Pre Embedding

Pre-Embedding Immunolabeling for Electron Microscopy:An Evaluation of Permeabilization Methods and MarkersBRUNO M. HUMBEL,* MARGO D.M. DE JONG, WALLY H. MULLER, AND ARIE J. VERKLEIJDepartment of Molecular Cell Biology, Institute for Biomembranes, Utrecht University, Utrecht, The Netherlands

KEY WORDS Brij; colloidal gold; diaminobenzidine; fluorochrome; peroxidase; Saponin; strepto-lysin; Triton; tyramide; ultrasmalls gold

ABSTRACT For scarce antigens or antigens which are embedded in a dense macromolecularstructure, on-section labeling, the first method of choice, is not always successful. Often, the antigencan be localized by immunofluorescence microscopy, usually by a pre-embedding labeling method.Most of these methods lead to loss of ultrastructural details and, hence, labeling at electronmicroscope resolution does not add essential information. The scope of this paper is to compare fivepermeabilization methods for pre-embedding labelling for electron microscopy. We aim for a methodthat is easy to use and suitable for routine investigations. For our ongoing work, special attention isgiven to labeling of the cell nucleus. Accessibility of cytoplasmic and nuclear antigens is monitoredwith a set of different marker antibodies.

From this investigation, we suggest that prefixation with formaldehyde/glutaraldehyde isnecessary to stabilize the ultrastructure before using a detergent (Triton X-100 or Brij 58) topermeabilize or remove the membranes. The experimental conditions for labeling should be checkedfirst with fluorescence or fluorescence-gold markers by fluorescence microscopy. Then eitherultrasmall gold particles (with or without fluorochrome) with silver enhancement or, if theultrasmall gold particles are obstructed, peroxidase markers are advised. The most promisingtechnique to localize scarce antigens with good contrast is the combination of a pre-embeddingperoxidase/tyramide-FITC or -biotin labeling followed by an on-section colloidal gold detection.Microsc. Res. Tech. 42:43–58, 1998. r 1998 Wiley-Liss, Inc.

INTRODUCTIONIn cell biology and biomedical sciences, immunolabel-

ing is an important tool to study the relationship ofstructure and function. For light microscopy, often apre-embedding immunolabeling method is followed ei-ther on permeabilized cells, thawed cryostat sections,rehydrated paraffin sections, or deplasticized methacry-late sections. This is true even for samples to beanalyzed by confocal (scanning laser) light microscopy.The location of the protein in question is visualizedeither by enzyme reaction products, e.g., peroxidase/diaminobenzidine, silver-enhanced gold particles, orfluorescent labels. In general, the fluorescence labelgives a more brilliant picture; often it also seems to bemore sensitive and is the method of choice for confocallight microscopy. The other markers have the advan-tage that the biological material can be counterstained,hence the label and the cellular structure can be seensimultaneously. Further, these preparations can bestored without fading of the label. Usually, the resultsof such pre-embedding techniques serve as the basis forsubsequent electron microscope investigations.

In electron microscopy, post-embedding immunolabel-ing methods, i.e., on-section labeling, are preferred. Foron-section labeling, the material can be prepared withoptimum preservation of the cellular ultrastructure if,e.g., preparation techniques based on cryofixation areused (Hohenberg et al., 1994; Humbel et al., 1983;Humbel and Muller, 1986; Menco, 1986; Muller andMoor, 1984; Robards and Sleytr, 1985; Steinbrecht and

Zierold, 1987; Studer et al., 1995). Gold markers arenow preferred for electron microscope localization stud-ies (Hayat, 1989–1991; Roth, 1989; Verkleij and Leunis-sen, 1989) because they are clearly distinguishablefrom biological structures.

Electron microscopic results, however, often do notreflect the label intensity expected from light micro-graphs. In pre-embedding methods, the structures areaccessible in three dimensions, whereas on sectionsonly the section surface is exposed to the antibodies(Stierhof et al., 1986). Further, fluorescent antibodiesseem to be more efficient in labeling than gold-taggedantibodies, even if the identical on-section preparationis used, e.g., cryosections (according to Tokuyasu, 1973,1986) either mounted on coverslips and labeled withfluorescent antibodies or mounted on grids and labeledwith colloidal gold particles (Humbel, unpublished re-sults). Figure 1 shows an example of ultrathin resinsections of Leishmania mexicana (Stierhof et al., 1991b)labeled for tubulin and either detected with a fluores-cent secondary antibody or the identical fluorescentantibody additionally tagged with ultrasmall gold par-ticles. The gold-tagged antibody exhibits a less intenseand a more patchy staining (Fig. 1: Humbel andStierhof, unpublished). The size of the gold particles

*Correspondence to: Bruno M. Humbel, Department of Molecular Cell Biology,Utrecht University, Padualaan 8, NL-3584 CH Utrecht, The Netherlands.E-mail: [email protected]

Received 18 March 1998; Accepted 1 April 1998

MICROSCOPY RESEARCH AND TECHNIQUE 42:43–58 (1998)

r 1998 WILEY-LISS, INC.

also seems to be important for labeling (Humbel andBiegelmann, 1992). The higher label density of ul-trasmall gold compared to larger gold particles sug-gests that the gold particles repulse each other. Thelarger the particles, the stronger the repulsion force.

In pre-embedding methods, the cells have to bepermeabilized either mechanically by sectioning orfreezing/thawing, or chemically by pore-forming pro-teins, detergents, or organic solvents to give the mark-

ers access to cytoplasmic or nuclear antigens. Duringpermeabilization processes the cellular membranes areperforated or completely removed and an uncontrollednumber of so-called soluble proteins may be redistrib-uted within the cell (Melan and Sluder, 1992) or lost.The consequence is a false labeling pattern or theprotein of interest escapes detection. On the otherhand, the loss of a part of the soluble proteins mayuncover an epitope of a structural protein resulting in apositive label. As an example, a lamin A/C antibody onlylabeled the nuclear lamina of Triton X-100 permeabi-lized cells (De Graaf et al., 1991) and not on Tokuyasucryosections (Tokuyasu, 1973, 1986) (Humbel, unpub-lished observations).

For in situ hybridization studies, three-dimensionalaccess to nucleic acids is very important. High copynucleic acids like 28S rRNA can clearly be labeled onsections (Sibon et al., 1995), whereas others are moredifficult or even impossible to detect, as, e.g., theEGF-receptor transcript (Sibon et al., 1994). Hence,although on-section labeling approaches are preferreddue to the superiority in preservation of the ultrastruc-ture, pre-embedding techniques are needed to locatescarce or masked antigens. In addition, pre-embeddinglabeling is the method of choice to study cellularinteractions in three dimensions by electron tomogra-phy (Koster et al., 1997; Mehlin et al., 1992).

Methods for pre-embedding immunolabeling arewidely used for light and electron microscopic investiga-tions, especially for biomedical studies. An example ofmechanical permeabilization is cryostat sectioning (Mat-suno et al., 1994; Yazama et al., 1997). During freezingof organs or pieces of tissue, ice crystals are formed,which disrupt the cell membranes. In light microscopy,Triton X-100 or Nonidet P40 are frequently used (DeGraaf et al., 1992; Langanger et al., 1984; Martone etal., 1996; Satijn et al., 1997; Sibon et al., 1994). Bothdetergents disrupt the bilayer structure by dissolvingthe lipids and membrane proteins (Helenius and Si-mons, 1975). For electron microscopy, less stringentdetergents such as Saponin are preferred (Andersson etal., 1990; Brown and Farquhar, 1989; Heuser, 1981;Louvard et al., 1982; Macville et al., 1995; Tanner et al.,1996). Saponin forms complexes with cholesterol thatleads to reversible pores in membranes (Brown andFarquhar, 1989). To avoid lipid removal by detergents,cells can be permeabilized by treatment with sodiumborohydride (Van Lookeren Campagne et al., 1992;Young and Furness, 1995).

A method to label ‘‘living’’ cells would be of specialinterest. Streptolysin, a cytolytic toxin of Streptococcuspyogenes, integrates into the cellular membranes andforms pores in vivo (Alouf, 1980). On ice, streptolysin-Owill bind to plasma membrane-associated cholesterol.Then excessive toxin is removed. At higher temperaturethe toxin-cholesterol complexes associate via lateraldiffusion to form oligomers. These oligomers form poresof about 35 nm in diameter (Leno et al., 1992). Thepores are large enough to allow free passage of cytoplas-mic proteins (Krijnse-Locker et al., 1994). Dundr andRaska (1993) applied the streptolysin method to labelactive transcription sites within the nucleolus. Theypermeabilized the plasma membrane of HeLa cells andadded bromouridine, which the RNA polymerases incor-porate into nascent RNA (Wansink et al., 1993).

Fig. 1. Ultrathin sections of Leishmania mexicana fixed with 2%(w/v) formaldehyde and 0.05% (v/v) glutaraldehyde and embedded inLowicryl HM20 (Stierhof et al., 1991b; gift of Dr. Y.-D. Stierhof) eithermounted on glass coverslips or grids, labeled for tubulin. The primaryantibody was detected with a goat anti-mouse FITC antibody (A) orwith the same antibody to which ultrasmall gold particles were bound(B,C). The labeling properties were imaged by fluorescence microscopy(A,B) or after silver enhancement by electron microscopy (C). Notethat the fluorescence signal of the Au-FITC antibody (B) is less intensethan of the identical FITC-antibody without gold tag (A). Further-more, the labeling pattern is more patchy, which correlates well withthe electron micrograph (C). These are unpublished results of Humbeland Stierhof (1997). A, B: Bar 5 10 µm; C: bar 5 1 µm.

44 B.M. HUMBEL ET AL.

We also considered the method published by Schliwaet al. (1981) for labeling ‘‘living’’ cells. They have shownthat, at least temporarily, the morphological integrityof cells can be maintained (Schliwa et al., 1981). After ashort Brij 58 treatment of living cells, only a fewso-called soluble proteins are lost (Schliwa et al., 1987)and even the loss of potassium ions is retarded in Brij58-opened cells (Kellermayer et al., 1986).

The scope of this article is to compare five permeabili-zation methods for pre-embedding labeling on theelectron microscope level based on: 1) pre-fixation/Triton X-100 (De Graaf et al., 1992); 2) Saponin (Mac-ville et al., 1995); 3) sodium borohydride (Van LookerenCampagne et al., 1992); 4) streptolysin-O (Dundr andRaska, 1993); and 5) Brij-opening (Schliwa et al., 1987).We assessed the accessibility of cytoplasmic and nuclearproteins (De Graaf, et al., 1991; Fu and Maniatis, 1990;Satijn, et al., 1997) for a set of different markerantibodies. The results should provide a basis forchoosing the most suitable method.

MATERIALS AND METHODSChemicals and Antibodies

Brij 58 (16004) was purchased from Fluka (FlukaChemicals, Buchs, Switzerland), Saponin (S-1252) andTriton X-100 (X-100) from Sigma (Sigma Chemical Co.,St. Louis, MO, USA) and streptolysin-O from Wellcome(Wellcome Laboratories, Beckenham, UK). Ethyleneglycol-bis(b-aminoethyl ether)N,N,N8,N8,-tetraaceticacid (EGTA; E-4378) was from Sigma, 2-amino-2-(hydroxymethyl)-1,3-propandiol (Tris; 708976) fromBoehringer (Boehringer Mannheim GmbH, Mannheim,Germany), piperazine-1,4-bis(2-ethanesulfonic acid)(PIPES; 10220) and 2-[4-(2-hydroxyethyl)-1-piperazi-nyl](2-ethanesulfonic acid) (HEPES; 1.10110.) both fromMerck (Merck, Darmstadt, Germany). Cell culture me-dia and plastic ware were purchased from Gibco BRL(Life Technologies, Breda, The Netherlands). All otherchemicals used were pro analysis grade from eitherMerck or Fluka.

The monoclonal mouse anti-a-tubulin antibodies werefrom Sigma (T-5168), supernatant of monoclonal mouseanti-SC-35 antibody from the group of Dr. T. Maniatis(Fu and Maniatis, 1990), the monoclonal mouse anti-AM88 was a gift of Dr. R van Driel (De Graaf et al.,1991), and the rabbit anti-polycomb antibodies (bmi/ring) were a gift from Drs. D. Satijn and A. Otte (Satijnet al., 1997). The rabbit anti-biotin antibodies werefrom Enzo (#43861; Enzo Diagnostics, Farmingdale,NY).

The following secondary antibodies were from Jack-son (Jackson Immunoresearch Laboratories, WestGrove, PA): goat anti-mouse-DTAF (dichlorotiazinylamino fluorescein; 11–015–044), goat anti-rabbit-FITC(Coons, 1956) (fluorescein isothiocyanate; 111–095–003), rabbit anti-mouse-peroxidase (Nakane and Pierce,1967) (315–035–044) and goat anti-rabbit-peroxidase(111–035–045). The ultrasmall gold particles, goat anti-mouse (880305) and goat anti-rabbit (880304) werefrom an experimental batch (Janssen Life SciencesProducts, Olen, Belgium, gift of Dr. J. Leunissen) andgoat anti-mouse Nanogold (#2001; Nanoprobe, StonyBrook, NY). The FITC-ultrasmall gold conjugated anti-bodies (Au-FITC) were from Nanoprobe (Powell et al.,1997) (goat anti-rabbit, #7004) and a custom-tailored

goat anti-mouse was a gift from Dr. J. Leunissen(Aurion, Wageningen, The Netherlands). All the othergold-tagged antibodies, goat anti-mouse 6 nm (GAMEM-6 nm), goat anti-rabbit 6 nm (GAR EM-6 nm), goatanti-mouse 10 nm (GAM EM-10 nm), and goat anti-rabbit 10 nm (GAR EM-10 nm), were purchased fromAurion. The FITC-tyramide and the biotin-tyramidewere a gift of R. van Gijlswijk and Dr. A. Raap.

Cell CultureOsteosarcoma cells (U2OS) were cultured in 75 cm2

cell culture flasks in Dulbecco’s modified Eagle’s me-dium (DMEM) supplemented with 10% fetal bovineserum in a 7% humidified CO2 atmosphere. For thelabeling experiments, the cells were seeded on 12-mmglass coverslips in 12-well culture dishes and grownovernight to 50–70% confluency. The cells were furthertreated as described below. If not stated otherwise, allincubations and washing steps were done in the 12-wellculture dishes at room temperature.

Permeabilization MethodsPrefixation and Triton X-100 Permeabilization.

The cells were rinsed twice with PBS (137 mM NaCl,2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH7.4) and fixed with 2% (w/v) formaldehyde in PBS (FAprefixed) for 15 minutes or with 2% (w/v) formaldehydeand 0.02% (v/v) glutaraldehyde in 200 mM HEPES (pH7.2; FA-GA prefixed) (Griffiths, 1993) for 30 minutes.After permeabilization with 0.5% (w/v) Triton X-100 inPBS for 5 minutes the cells were washed twice withPBS. In one experiment, Triton X-100 was replaced by0.2% (w/v) Brij 58.

Saponin Permeabilization (Macville et al., 1995).The coverslips were rinsed twice with PBS and fixedwith 1% (w/v) formaldehyde and 0.02% (v/v) glutaralde-hyde for 30 minutes. Permeabilization was done with0.1% (w/v) Saponin in PBS for 30 minutes.

Sodium Borohydride Permeabilization (VanLookeren Campagne et al., 1992). The cells wererinsed twice with PBS, then fixed with 4% (w/v) formal-dehyde in 0.1 M sodium cacodylate buffer, pH 7.6, for 30minutes at room temperature and permeabilized twicewith 0.1% (w/v) sodium borohydride in PBS for 5minutes at room temperature. In the experiment tocheck which of the components was responsible forperforating the plasma membrane, incubations weredone with either of the reactants sodium cacodylatebuffer, formaldehyde in sodium cacodylate buffer, orsodium borohydride in PBS. Membrane leakage wasmonitored with propidium iodide, a stain for nucleicacids (Tas and Westerneng, 1981).

Streptolysin-O Permeabilization (Dundr andRaska, 1993; Krijnse-Locker et al., 1994). Cellsgrown on coverslips were rinsed once with PBS andonce with SLO-buffer (25 mM HEPES, pH 7.0, 50 mMKCl, 5 mM MgCl2, 2 mM EGTA). Then they wereincubated with 1 U/ml streptolysin-O in SLO-buffersupplemented with a cocktail of protease inhibitors(Complete, Boehringer) and 7 mM mercaptoethanol for30 minutes at 37°C. Thereafter, the cells were fixed for30 minutes in 4% (w/v) formaldehyde at room tempera-ture.

45PRE-EMBEDDING IMMUNOLABELING FOR EM

Brij 58-Opening. Cells grown on coverslips werewashed twice with PBS and incubated for 5 minutes atroom temperature with 0.2% (w/v) Brij 58 in PHEMbuffer (60 mM PIPES, pH 6.9, 25 mM HEPES, 10 mMEGTA, 2 mM MgCl2) supplemented with the cocktail ofprotease inhibitors and 7 mM mercaptoethanol. Thecells were fixed with 4% (w/v) formaldehyde in PHEM-buffer for 30 minutes at room temperature (Schliwa etal., 1981).

In all experiments formaldehyde was freshly pre-pared by de-polymerizing para-formaldehyde.

ImmunolabelingThe permeabilized and fixed cells were washed twice

with PBS and incubated twice with PBG (PBS, 0.5%(w/v) bovine serum albumin, 0.045% (w/v) cold-waterfish gelatin (Birrell et al., 1987)) for 5 minutes to blockprotein binding sites. The coverslips were removedfrom the 12-well dishes and placed on a piece ofparafilm in a moist chamber. The diluted primaryantibodies mouse anti-tubulin (1:1,000), mouse anti-AM88 (1:1,000), mouse anti-SC35 (1:5), rabbit anti-bmi/ring (1:500), or PBG (as a control) were pipetted ontothe coverslips and incubated for 1 hour at room tempera-ture. The Saponin preparations were incubated for 2hours at 37°C. Then the coverslips were put back on the12-well plates and washed six times for at least 5minutes. The plates were rocked on a rotary shaker at150 rpm. The coverslips were put back on the parafilmand incubated with the secondary antibodies overnightin the moist chamber. Up to this point, 0.1% Saponinwas added to all the solutions of Saponin-permeabilizedcells. The coverslips were put back in the 12-well platesand washed four times with PBG and four times withPBS for at least 15 minutes each step on a rotaryshaker.

Visualization for Light MicroscopyA coverslip (24x50 mm) with on-grown living cells

was inverted on a glass slide and immediately photo-graphed. The FITC and Au-FITC labeled cells weredirectly mounted on glass slides with moviol (Rodriguezand Deinhardt, 1960) containing para-phenylene di-amine (Johnson and de C. Nogueira Araujo, 1981) andphotographed with a Leitz Orthoplan (Ernst LeitzWetzlar GmbH, Wetzlar, Germany) microscope using aPLANAPO 63/1.4 OEL PHACO 4 objective and the I3filter set (BP450–490, RKP510; LP515).

The gold-labeled cells and Au-FITC-labeled cells (af-ter being examined with fluorescence microscopy) werefixed with 1% (v/v) glutaraldehyde in PBS for 30minutes at room temperature. Silver enhancement wasdone according to the method of Danscher (1981, 1984;Stierhof et al., 1991a, 1995) for 40 minutes. The peroxi-dase-labeled cells were rinsed in 0.1 M Tris/HCl, pH8.8, and 10 mM imidazole and incubated in the samebuffer supplemented with 0.001% (v/v) H2O2 and 0.5mg/ml 3,38diaminobenzidine (DAB) for 20 minutes inthe dark (Macville et al., 1995). DAB can be stored inthe freezer as a stock solution of 100 mg/ml in dimethylformamid. Alternatively, peroxidase can also be devel-oped with FITC-tyramide or biotin-tyramide (Van Gijls-wijk et al., 1996, 1997) under the same conditions asDAB. Rinsing once with the buffer and once with PBSstopped the reaction. The coverslips with the gold and

peroxidase/DAB labeled cells were mounted on glassslides with moviol and photographed with a LeitzOrthoplan microscope in the bright field and phasecontrast mode. The FITC-tyramide labeled probes werephotographed in the fluorescence mode and, thereafter,together with the tyramide-biotin probes embedded inLowicryl HM20 (see below).

Electron MicroscopyAfter light microscopical investigations, the cover-

slips were soaked in PBS for 2–8 hours and removedfrom the glass slides. The peroxidase/DAB-labeledsamples were postfixed with 1% (v/v) glutaraldehyde inPBS and 1% (w/v) osmium tetroxide in double-distilledwater. After dehydration in a graded series of ethanolthe cells were embedded in Epon. For flat embedding,the bottom of BEEM capsules 3 (G362; PLANO, W.Plannet GmbH, Marburg, Germany) was cut off and thecapsules were put on the cell side of the coverslips.Overnight a thin layer of Epon was polymerized to sealthe capsules to the coverslips. After introducing asample label the capsules were filled with Epon andpolymerized for another 2 days. The dish containing thecoverslips was immersed in liquid nitrogen until crack-ing, then the capsules were broken off. Remaining glasswas removed with 40% (v/v) hydrofluoric acid.

For Lowicryl embedding (Carlemalm et al., 1982;Simon et al., 1987), after dehydration the cells wereinfiltrated with Lowicryl HM20 (80% (w/w) monomer,20% (w/w) cross-linker and 0.5% (w/w) initiator). Thecoverslips were placed on a glass slide wrapped inaluminum foil (Fig. 2). A BEEM capsule 3 was filledwith fresh Lowicryl and frozen in liquid nitrogen andput upside down onto the coverslip (Fig. 2). The glassslide was placed in a CS Auto freeze-substitutionapparatus (Reichert-Jung, Vienna, Austria) cooled to-40°C. Two hours later, after adaptation to the tempera-ture, the resin was polymerized under UV illuminationfor 1 day at -40°C and 1 day at room temperature.Thereafter, the capsules were removed from the glassslide by immersion in liquid nitrogen. Under theseconditions, the glass cracks and the Lowicryl blocks canbe lifted off. The biotin-tyramide polymer was detectedwith a rabbit anti-biotin antibody and an ultrasmallgold or 10 nm colloidal gold goat anti-rabbit antibody onultrathin Lowicryl sections.

Sections of 90 nm or 300 nm were cut parallel to thecells. Except for the peroxidase/DAB-labeled cells, sec-tions were stained with 2% (w/v) aqueous uranyl ac-etate and 0.4% (w/v) lead citrate (Venable and Cogge-shall, 1965) 10 minutes each. The sections wereexamined with an EM 420 electron microscope (PhilipsElectron Optics, Eindhoven, The Netherlands) at 60 kVacceleration voltage using a 20 µm aperture.

RESULTSStructural Preservation Evaluated

by Light MicroscopyThe structural appearance of the cells was evaluated

by phase contrast light microscopy and by the appear-ance of the labeled microtubules by fluorescence micros-copy.

The morphology of cells prefixed with formaldehydeor formaldehyde/glutaraldehyde and permeabilized withTriton X-100 (Fig. 4A,B) or after Saponin permeabiliza-


tion (Fig. 4C) did not change significantly compared toliving cells (Fig. 3). The organelles were smaller and thecytoskeleton was more distinct. The nucleoplasm of theFA prefixed/Triton X-100 (Fig. 4A) and the Saponin(Fig. 4C) permeabilized cells was slightly granular. Thevery sensitive microtubular network in the three prepa-

rations show a nice, smooth curvature, as expected (Fig.5A,B,C).

To establish which component of the sodium borohy-dride method (Van Lookeren Campagne et al., 1992)causes the permeabilization, cells grown on coverslipswere exposed to the individual components. Permeabili-zation was assessed by penetration of propidium iodide.Buffer-treated cells were impermeable (not shown).After formaldehyde fixation, some propidium iodidecould pass the membrane barrier, indicated by a lightlabeling of the nucleic acids (Fig. 6A). Only if followedby sodium borohydride treatment were the cells com-pletely permeable for the dye (Fig. 6B). Treatment withsodium borohydride without prefixation was so roughthat all the cells were removed from the coverslips (notshown). Hence, only the combination of fixation fol-lowed by sodium borohydride treatment led to permeabi-lized cells. These results indicate that borohydridetreatment opens the plasma membrane, but not formal-dehyde. Sodium borohydride permeabilized cells stillhave the contours of living cells but fewer organellesare visible and the cytoplasm has become granulararound the nucleus (Fig. 4D). It looks as if the cytoplas-

Fig. 2. (A) BEEM capsules were filled with fresh resin and frozenin liquid nitrogen so that the capsules could be safely turned upsidedown and placed with the open side on the coverslips. (B) For flatembedding into Lowicryl, the coverslips with resin-infiltrated cellswere placed on a glass slide, which is wrapped in a trough ofaluminium foil. (C) A bottom view of the polymerized sample on thecoverslip.

Fig. 3. Living cells photographed in phase contrast. Two micro-graphs showing the same group of cells, focused on the nuclei (A) andon the surface of the coverslip (B). Different features, such as rufflingcell membrane, vesicles, the nuclear envelope, nucleoli and largenuclear bodies, are clearly seen. Bar 5 20 µm.


mic structures have been dug up. The tubulin labelingshown in Figure 5D and labeling of the nuclear proteinSC-35 (not shown) demonstrate that the holes are largeenough to let antibodies pass, but permeabilization isnot equally efficient for different cells. Even in oneindividual cell, only a part of the tubulin network islabeled (Fig. 5D).

Most of the permeabilized cells at room temperaturewith streptolysin-O were collapsed (Fig. 4E). Only thesubpopulation of large flat cells seemed to have re-tained some nuclear structures. The microtubuli net-work appeared crumpled (Fig. 5E).

In some preparations of Brij 58-opened cells (Fig. 4F),the structural appearance is close to that of prefixedand Triton X-100 or Saponin-treated cells. In manycells, a halo around the nucleus could be observed,

suggesting that the cytoskeleton has come off the cellnucleus (Fig. 4F). This impression was confirmed byelectron microscopy (data not shown). The microtubu-lar network shows little sign of collapse (Fig. 5F): it stillextends through the whole cell, but the individualmicrotubules are wavy, rather than having a tautcurvature. In addition, the preparation is not easilyreproducible. From these results, we conclude thatprefixation is favorable, as it leads to more reproducibleresults.

Accessibility of Cytoplasmicand Nuclear Proteins

In Table 1, the labeling results of tubulin and thenuclear antigens are summarized. The cytoplasm andthe nucleus of cells fixed with formaldehyde and perme-

Fig. 4. Cells prepared using the different permeabilization methods were investigated by phasecontrast microscopy. (A) Formaldehyde and (B) formaldehyde/glutaraldehyde prefixation followed byTriton X-100, (C) Saponin, (D) sodium borohydride, (E) streptolysin-O, and (F) Brij 58-opened. Bar 520 µm.


abilized with Triton X-100 were easily accessible for theprimary antibodies and the marker antibodies. The 10nm gold probe showed a reduced labeling intensity. Thesame is true for cells fixed with formaldehyde andglutaraldehyde (Fig. 7). Only the larger gold probes, 6nm and 10 nm, were no longer able to access thenucleus (Fig. 7E,F). The microtubular network wasweakly labeled and, in the case of 10 nm colloidal goldparticles, the access of the network was clearly ham-pered.

Saponin-permeabilized cells gave essentially the sameresults in terms of tubulin labeling as described forTriton X-100. The access of the nucleus was clearlyreduced for FITC-tagged antibodies. Only in one casedid the peroxidase-tagged antibodies label a nuclear

antigen. All the gold-tagged antibodies were excludedfrom the nucleus.

From the labeling pattern obtained in sodium borohy-dride-permeabilized cells, we conclude that the cellmembrane of only about half of the cells is perforatedenough for antibody penetration. The labeling of themicrotubular network is more or less the same with allthe markers. Hence, if permeabilization is sufficient forantibodies to pass, it is also sufficient for the marker.Only FITC-tagged antibodies can penetrate the nucleusand localize nuclear proteins. Even then, only a fewcells showed a good labeling pattern; most, however, didnot label at all, supporting the notion that only abouthalf of the cells are permeable enough for antibodypenetration.

Fig. 5. Cells prepared using the different permeabilization methods were labeled for tubulin andvisualized with an FITC-tagged secondary antibody. (A) Formaldehyde and (B) formaldehyde/glutaraldehyde prefixation followed by Triton X-100, (C) Saponin, (D) sodium borohydride, (E) streptoly-sin-O, and (F) Brij 58-opened. Bar 5 20 µm.


In our hands, streptolysin-O treatment at 37°C had adeleterious effect on the cellular structure and, there-fore, the labeling was not further evaluated.

In Brij 58-opened cells all the markers label themicrotubular network. However, only FITC- and peroxi-dase-tagged antibodies can access nuclear proteins;but, all the gold probes were excluded.

From Table 1 some conclusions on the labeling prop-erties of the secondary antibodies can be drawn. FITC-tagged antibodies are obviously so small that they canaccess all the primary antibodies. Peroxidase-taggedantibodies, increasing in size by about 40 kD (Griffiths,1993), have a somewhat reduced penetration capacitycompared to FITC-tagged antibodies. Still, they aremore efficient than the ultrasmall gold probes. Thus, incritical cases peroxidase labels are preferred to ul-trasmall gold labels. The real cut-off, however, is at thelevel of the ultrasmall gold particles. The detectionefficiency of larger colloidal gold particles is clearlyinferior; therefore, they are not suited for pre-embed-ding labeling of nuclear proteins.

Ultrastructural Preservation and LabelingEvaluated by Electron Microscopy

In our hands, the most successful and reproduciblemethod is based on prefixation with formaldehyde/

glutaraldehyde (FA-GA prefixed) and permeabilizationwith Triton X-100 (Fig. 8A–C) or Brij 58 (Fig. 8D–F).The cellular organelles can still be recognized; hence, alabel relates to ultrastructural details. The cytoplasmappears to be riddled with holes after detergent treat-ment (Fig. 8B,E). The holes seem to be somewhat largerin regions with few organelles and very fine in the areaof mitochondria (Fig. 8A,B). In prefixed and Brij 58-treated cells, the holes also seem somewhat larger (Fig.8E, note also the inverted contrast of the mitochondriaand that the membranes of the cristae are still visible).The state of preservation varies from cell to cell; thus,extraction is not even for all the cells. The nucleoplasmof prefixed and Triton X-100-permeabilized cells be-comes grainy and the area of heterochromatin can nolonger be seen. Other features, such as nucleoli (Nu)with the fibrillar centers and nuclear bodies (Nb), arestill recognizable. The nucleoplasm of prefixed and Brij58-permeabilized cells is denser and seems better pre-served than that of Triton X-100-treated cells (Fig.8C,F). Again, the contrast is inverted, which rendersthe structural details less recognizable (Fig. 8F), e.g.,for heterochromatin. Histochemical staining tech-niques for DNA (Testillano et al., 1991) and RNA(Bernhard, 1969; Monneron and Bernhard, 1969) wouldbe needed. Structures such as nucleolus (Nu) withfibrillar centers and clusters of interchromatin gran-ules (Ig) can easily be recognized (Fig 8C,F).

Fig. 6. Cells were fixed with formaldehyde in cacodylate buffer (A)and treated with sodium borohydride (B). Permeabilization wasmonitored with propidium iodide. Bar 5 20 µm.

TABLE 1. Accessibility of cytoplasmic and nucleus proteins

Marker FITC PO Au-FITC US 6 nm 10 nm

Triton X-100 permeabilization (formaldehyde pre-fixed)cytoplasm

tubulin 111 11 11 11 11 1nucleus

AM88 111 11 11 11 11 1bmi/ring 111 11 11 11 11 1

Triton X-100 permeabilization (formaldehyde/glutaraldehyde pre-fixed)

cytoplasmtubulin 111 11 1 11 1 1/2

nucleusSC-35 11 11 1 11 2 2bmi/ring 11 11 1/2 1/2 2 2

Saponin permeabilizationcytoplasm

tubulin 111 11 1 11 1 1/2nucleus

SC-35 1 2 2 2 2 2bmi/ring 1/2 2 2 2 2 2

Sodium borohydride permeabilizationcytoplasm

tubulin 1/2 1/2 1/2 1/2 1/2 1/2nucleus

SC-35 1/2 1/2 2 2 2 2bmi/ring 1/2 2 2 2 2 2

Brij 58-openedcytoplasm

tubulin 111 11 11 11 11 11nucleus

SC-35 11 11 2 2 2 2bmi/ring 11 11 1 2 2 2

Cells were prepared according to the different permeabilization methods de-scribed in Materials and Methods. To evaluate the accessibility within the cells,one cytoplasmic antigen: tubulin; and three nuclear antigens: a nuclear matrixprotein AM88, the essential splicing factor SC-35, and a member of the polycombgroup bmi/ring; were labeled. The label efficiency of the different markers wasscored by light microscopy. 111 high label intensity, 11 good label efficiency, 1sufficient label efficiency, 1/2 debatable labeling, 2 no label.


A further proof for the denser nucleoplasm is shownin Figure 9. Cells prefixed with formaldehyde/glutaral-dehyde were permeabilized with Brij 58 and labeled forthe splicing factor SC-35. The label pattern could beestablished with FITC- (Fig. 9A) and peroxidase-taggedsecondary antibodies (Fig. 9B); however, not with theultrasmall gold particles (Fig. 9C). Again, the goldparticles prove to be bulkier than peroxidase.

Pre-embedding is the method of choice for the studyof cellular interactions in three dimensions by electrontomography. Here, we labeled the essential splicingfactor SC-35 and visualized the primary antibodieseither with peroxidase/DAB (Fig. 10A) or silver-enhanced ultrasmall gold particles (Fig. 10B). The mostobvious difference is the labeling density. The clusters

of interchromatin granules are completely covered withDAB, while the individual gold particles do not obscureunderlying structures. For the peroxidase/DAB method,the sections cannot be stained, as stain obscures thelabel, and the ultrastructural details are less clearlyvisible. The distribution pattern of gold particles inthree dimensions (Fig. 10B) demonstrates why a muchlower label density for on-section labeling methods hasto be expected. In one plane of the example shown, onlya few particles are present. Three-dimensional viewingalso reveals much more clearly the diffusion gradient ofthe DAB polymerization product (Fig. 10A). Both of themicrographs show that the label can be related tonuclear domains, e.g., nucleolus and nuclear bodies.

Fig. 7. Cells were prefixed with formaldehyde and glutaraldehyde,then permeabilized with Triton X-100. A nuclear protein, the essentialsplicing factor SC-35, was identically labeled with a monoclonalprimary antibody. The labeling was visualized with FITC (A), peroxi-

dase (B), Au-FITC (C), ultrasmall gold (D), 6 nm (E) and 10 nm gold(F) tagged secondary antibodies. The gold particles were enlarged bysilver enhancement. A and C are fluorescence and B, D–F bright-fieldimages. Bar 5 20 µm.


Fig. 8. The two most successful methods were based on formalde-hyde/glutaraldehyde prefixation followed by permeabilization witheither Triton X-100 (A–C) or Brij 58 (D–F). In both the preparationsthe cytoplasm and the nucleus are rather dense and mitochondria (m)and vesicles (v) are visible. In the nucleus the nucleoli (Nu) with the

fibrillar center, clusters of interchromatin granules (Ig) and nuclearbodies (Nb) are observed. In Brij 58-treated cells the contrast in thenucleus and the mitochondria is inverted. (A,D) Whole cell at lowmagnification, bar 5 5 µm. (B,E) A portion of the cytoplasm; (C,F) aportion of the nucleus at higher magnification; bar 5 1 µm.

The intense label after pre-embedding labeling withperoxidase suggests combining pre-embedding labelingwith post-embedding detection. In a mixed technique,one would profit from the higher labeling efficiencywithout the disadvantage of the low contrast of theDAB reaction product. DAB is replaced by the recentlyestablished tyramide probes (Van Gijlswijk et al., 1996,1997). In whole mount preparations the label patternand efficiency is checked by light microscopy usingFITC-tyramide (Fig. 11A). For electron microscopy,biotin-tyramide was used. The labeling procedure iscontrolled stepwise. First, the distribution of the biotinis shown on ultrathin Lowicryl sections by fluorescencemicroscopy (Fig. 11B); thereafter, subsequent sectionswere labeled with an ultrasmall (Fig 11C) or a 10 nmgold-tagged antibody (Fig. 11D) in two steps. After goldlabeling, the sections can be stained with uranyl ac-etate and lead citrate without fear of covering the weakcontrast of the label product (Fig. 11C,D).

DISCUSSIONIn this article, we discuss different permeabilization

methods in respect to their ultrastructural quality andthe labeling efficiency. The most appealing methods arethose that open ‘‘living’’ cells and make them permeableto drugs and, hopefully, for antibodies and markers. Inour investigation, we concluded that both methodsinvestigated, streptolysin-O pore formation and Brij58-opening (without prefixation), are not suited forroutine use. In fact, one has to expect that if the cellsare as well preserved as described by Schliwa et al.(1981, 1987), movement of the antibodies in the cell isgreatly obstructed. Indeed, the nucleus was less acces-sible after protease inhibitors were added to the extrac-tion buffer. Dundr and Raska (1993) obtained excellentresults using the streptolysin-O technique. They la-beled nascent transcript with bromouridine. Nuclearactivity, e.g., RNA synthesis, could continue, although

the plasma membrane was porous. The nucleoplasmwas well preserved and fine localization of rRNAsynthe-sis in the nucleoli could be demonstrated. As reportedby Leno et al. (1992) the nuclear membranes were stillintact and non-nuclear proteins excluded. The strepto-lysin-O method undoubtedly has its merits for cytoplas-mic localization studies (Krijnse-Locker et al., 1994).For immunolabeling of nuclear proteins, the streptoly-sin method should be used at higher temperatures toaccess nuclear membranes as well. In addition, themicrotubules disassemble at 0°C. The cellular integrityis destroyed if the streptolysin method is applied athigher temperature. From our results, we conclude thatboth methods to permeabilize or open ‘‘living’’ cells arenot suited for immunolabeling of nuclear antigens foreach cell type the method has to be optimized and underoptimum conditions the cell nucleus is not accessible forlarge molecules, e.g., antibodies.

For routine applications, fixation prior to permeabili-zation is favorable, as the structures can be fixed to adegree defined by the fixation process. Very strongfixatives, e.g., high concentrations of glutaraldehyde,likely also prevent free movement of antibodies withinthe cells. Formaldehyde fixation is widely used for(confocal) light microscope investigations. Under opti-mum conditions, i.e., high concentrations, formalde-hyde can preserve ultrastructural details and is usedfor cryosection labeling (Griffiths, 1993). In combina-tion with Triton X-100 as a detergent, cytoplasmicproteins are lost or can be relocated (Melan and Sluder,1992). In addition, the nucleoplasm loses its fine struc-ture and changes into a fibrillar network (data notshown). We suspect that not just soluble proteins arelost to reveal the nucleic acid-protein network, but alsothat the stretches of DNA and RNA are dislocated. Oneshould bear in mind that these displacements might besignificant enough to influence the labeling patterneven at the level of fluorescence microscopy.

Fig. 9. Cells prefixed with formaldehyde/glutaraldehyde and permeabilized with Brij 58 were labeledfor SC-35. The primary antibody was detected with (A) FITC, (B) peroxidase DAB, and (C) ultrasmall goldparticles. Bar 5 20 µm.


Fig. 10. Stereo micrographs of cells prefixed with formaldehyde/glutaraldehyde and permeabilized with Triton X-100 and labeled forSC-35. The primary antibody was detected either with peroxidase/DAB (A) or silver-enhanced ultrasmall gold markers (B). In theperoxidase/DAB preparation (A), the whole volume of the clusters ofinterchromatin granules is intensely labeled. Note the diffusion halo

between two clusters of interchromatin granules and the blurring ofthe ultrastructure. In the colloidal gold-labeled sections (B) ultrastruc-tural details can be seen clearly without obscuring the distinctsilver-enhanced gold marker. Careful inspection of the stereo imageshows that only a few gold particles lay within one horizontal plane.Bar 5 1 µm.


Fig. 11. Cells prefixed with formaldehyde/glutaraldehyde and per-meabilized with Triton X-100 were labeled for SC-35. The primaryantibody was revealed by peroxidase/FITC-tyramide (A) or peroxidase/biotin-tyramide (B–D) reaction. Ultrathin sections of biotin-tyramide

developed cells were mounted on glass coverslips (B) or grids andvisualized by FITC (B), ultrasmall gold (C), or 10 nm colloidal goldparticles (D). Note the dense labeling on the interchromatin granules.Bars 5 20 µm and 1 µm, respectively.


In general, mild fixation with formaldehyde is pre-ferred because it interferes least with antibody binding.Therefore, we investigated the combination of formalde-hyde fixation with borohydride as an agent to pierce themembranes. First, it had to be established which of thecomponents of the method described by van LookerenCampagne et al. (1992) caused leakage. It was sus-pected that formaldehyde in cacodylate buffer might besufficient to render plasma membranes permeable. Asindicated by propidium iodide labeling of nucleic acids(Fig. 6A), the membranes indeed become slightly perme-able, but only borohydride treatment makes holes largeenough to give efficient access to the dye. Antibodies canalso reach cytoplasmic structures, which seem to lay ontop of the cells under the surface of the membranes(Fig. 5D). Deeper layers or those close to the coverslipare not accessible. Close inspection of the cells in phasecontrast microscopy suggest that borohydride digs upthe cytoplasm from the apical side. The borohydridemethod may well be suited for the long and thindendrites or nerve growth cones (Van Lookeren Cam-pagne et al., 1992) but not for bulkier specimens, exceptperhaps in combination with an ethanol wash (Youngand Furness, 1995).

Saponin is suggested as the method of choice forelectron microscope pre-embedding labeling and manyexcellent studies have been published (Louvard et al.,1982; Macville et al., 1995; Griffiths, 1993, and refer-ences therein). As demonstrated (Figs. 4, 5C), thecellular structure is well preserved and cytoplasmicantigens are easily accessed. The ultrastructural mor-phology was considerably improved compared to theformaldehyde prefixed and Triton X-100-extracted cells.The nuclear antigens, however, were not accessible(Table 1) for most of the markers. The reason for this isnot yet clear, considering the excellent detection of aviral transcript by Macville et al. (1995). There are twoexplanations to consider: either the in situ hybridiza-tion process opens the nuclear structures more or theproteins labeled in this report are tightly bound to othermacromolecules, which first have to be removed beforethey can be accessed by an antibody.

In our hands, the best and easiest to use method isprefixation with formaldehyde and glutaraldehyde. Per-meabilization is either done with Triton X-100 or Brij58. In both cases, preservation of the ultrastructure isgood enough to correlate a label with cellular compo-nents (Fig. 8). Most of the features of the nucleus, suchas nucleolus with its substructures, clusters of interchro-matin granules, and nuclear bodies, can be identified.Still, the nuclear meshwork is loose enough to giveaccess to the antibodies. After Triton X-100 permeabili-zation, all small markers (FITC, peroxidase, and ul-trasmall colloidal gold particles) can enter the nucleus(Figs. 7, 10); only the 6 and 10 nm particles areexcluded, confirming previously published results (DeGraaf et al., 1991). As suggested by the electron micro-graphs (Fig. 8F), the nucleoplasm of Brij 58-permeabi-lized cells is denser. The splicing factor SC-35 couldonly be detected with FITC- or peroxidase-tagged anti-bodies and not with ultrasmall gold particles (Fig. 9).From these results, we conclude that even the smallestgold markers cannot move within a cell as easily as afluorescence or peroxidase marker. Therefore, in criti-

cal situations the use of peroxidase/DAB might reveal alabeling which otherwise would escape our attention.

Both of the markers, peroxidase/DAB and ultrasmallgold particles, may be used for three-dimensional local-ization studies. The stereo micrographs (Fig. 10) clearlyshow the advantages and disadvantages of the twomarkers. Peroxidase/DAB seems more efficient. Theentire volume of the clusters of interchromatin gran-ules is intensely labeled. On the other hand, there isdiffusion of the polymer (note the less dark area be-tween two clusters of interchromatin granules). Viewedin stereo, the diffusion halo is more visible. The nucleo-plasm is weakly stained. In the stereo view, the struc-tures have a slightly blurred appearance. Peroxidase/DAB preparation should not be stained with uraniumand lead ions in order not to obscure the label. Incontrast, the colloidal gold-labeled sections may becontrasted. The ultrastructural details can clearly beseen without obscuring the distinct silver-enhancedgold marker. Viewed in stereo, the structural detailsare clearly visible without blurring. On the other hand,one has to accept reduced label density. The location ofthe clusters of interchromatin granules is unambigu-ously revealed. Careful inspection of the stereo imageshows that only a few gold particles lay within onehorizontal plane. This observation coincides with andmay explain the low label efficiency observed by on-section labeling experiments.

The described results suggest combining high labelefficiency and better penetration of enzyme markerswith the clear distinction of colloidal gold markers onwell-contrasted sections. In addition, the enzyme labelcan be used as an enhancement step. From one boundperoxidase molecule, a controllable amount of second-ary ‘‘antigen’’ can be produced. The recently introducedtyramide technique (Van Gijlswijk et al., 1996) canserve as the link between the two labeling methods. Ina pre-embedding approach, the antigen is detected byperoxidase antibodies and a polymer of tyramide mol-ecules, to which either biotin or FITC is bound. FITChas the advantage that the label pattern, the amount oftyramide deposition, and the degree of diffusion can bedirectly monitored with the fluorescence microscope.The reaction time determines the label intensity andthe viscosity of the development solution controls thediffusion radius (Van Gijlswijk et al., 1997). Thereafter,the biotin or FITC molecules instead of the antigen canbe visualized by on-section labeling with a colloidal goldmarker. Thus, high label efficiency even of scarceantigens and good visualization can be combined.

Of course, labeling with enzyme products will compro-mise the spatial resolution. However, we should notforget that pre-embedding labeling as such is already acompromise—the cellular ultrastructure is influencedin that it has to give way to antibodies and markers.Proteins may be relocated or lost. In terms of spatialresolution, the pre-embedding labeling technique issomewhere between light and electron microscopy be-cause the ultrastructure can not be preserved to highresolution. Often, however, pre-embedding labeling isthe only method of choice to locate scarce or maskedantigens and it opens the possibility of studying cellu-lar interactions in three dimensions. In addition, it canlink light and electron microscopic investigations.


Based on our results, the routine pre-embeddinglabeling method of choice is prefixation with formalde-hyde/glutaraldehyde and permeabilization with TritonX-100 or Brij 58. For detection, either ultrasmall goldparticles (with or without fluorochrome) with silverenhancement or if the ultrasmall gold particles areobstructed, peroxidase markers are advised. The mostpromising technique to localize scarce antigens withgood contrast is the combination of a pre-embeddingperoxidase/tyramide-FITC or -biotin labeling followedby an on-section colloidal gold detection.

ACKNOWLEDGMENTSWe thank Drs. R. van Driel, D. Satijn and A. Otte and

Dr. J. Leunissen for their generous gift of antibodiesand Dr. T. Raap and R. van Gijlswijk for the tyramideconjugates. We thank Ronald Leito for Figure 2 andFrits Kindt and his colleagues for fast and professionalphotographic work. We thank Dr. H. Schwarz and Dr.Y.-D. Stierhof Schwarz for their continuous interest anddiscussions and for careful reading of the manuscript.Dr. Stierhof also supplied the Lowicryl-embedded Leish-mania mexicana cells.

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