. While this approach is very important fordetermining the distribution of antigens, its usefulnessfor quantitative analysis is limited.

Colloidal gold immunolabeling, like most techniquesand methodologies, has its limitations. Nonetheless, itsuse has provided unique information and has been amajor contributor to our understanding of cell structureand function.

ULTRASMALL GOLD IMMUNOPROBESThe recognition that enhanced labeling efficiency and

possibly greater penetration into samples are associ-ated with smaller immunogold particles has led investi-gators to develop ultrasmall gold probes. Another rea-son for the development of very small gold probesrelates to the need for high-resolution labeling systems(e.g., detection of single protein subunits within acomplex such as a viral capsoid).

Small colloidal gold particles in the range of 1–3 nmhave been prepared and used for immunocytochemicalapplications (Baschong et al., 1985; Baschong andWrigley, 1990; Chan et al., 1990; De Valck, 1991; Slotand Geuze, 1985; Van de Plas and Leunissen, 1993).

Another approach has been the use of metal clustercompounds (i.e., gold clusters) that are in the 0.8–1.4nm range as immunolabels (Hainfeld, 1987, 1988;Hainfeld and Furuya, 1992). Despite potential advan-tages associated with enhanced labeling and penetra-tion, ultrasmall probes have been used less frequentlythan the larger colloidal gold particles (i.e., $5 nm indiameter). A major reason for this is the fact that thesmallest of these gold particles (i.e., <1 nm) are essen-tially impossible to detect in routine cell or tissuepreparations by conventional transmission electronmicroscopy. However, they can be detected readily insimpler preparations, such as isolated macromolecules,and with sophisticated imaging equipment (e.g., dark-field scanning transmission electron microscopy) (Hain-feld and Furuya, 1992).

The practical application of ultrasmall immunogoldprobes requires that the particles be converted to alarger size for visualization. This is typically accom-plished by means of silver enhancement (see below fordiscussion of silver enhancement). To use ultrasmallgold and then render it larger may seem counterintui-tive. However, it serves as a means to take advantage ofthe most useful attributes of the ultrasmall gold (i.e.,potential for increased labeling efficiency and penetra-tion into samples). Examples of immunocytochemicalapplications from our own work with ultrasmall immu-nogold will serve to illustrate these points.

We used 1.4-nm Nanogoldt (NG) particles to localizemarker proteins to cytoplasmic granules of humanneutrophils in ultrathin cryosections (Takizawa andRobinson, 1994). The localization of lactoferrin, a markerfor the so-called specific granules, compared favorablyto that observed with colloidal gold probes (Fig. 1). Asnoted earlier, silver enhancement was required todetect NG. In these experiments, we used the silverenhancement technique developed by Burry and associ-ates (see below). This procedure, which was carried outat pH 6, did not perturb the ultrastructural appearanceof the delicate ultrathin cryosections. Moreover, thisprocedure was gentle enough that a second antigencould be detected with colloidal gold following silverenhancement of NG (Fig. 2). Thus, NG and silverenhancement can be used in concert with colloidal goldin double-labeling experiments.

One of the major premises behind the uses of ul-trasmall gold particles has been that there will beincreased penetration of these probes into specimenscompared to colloidal gold. We tested this directly bylocalizing lactoferrin in ‘‘thick’’ cryosections (1–2 µm) offixed neutrophils with NG, 5 nm, and 10-nm colloidal

TABLE 1. Summary of studies showing that the size of immunogold can influence labeling density

Biological material Molecule detectedSize of gold

(nm) Reference

Pituitary Growth hormone 5, 15, 40 Lackie et al. (1985)Liver Catalase 5, 12, 18, 26, 38 Yokota (1988)Heart Atrial natriuretic peptide 5, 15, 30, 40 Gu and D’Andrea (1989)Pancreas Amylase 5, 15 Ghitescu and Bendayan (1990)Pancreas Amylase 5, 10 Slot and Geuze (1981)Intestine Aminopeptidase 5, 10 Hansen et al. (1992)Skeletal muscle Calcium ATPase 1, 5, 10 Dulhunty et al. (1993)Platelets WGA-binding sites 32, 50 Horisberger (1981)S. aureus Protein A 6, 20 Kehle and Herzog (1987)Neutrophils Lactoferrin 5, 10, 15 Takizawa and Robinson (1994)

15ULTRASMALL IMMUNOGOLD PARTICLES

gold (Takizawa and Robinson, 1994). We found that10-nm gold was restricted to the cut surface of specificgranules. Labeling with 5-nm gold was more extensivethan with 10-nm gold, but it, too, was primarily at thecut surface, with little label in the granule matrix. On

the other hand, labeling with NG occurred throughoutthe matrix of specific granules. In addition, specificgranules were labeled in the entire specimen, not justat the cut surface (Fig. 3). A summary of these results isdiagrammed in Figure 4.

We compared the labeling efficiency of NG to commer-cially available 1 nm colloidal gold for the localization ofmitotic microtubules (Vandre and Burry, 1992). It wasfound that labeling with NG was more intensive thanwith the 1-nm gold. The reason for this discrepancyappears to be due to dissociation of the 1-nm gold fromantibodies so that unbound antibodies compete withgold-associated antibodies, thus reducing the signalfrom the gold (Fig. 5). Proteins, such as the secondaryantibody used in this case, bind to colloidal gold byadsorption. Antibodies are, therefore, more susceptibleto loss from the colloidal gold particles than from NG,since the association with NG is through a covalentlinkage.

Fig. 1. Localization of lactoferrin in the specific granules of humanneutrophils in ultrathin cryosections by electron microscopy. (A)Lactoferrin was detected in the specific granules with 15-nm IgGcolloidal gold as secondary antibody (arrowheads). Lactoferrin-negative granules (*) are also evident; these are probably azurophilgranules. (B) Lactoferrin was detected with 1.4-nm NG-Fab as second-ary antibody followed by silver enhancement of the gold (arrowheads);negative granules (*) are also evident. (C) Control sample incubated asin (B) except the primary antibody was omitted; note the absence oflabeling. For details concerning methodology, see Takizawa andRobinson (1994). Bar 5 0.5 µm.

Fig. 2. Localization of complement decay accelerating factor (DAF)in ultrathin cryosections of quiescent human neutrophils. (A) Lowmagnification electron micrograph showing that, in resting neutro-phils, DAF exists primarily in intracellular compartments. (B) Highermagnification electron micrograph in which DAF was localized tosmall granule-like structures with 1.4-nm gold followed by silverenhancement (single arrows). Lactoferrin was localized in the specificgranules on the same section with 15-nm colloidal gold (doublearrows). Granules devoid of DAF or lactoferrin labeling are present (*).This illustrates that 1.4-nm gold with silver enhancement and colloi-dal gold can be used together for double-labeling purposes. For detailsconcerning methodology, see Takizawa and Robinson (1994). Bar 51.0 µm.


The advantages and disadvantages of antibody-labeled NG compared to colloidal gold are summarizedbelow. The advantages of NG are:

1. They are uniform in size since they are discretecompounds (1.4-nm gold core).

2. They can be coupled to various macromolecules.When coupled to Fab’ fragments they are among thesmallest particulate probes available.

3. They are covalent conjugates and more stable thanIgG-labeled colloidal gold probes.

4. They are not prone to aggregation.5. They can be chromatographically purified so that

single gold-antibody conjugates can be isolated.6. They can penetrate further into cells and tissues

than larger colloidal gold particles.7. They can be used for either light or electron micros-

copy by adjusting the time of silver enhancement.8. They can be conjugated with fluorochromes to gener-

ate unique probes for correlative microscopy.

The disadvantages of NG are:

1. They require silver enhancement for routine visual-ization.

2. The size of the particles is not uniform followingsilver enhancement.

CORRELATIVE MICROSCOPY ANDULTRASMALL GOLD PROBES

Correlative microscopy refers to situations in which asingle sample is examined by two or more imagingtechniques (e.g., fluorescence and electron microscopy).Observing multiple structures or molecules within asingle sample with the same imaging technique (e.g.,fluorescence microscopy) can also be considered correla-tive microscopy. Such an integrated approach to micros-copy can be a useful experimental tool. Moreover,correlative microscopy can provide additional insightinto biological questions not gained by a single imagingprocedure.

Immunoprobes labeled with fluorescent or particu-late markers represent two of the most powerful re-porter systems available for immunocytochemistry. Par-

Fig. 3. Comparison of the degree of penetration of three different-sized immunogold probes into paraformaldehyde-fixed and cryosec-tioned neutrophils. The cryosections were 1–2 µm in thickness.Following immunolabeling, the samples were embedded in Epon; thinsections were cut and stained with heavy metals. For experimentaldetails, see Takizawa and Robinson (1994). (A) Portion of a cryosec-tioned neutrophil in which localization of lactoferrin was determinedwith 1.4-nm gold-Fab followed by silver enhancement. Lactoferrin-positive granules are detected throughout the thickness of the section(arrows) in addition to those at the cut surface (arrowheads). (B)Portion of a cryosectioned neutrophil in which the localization oflactoferrin was determined with 5-nm gold-IgG. The granules displayheavy labeling at the cut surface (arrows); however, granules awayfrom the cut surface remain unlabeled. (C) Portion of a cryosectionedneutrophil in which localization of lactoferrin was determined with10-nm gold-IgG. Labeling is restricted to the cut surface (arrowheads),with little if any penetration of the marker into the granules. Note alsothat the labeling density achieved with 10-nm gold is less than with5-nm gold. Figure reproduced with permission from T. Takizawa andJ.M. Robinson (1994). Bar 5 0.1 µm.

Fig. 4. Diagram summarizing our results comparing the penetra-tion of various sized immunogold probes into ‘‘thick’’ cryosections.Lactoferrin was localized as in Figure 3. The oval structures representspecific granules in neutrophils. They are either at the cut surface orthe interior portion of the cryosections. The dots represent theimmunogold particles; their distribution indicates the degree of pen-etration into the sections. Labeling with 5- and 10-nm colloidal goldwas restricted to specific granules at the cut surface; there was agreater degree of labeling with 5-nm gold compared to 10-nm gold. Thelabeling with 1.4-nm gold was present in specific granules throughoutthe sections; penetration of the Fab-labeled probe appeared somewhatgreater than that of the IgG-labeled probe.


ticulate immunoprobes permit high-resolution detectionof molecules at the ultrastructural level. Fluorescentimmunoprobes offer the advantages afforded by high-resolution optical and confocal microscopes. In addi-tion, the availability of numerous fluorochromes withdifferent spectral properties is important for multiplelabeling and correlative microscopy.

The marriage of fluorochromes to colloidal gold wouldappear to represent an ideal probe for correlativemicroscopy. However, there are few reports of fluores-cent-colloidal gold immunoprobes. Roth and associates(1980) successfully prepared a 20-nm protein A-fluorescein complex; however, this approach to probedesign has not become a generally applied method. Thereason for the paucity of such probes appears to be dueto the diminished or complete loss of the fluorescencesignal resulting from the association of fluorochromesand proteins with colloidal gold (Goodman et al., 1991;

Powell et al., 1997). As a result, alternative methodshave been developed to couple fluorescence and electronmicroscopy. Colloidal gold particles have been conju-gated to fluorescent latex beads for neuronal tracttracing by light and electron microscopy (Quattrochi etal., 1987). While useful for that purpose, this probelacks the resolution and specificity required for exami-nation of single cell preparations. In other situations,fluorescent and gold immunoprobes have been used inthe same sample (e.g., Van Dongen et al., 1985; Sun etal., 1995). However, in these cases separate probes wereused (i.e., the gold immunoprobe was not labeled with afluorochrome).

Fluorescence photooxidation of diaminobenzidine(DAB) represents another approach to correlate fluores-cence and electron microscopy (Maranto, 1982). In thisprocedure, DAB is present in the cell or tissue sectionwhen a specific fluorochrome is excited by light at the

Fig. 5. Light micrographs comparing the labeling intensities ofspindle MTs by different ultrasmall gold-labeled antibody probes asdetected by silver enhancement and indirect immunofluorescence.LLC-PK cells were incubated with primary anti-tubulin antibodyfollowed by the secondary gold-labeled antibodies (A,B) AuroProbeOne or (B,C) Nanogold Fab’ (note that this was Nanogold, notFluoroNanogold). Either the gold on the secondary antibodies wasdetected directly by silver enhancement (A,C) or the secondaryantibodies themselves were detected by indirect immunofluorescence

with a tertiary fluorescein-conjugated antibody (B,D). The indirectimmunofluorescence labeling was similar for each secondary probe.However, the AuroProbe One sample did not show labeling compa-rable to the Nanogold labeling when detected by silver enhancement.The diagram summarizes the interpretation of these results for MTlabeling. These staining patterns indicate that unlabeled (i.e., lackinggold) secondary antibodies were present in the AuroProbe One sample;this does not appear to be the case for Nanogold. For details concern-ing methodology, see Vandre and Burry (1992). Bar 5 10 µm.


appropriate wavelength. DAB becomes oxidized in theprocess and precipitates (presumably this is mediatedby the free radicals generated during fluorescencedecay). With this procedure, localization can be achievedby both light and electron microscopy. Several differentfluorescent compounds have been used in this manner(e.g., Deernick et al., 1994; Lubke, 1993; Pagano et al.,1989; Sandell and Masland, 1988). While this has beena useful technique, there is a potential problem associ-ated with the use of DAB. In immunocytochemicalexperiments, DAB reaction product has drifted fromthe site of the antigen, even when the antigen was apoint source (Courtoy et al., 1983).

The ability to conjugate fluorochromes to NG is veryimportant for the development of unique immuno-probes. These reagents, called FluoroNanogoldt (FNG),are particularly useful for correlative microscopy. WithFNG, a single probe has a gold particle (metal cluster)and a fluorescent tag. For routine localization of FNGby electron microscopy, silver enhancement is required.While silver enhancement of FNG may be consideredsomewhat equivalent to photooxidation of DAB, thereshould be more precise localization with FNG, sincereaction product drift is less apt to occur than withDAB. In our experiments with FNG, we employed themicrotubules of human phagocytic leukocytes as themodel system. The microtubules of these leukocytes,particularly neutrophils, have been difficult to study byimmunocytochemical means. However, we developed apreparative procedure which permits the reliable detec-tion of microtubules in these cells by immunocytochem-istry (Ding et al., 1995). We employed this preparativeprocedure and FNG as the secondary antibody toexamine leukocyte microtubules by light and electronmicroscopy (Robinson and Vandre, 1997). In these

Fig. 6. Localization of microtubules in human neutrophils withFNG: detection with different imaging modalities in the opticalmicroscope. The same two neutrophils are shown in each panel. Afterlabeling the cells with FNG, the sample was incubated in silverenhancement solution for 2 minutes. The sample was then mounted ona glass slide and observed. For experimental details, see Robinson andVandre (1997). The same individual microtubules are marked aspoints of reference in each panel (arrowheads). A region at which athin cellular process is located is shown (open arrows). (A) Thefluorescence pattern derived from the immunolabeled microtubules. Itis noteworthy that the fluorescence signal is still present after thisperiod of silver enhancement. Longer periods of silver enhancementresult in loss of the fluorescence signal (not shown). (B) The brightfieldpattern of immunolabeling after silver enhancement. Individual micro-tubules are readily apparent and the detail shown in brightfield iscomparable to that of the fluorescence image. (C) The DIC image of thesilver-enhanced FNG. The microtubules are readily apparent and canbe examined in the context of the entire cell in the same image. Thislatter attribute is useful but cannot be achieved in the fluorescence orbrightfield images. After silver enhancement of the FNG-labeledtubulin, the microtubules were readily visualized with DIC opticswithout any form of electronic enhancement. The microtubules werenot visible under these conditions without silver enhancement of FNG.(D) The distribution of the silver-enhanced FNG when observed withphase-contrast optics. As with the DIC example, the microtubules canbe examined in the context of the entire cell in the same image. (E) Thedistribution of silver-enhanced FNG when examined by epipolariza-tion optics. In this case the areas of close association of the cell withthe substratum can be examined because they appear as dark areas atthe cell periphery and in the thin projection from the upper cell (openarrows). Microtubules are excluded from this cellular projection.Figure reproduced with permission from J.M. Robinson and D.D.Vandre (1997). Bar 5 10 µm.


studies, we found that the fluorescence signal fromFNG gave microtubule labeling comparable to conven-tional fluorescently labeled secondary antibodies. Micro-tubules were also readily detected with FNG followingthe silver enhancement procedure. It should be notedthat the silver enhancement conditions are very impor-tant. For example, the time of the silver enhancementreaction was critical. With short enhancement times,the fluorescent signal was retained even though silverdeposition was evident (Fig. 6). Longer silver enhance-ment time, which may be optimal for brightfield imag-ing of microtubules, leads to quenching of the fluores-cence signal. Prolonged silver enhancement times leadto high background signal, which degrades the immuno-cytochemical results (Robinson and Vandre, 1997).

These experiments were carried out on leukocytesthat were initially fixed (0.7% glutaraldehyde) andsubsequently permeabilized with detergent (for details,see Ding et al., 1995). Microtubules in these cells werereadily detected with FNG; on the other hand, 5-nmcolloidal gold failed to detect these cytoskeletal ele-ments (Fig. 7). However, 5-nm colloidal and FNG wereequally effective in labeling microtubules when cellswere permeabilized prior to fixation (Fig. 8). Theseresults provide direct evidence showing that ultrasmallgold probes penetrate into cells under conditions thatpreclude penetration of colloidal gold particles that are5 nm or greater in diameter.

We have shown the utility of FNG as a secondaryantibody for the immunocytochemical detection of micro-tubules with several modes of optical microscopy. Inaddition, the reflected light signal from the silver-enhanced FNG has been detected by confocal micros-copy (Robinson and Vandre, 1997). We have also foundthis probe well suited to the ultrastructural demonstra-tion of these cytoskeletal elements following silverenhancement (Fig. 9). These microtubules were in-tensely labeled, thus indicating the ready access of allthe labeling reagents. We herein present additionalevidence showing the high degree of penetration ofFNG. Tubulin within leukocyte centrioles was demon-strated using FNG as the secondary antibody (Fig. 10).It should be noted that this centriole labeling wasachieved by preembedding immunocytochemistry incells that were fixed prior to detergent permeabiliza-tion. To the best of our knowledge, tubulin within thesestructures has been refractory to immunolabeling withcolloidal gold in preembedding immunocytochemistry,even when cells were permeabilized prior to fixation(e.g., Geuens et al., 1986).

SILVER ENHANCEMENT OF IMMUNOGOLDAs noted above, routine detection of ultrasmall gold

particles requires silver enhancement. Methods for thedetection of gold within tissues were introduced byDanscher (1981) and later adapted for demonstration ofimmunogold (Holgate et al., 1983). The Danscher silver-enhancement solution was shown to be effective forultrasmall gold particles in preembedding immunocyto-chemistry (Lah et al., 1990). However, the ultrastruc-tural appearance of the cells were compromised follow-ing this procedure.

The original Danscher solution was acidic (pH 3.5).The acidity of the solution was thought to be one of thereasons for the poor ultrastructural detail in samples

subjected to silver enhancement. Subsequent work hasfocused on buffering the reaction at higher pH whilestill permitting controllable silver enhancement withdifferent reducing agents (Burry et al., 1992; Burry,1995; Gilerovitch et al., 1995). The silver enhancementsolution that we currently use has a final pH of 6.0,with n-propylgallate as a reducing agent, and results ingood cellular morphology following its use in preembed-ding immunocytochemistry (Burry, 1995).

SUMMARYImmunocytochemistry is a powerful approach for

obtaining site-specific information concerning the distri-

Fig. 7. Comparison of the labeling efficiency of FNG and 5-nmimmunogold particles in leukocytes at the light microscope level. Allcells are from the same cell preparation and were treated in exactlythe same manner (i.e., fixation, permeabilization, primary antibodyincubation, silver enhancement schedule) except for the size of thesecondary immunoprobes. For experimental details, see Robinson andVandre (1997). (A,B) The distribution of microtubules (arrowheads) inhuman monocytes as detected by FNG. The full complement ofmicrotubules is demonstrated with this technique. (C,D) The level ofmicrotubule detection achieved with two different commercial 5-nmimmunogold particles. There is little if any immunolabeling of themicrotubules (arrowheads) in these cells. (C’,D’) Companion DICimages of the same cells shown in C and D. Figure reproduced withpermission from J.M. Robinson and D.D. Vandre (1997). Bar 5 10 µm.


bution of molecules in biological material. Immunocyto-chemistry has been enriched by numerous modifica-tions that have been added to the basic concept ofvisualizing antigen–antibody interactions in situ. Thesemodifications relate, for the most part, to advances inspecimen preparation and reporter systems for thedetection of antigen–antibody interactions. In this re-view, we discuss gold cluster compounds conjugated tospecies-specific antibodies for use as secondary antibod-ies in indirect immunocytochemical studies. We presentadvantages and disadvantages to the use of theseprobes and compare them to colloidal gold immuno-probes. In addition, we discuss a recently describedfluorescently labeled gold cluster immunoprobe and its

application to correlative microscopy. Results pre-sented herein support our contention that these immu-noprobes are well suited to immunocytochemistry andoutperform colloidal gold in certain applications. These

Fig. 8. Comparison of the efficiency of FNG and colloidal goldparticles for immunolabeling of microtubules in LLC-PK cells at thelight microscope level: effect of the sequence of fixation and cellpermeabilization. For experimental details, see Robinson and Vandre(1997). (A,B) The distribution of microtubules in cells detected byfluorescence microscopy using FNG-Fab and FITC-IgG, respectively.In this case the samples were fixed before permeabilization. (C) Thedistribution of microtubules in an LLC-PK cell detected with FNG andsilver enhancement, as observed by phase-contrast optics. In this casethe sample was fixed before permeabilization. (D) Lack of labeling ofmicrotubules when 5-nm colloidal gold and silver enhancement wereused in samples fixed before permeabilization and imaged by phase-contrast microscopy. (E,F) The distribution of microtubules in LLC-PKcells detected by FNG and 5-nm colloidal gold, respectively. In thiscase the samples were permeabilized before fixation. The 5-nm goldefficiently labeled microtubules under these conditions when observedby phase-contrast microscopy. (G–I) The distribution of microtubulesand chromatin in a dividing LLC-PK cells. (G) Brightfield micrographof an LLC-PK cell during anaphase. (H) Fluorescence micrograph ofthe same cells shown in (G) that were labeled with DAPI to demon-strate DNA. (I) Simultaneous brightfield and fluorescence image of thesame cells shown in (G). The fluorescence micrograph of DAPI-stainedchromosomes demonstrates that this method is compatible with thesilver enhancement procedure. Note the lagging chromosome frag-ment (arrowheads) in H and I. Reproduced with permission from J.M.Robinson and D.D. Vandre (1997). Bar 5 10 µm.

Fig. 9. Localization of microtubules in a human monocyte withFNG and silver enhancement at the EM level. Low magnificationelectron micrograph of a thin section showing the distribution ofmicrotubules (arrowheads) within the cell. Inset. A higher magnifica-tion image of the microtubules shown between the brackets in thelower magnification image. Note the uniform labeling along theindividual microtubules. There is some cytoplasmic labeling awayfrom the microtubules (brackets). This is probably specific labelingand represents the pool of unpolymerized tubulin not extracted withthe fixation and permeabilization protocols used to prepare this cell.For experimental details, see Robinson and Vandre (1997). Bar 50.5 µm.


probes should prove to be useful in numerous applica-tions.

REFERENCESAlbrecht, R.M., Olorundare, O.E., Simmons, S.R., Loftus, J.C., and

Mosher, D.F. (1992) Use of correlative microscopy with colloidal goldlabeling to demonstrate platelet receptor distribution and move-ment. Methods Enzymol., 215:456–479.

Baschong, W., and Wrigley, N.G. (1990) Small colloidal gold conjugatedto Fab fragments or to immunoglobulin G as high-resolution labelsfor electron microscopy: A technical overview. J. Electron Microsc.Tech., 14:313–323.

Baschong, W., Lucocq, J.J., and Roth, J. (1985) ‘‘Thiocyanate gold’’:Small (2–3 nm) colloidal gold for affinity cytochemical labelling inelectron microscopy. Histochemistry, 83:409–411.

Bendayan, M. (1995) Colloidal gold post-embedding immunocytochem-istry. Prog. Histochem. Cytochem., 29:1–159.

Burry, R.W. (1995) Pre-embedding immunocytochemistry with silver-enhanced small gold particles. In: Immunogold-Silver Staining:Principles, Methods, and Applications. M.A. Hayat, ed. CRC Press,Boca Raton, FL, pp. 217–230.

Burry, R.W., Vandre, D.D., and Hayes, D.M. (1992) Silver enhance-ment of gold antibody probes in pre-embedding electron microscopicimmunocytochemistry. J. Histochem. Cytochem., 40:1849–1856.

Chan, J., Aoki, C., and Pickel, V.M. (1990) Optimization of differentialimmunogold-silver and peroxidase labeling with maintenance ofultrastructure in brain sections before plastic embedding. J. Neuro-sci. Methods, 33:113–127.

Coons, A.H., Creech, H.J., Jones, R.N., and Berliner, E. (1942) Thedemonstration of pneumococcal antigen in tissues by the use offluorescent antibody. J. Immunol., 45:159–170.

Courtoy, P.J., Picton, D.H., and Farquhar, M.G. (1983) Resolution andlimitations of the immunoperoxidase procedure in the localization ofextracellular matrix antigens. J. Histochem. Cytochem., 31:945–951.

Danscher, G. (1981) Localization of gold in biological tissue. Photo-chemical method for light and electron microscopy. Histochemistry,71:81–88.

Deernick, T.J., Martone, M.E., Lev-Ram, V., Green, D.P.L., Tsien, R.Y.,Spector, D.L., Huang, S., and Ellisman, M.H. (1994) Fluorescencephotooxidation with eosin: A method for high resolution immunolo-

calization and in situ hybridization detection for light and electronmicroscopy. J. Cell Biol., 126:901–910.

De Valck, V., Renmans, W., Segers, E., Leunissen, J., and De Waele, M.(1991) Light microscopical detection of leukocyte cell surface anti-gens with a one-nanometer gold probe. Histochemistry, 95:483–490.

Ding, M., Robinson, J.M., Behrens, B.C., and Vandre, D.D. (1995) Themicrotubule cytoskeleton in human phagocytic leukocytes is ahighly dynamic structure. Eur. J. Cell Biol., 66:234–245.

Dulhunty, A.F., Junankar, P.A., and Stanhope, C. (1993) Immunogoldlabeling of calcium ATPase in sarcoplasmic reticulum of skeletalmuscle: Use of 1-nm, 5-nm, and 10-nm gold. J. Histochem. Cyto-chem., 41:1459–1466.

Erlandsen, S.L., Hasslen, S.R., and Nelson, R.D. (1993) Detection andspatial distribution of the b2 integrin (mac-1) and L-slectin(LECAM-1) adherence receptors on human neutrophils by highresolution field emission SEM. J. Histochem. Cytochem., 41:327–333.

Faulk, W.P., and Taylor, G.M. (1971) An immunocolloid method for theelectron microscope. Immunochemistry, 8:1081–1083.

Frens, G. (1973) Controlled nucleation for the regulation of theparticle size in monodisperse gold suspension. Nature (Phys. Sci.),241:20–22.

Geoghegan, W.D., and Ackerman, G.A. (1977) Adsorption of horserad-ish peroxidase, ovomucoid and anti-immunoglobulin to colloidalgold for the indirect detection of conconavalin A, wheat germagglutinin and goat anti-human immunoglobulin G on cell surfacesat the electron microscopic level: A new method, theory and applica-tion. J. Histochem. Cytochem., 25:1187–1200.

Geuens, G., Gundersen, G.G., Nuydens, R., Cornelissen, F., Bulinski,J.C., and deBrabander, M. (1986) Ultrastructural colocalization oftyrosinated and detyrosinated a-tubulin in interphase and mitoticcells. J. Cell Biol., 103:1883–1893.

Ghitescu, L., and Bendayan, M. (1990) Immunolabeling efficiency ofprotein A-gold complexes. J. Histochem. Cytochem., 38:1523–1530.

Gilerovitch, H.G., Bishop, G.A., King, J.S., and Burry, R.D. (1995) Theuse of electron microscopic immunocytochemistry with silver-enhanced 1.4-nm gold particles to localize GAD in the cerebellarnuclei. J. Histochem. Cytochem., 43:337–343.

Goodman, S.L., Park, K., and Albrecht, R.M. (1991) A correlativeapproach to colloidal gold labeling with video-enhanced light micros-copy, low voltage scanning electron microscopy, and high-voltageelectron microscopy. In: Colloidal Gold: Principles, Methods, andApplications. Vol. 3. M.A. Hyatt, ed. Academic Press, San Diego, pp.369–409.

Griffiths, G., and Hoppeler, H. (1986) Quantitation in immunocyto-chemistry: Correlation of immunogold labeling to absolute numberof membrane antigens. J. Histochem. Cytochem., 34:1389–1398.

Gu, J., and D’Andrea, M. (1989) Comparison of detecting sensitivenessof different sizes of gold particles with electron-microscopic immuno-gold staining using atrial natriuretic peptide in rat atria as a model.Am. J. Anat., 185:264–270.

Hainfeld, J.F. (1987) A small gold-conjugated antibody label: Improvedresolution for electron microscopy. Science, 263:450–453.

Hainfeld, J.F. (1988) Gold cluster-labeled antibodies. Nature, 333:281–282.

Hainfeld, J.F., and Furuya, F.R. (1992) A 1.4-nm gold cluster cova-lently attached to antibodies improves immunolabeling. J. Histo-chem. Cytochem., 40:177–184.

Hansen, G.H., Wetterberg, L-L., Sjostrom, H., and Noren, O. (1992)Immunogold labeling is a quantitative method as demonstrated bystudies on aminopeptidase N in microvillar membrane vesicles.Histochem. J., 24:132–136.

Hermann, R., Walther, P., and Muller, M. (1996) Immunogold labelingin scanning electron microscopy. Histochem. Cell Biol., 106:31–39.

Holgate, C.S., Jackson, P.J., Cowen, P.N., and Bird, C.C. (1983)Immunogold-silver staining: New method of immunostaining withenhanced sensitivity. J. Histochem. Cytochem., 31:938–944.

Horisberger, M. (1981) Colloidal gold: A cytochemical marker for lightand fluorescence microscopy and transmission electron microscopy.Scan. Electron Microsc., 2:9–31.

Horisberger, M. (1992) Colloidal gold and its application in cell biology.Int. Rev. Cytol., 136:227–287.

Hortsch, M., Griffiths, G., and Meyer, D.I. (1985) Restriction of dockingprotein to the rough endoplasmic reticulum: Immunocytochemicallocalization in rat liver. Eur. J. Cell Biol., 38:271–279.

Howell, K.E., Reuter-Carlson, U., Devaney, E., Luzio, J.P., and Fuller,S.D. (1987) One antigen, one gold? A quantitative analysis ofimmunogold labeling of plasma membrane 58-nucleotidase in frozenthin sections. Eur. J. Cell Biol., 44:318–327.

Kehle, T., and Herzog, V. (1987) Interactions between protein-gold

Fig. 10. Localization of tubulin within centrioles of human neutro-phils with FNG and silver enhancement. Cells prepared in the samemanner as in Figure 9. (A) A centriole pair in which the lower one wascut in cross-section while the upper one was cut transversely. Notethat each half of the centriole is heavily labeled for tubulin. Eachcentriole pair is known to have nine sets of triplet microtubules in abarrel-like arrangement. Three rows of triplet microtubules areevident in the transversely sectioned centriole barrel (arrows). (B)Higher magnification of a cross-sectioned centriole. The nine sets ofmicrotubules are evident (numbers). This pattern of labeling is anexquisite example demonstrating the high degree of penetrationachieved with FNG. Labeling of tubulin within centrioles has not beenreadily detected with colloidal gold probes in preembedding immuno-cytochemistry. Bar 5 0.5 µm.


complexes and cell surfaces: A method for precise quantitation. Eur.J. Cell Biol., 45:80–87.

Lackie, P.M., Hennessy, R.J., Hacker, G.W., and Polak, J.M. (1985)Investigation of immunogold-silver staining by electron microscopy.Histochemistry, 83:545–550.

Lah, J.J., Hayes, D.M., and Burry, R.W. (1990) A neutral pH silverdevelopment method for the visualization of 1 nanometer goldparticles in pre-embedding electron microscopic immunocytochemis-try. J. Histochem. Cytochem., 38:503–508.

Lubke, J. (1993) Photoconversion of diaminobenzidine with differentfluorescent neuronal markers into a light and electron microscopicproduct. Microsc. Res. Tech., 24:2–14.

Maranto, A.R. (1982) Neuronal mapping: A photooxidation reactionmakes Lucifer yellow useful for electron microscopy. Science, 217:953–955.

Mason, T.C., Phifer, R.F., Spicer, S.S., Swallow, R.A., and Deskin, R.B.(1969) An immunoglobulin-enzyme bridge method for localizingtissue antigens. J. Histochem. Cytochem., 17:563–569.

Nakane, P.K., and Pierce, G.B. Jr. (1966) Enzyme-labeled antibodies:Preparation and application for the localization of antigens. J.Histochem. Cytochem., 14:929–931.

Opins, A., Geuze, H.J., and Slot, J.W. (1994) Cryosubstitution dehydra-tion of aldehyde-fixed tissue: A favorable approach to quantitativeimmunocytochemistry. J. Histochem. Cytochem., 42:497–503.

Pagano, R.E., Sepanski, M.A., and Martin, O.C. (1989) Moleculartrapping of fluorescent ceramide analogue at the Golgi apparatus offixed cells: Interaction with endogenous lipids provides a trans-Golgi marker for both light and electron microscopy. J. Cell Biol.,109:2067–2079.

Powell, R.D., Halsey, C.M.R., Spector, D.L., Kaurin, S.L., McCann, J.,and Hainfeld, J.F. (1997) A covalent fluorescent-gold immunoprobe:Simultaneous detection of a pre-mRNA splicing factor by light andelectron microscopy. J. Histochem. Cytochem., 45:947–956.

Quattrochi, J.J., Madison, R., Sidman, R.L., and Kljavin, I. (1987)Colloidal gold fluorescent microspheres: A new retrograde markervisualized by light and electron microscopy. Exp. Neurol., 96:219–224.

Robinson, J.M., and Vandre, D.D. (1997) Efficient immunocytochemi-cal labeling of leukocyte microtubules with FluoroNanogold: Animportant tool for correlative microscopy. J. Histochem. Cytochem.,45:631–642.

Romano, E.L., and Romano, M. (1977) Staphylococcal protein A boundto colloidal gold: A useful reagent to label antigen-antibody sites inelectron microscopy. Immunochemistry, 14:711–715.

Romano, E.L., Stolinski, C., and Hughes-Jones, N.C. (1974) Anantiglobulin reagent labeled with colloidal gold for use in electronmicroscopy. Immunochemistry, 11:521–522.

Roth, J. (1996) The silver anniversary of gold: 25 years of the colloidalgold marker system for immunocytochemistry and histochemistry.Histochem. Cell Biol., 106:1–8.

Roth, J., Bendayan, M., and Orci, L. (1978) Ultrastructural localiza-tion of intracellular antigens by use of protein A-gold complex. J.Histochem. Cytochem., 26:1074–1081.

Roth, J., Bendayan, M., and Orci, L. (1980) FITC-protein A-goldcomplex for light and electron microscopic immunocytochemistry. J.Histochem. Cytochem., 28:55–57.

Sandell, J.H., and Masland, R.H. (1988) Photoconversion of somefluorescent markers to a diaminobenzidine product. J. Histochem.Cytochem., 36:555–559.

Singer, S.J. (1959) Preparation of an electron dense antibody conju-gate. Nature, 183:1523–1524.

Slot, J.W., and Geuze, H.J. (1981) Sizing of protein A-colloidal goldprobes for immunoelectron microscopy. J. Cell Biol., 90:533–536.

Slot, J.W., and Geuze, H.J. (1985) A new method of preparing goldprobes for multiple-labeling cytochemistry. Eur. J. Cell Biol., 38:87–93.

Slot, J.W., Posthuma, G., Chang, L-Y., Crapo, J.D., and Geuze, H.J.(1989) Quantitative aspects of immunogold labeling in embeddedand in nonembedded sections. Am. J. Anat., 185:271–281.

Sternberger, L.A., and Cuculis, J.J. (1969) Method for enzymaticintensification of the immunocytochemical reaction without use oflabeled antibodies. J. Histochem. Cytochem., 17:190.

Sternberger, L.A., Hardy, P.H. Jr., Cuculis, J.J., and Meyer, H.G.(1970) The unlabeled antibody enzyme method of immunohistochem-istry. Preparation and properties of soluble antigen-antibody com-plex (horseradish peroxidase-antihorseradish peroxidase) and itsuse in identification of spirochetes. J. Histochem. Cytochem., 18:315–333.

Stirling, J.W. (1993) Controls for immunogold labeling. J. Histochem.Cytochem., 41:1869–1870.

Sun, X.J., Tolbert, L.P., and Hildebrand, J.G. (1995) Using laserscanning confocal microscopy as a guide for electron microscopicstudy: A simple method for correlation of light and electron micros-copy. J. Histochem. Cytochem., 43:329–335.

Takizawa, T., and Robinson, J.M. (1994) Use of 1.4-nm immunogoldparticles for immunocytochemistry on ultra-thin cryosections. J.Histochem. Cytochem., 42:1615–1623.

Van de Plas, P., and Leunissen, J.L.M. (1993) Ultrasmall gold probes:Characteristics and use in immuno(cyto)chemical studies. MethodsCell Biol., 37:241–257.

Van Dongen, J.J., Hooijkaas, H., Comans-Bitter, W.M., Benne, K., VanOs, T.M., and De Josselin de Jong, J. (1985) Triple immunologicalstaining with colloidal gold, fluorescein, and rhodamine as labels. J.Immunol. Methods, 80:1–6.

Vandre, D.D., and Burry, R.W. (1992) Immunoelectron microscopiclocalization of phosphoproteins associated with the mitotic spindle.J. Histochem. Cytochem., 40:1837–1847.

Wang, B-L., Scopsi, L., Nielsen, M.H., and Larsson, L.-I. (1985)Simplified purification and testing of colloidal gold probes. Histo-chemistry, 83:109–115.

Weller, T.H., and Coons, A.H. (1954) Fluorescent antibody studies withagents of varicella and herpes zoster propagated in vitro. Proc. Soc.Exp. Biol., 86:789–794.

Yokota, S. (1988) Effect of particle size on labeling density for catalasein protein A-gold immunocytochemistry. J. Histochem. Cytochem.,36:107–109.


Documents

Ultrasmall immunogold particles: Important probes for immunocytochemistry