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http://jhc.sagepub.com/content/45/1/119Theonline version of this article can be found at:

DOI: 10.1177/002215549704500115

1997 45: 119J Histochem CytochemG. Nicolas, F. Gaill and L. Zylberberg

Microscopy After Cryotechnical ProcessingSitu Localization of Two Fibrillar Collagens in Two Compact Connective Tissues by Immunoelec

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Volume 45(1): 119128, 1997

The Journal of Histochemistry & Cytochemistry

The Histochem ica l Societ y, Inc. 002 2-1554/97/$3.30 119

ARTICLE

In Situ Localization of Two Fibrillar Collagens in Two CompactConnective Tissues by Immunoelectron Microscopy AfterCryotechnical Processing

G. Nicola s, F. G a ill, an d L. Zylberb erg

Centre de Microscopie Electroniq ue, UPMC (GN), La bo rat oire d e Biolog ie Ma rine, CNRS UPR 9042 Roscoff an d UPMC (FG),Laboratoire dAnatomie Compare, CNRS URA 1137, Universit Paris 7-Denis Diderot (LZ), Paris, France

SUMMARY Tw o f ibrillar collag ens, the w orm cuticular collage n a nd the vertebrat e Type I

fish scale collag en, bo th o rga nized in a compa ct tissue, were localized b y immuno go ld elec-tron microscopy in resin sections a fte r freeze-fixation an d freeze-substitut ion. Identifica-

tion of these tw o f ibri llar collag ens fai led w ith the use of postembedd ing labeling af t er

convent iona l electron microscopic processing . Positive labe ling o f th e Type I colla g en w a s

observed in sections of fish scales freeze-fixed by either slam-freezing or high-pressure

freezing, f reeze-substituted in acetone w ith or w ithout osmium t etroxide, and embedded

in LR White. The w orm cuticular collag en w a s det ected in sections of cuticle th a t w ere

freeze-fixed, f reeze-substitut ed (necessarily with o smium t etroxide a dde d t o a cetone), and

embed ded in either LR White o r Epon. It w as a lso d etected in specimens pre-fixed by a lde-

hyde s befo re freez e-fixat ion. The Type I fish scale colla ge n a ppea rs to be m ore sensitive

tha n the f ibrillar cuticular collag en of w orms to t he procedures employed for postemb ed-

ding immunoelectron microcopy. Our results have shown that freeze-fixation and freeze-

substitution preserved the antigenicity of the fibrillar collagens organized in a compact

three-dimensiona l net w ork, w hereas immuno labeling fa iled a fte r convent ional electron

microscopic procedures. These cryostab ilization techniques a ppea r to be of value t o im-

prove the immunolocalization of collage ns.

(J Histochem Cytochem 45:119128, 1997)

I

n so me connective tissues, fibrillar collagens orga-nized in highly ordered arrangements form compactstructures comparable to plywood. Such spatial ar-rangements of the collagenous network were de-scribed in t he cuticle of marine invertebrates (G aill etal., 1994) and the basal plate of the scales of bony fish(Giraud et a l., 1978). The presence of these denselypacked collagen fibrils forming plywood-like struc-

tures in the integument of invertebrates and verte-brates can be related to the protective functions ofthese connective tissues. Biophysical and biochemicalproperties of connective tissues are at least in par t d e-termined by the macromolecular organization of the

different types of fibrillar collagens identified in extra -cellular mat rices (Kielty et a l., 1993; Van der Rest andGarrone, 1991; Mayne and Burgeson, 1987). Dataabout their distribution rely on electron microscopycombined w ith immunolabeling (reviewed by B irk andLinsenmayer, 1994). It is necessary to examine col-lagen distribution without prior disruption, displace-ment, or extraction of the matrix components. The

limitation of immunoelectron microscopic studies isrelated to the problem of preserving tissues after pro-cessing in a stat e as close to na tive as possible.

Conventional methods using aqueous fixations, de-hydration, and embedding induce changes in thestructure and composition of the tissues, including ex-traction, precipitation, displacement, and loss of anti-genicity. To overcome the problems induced by chem-ical fixation and dehydration, alternative methods oftissue processing have been developed (Kellenbergerand Hayat, 1991). Pre-embedding immunolabeling

Correspondence to: Dr. G isle Nicolas, C entre Interuniversitairede Microscopie Electronique (CIME), Case 197, Universit Pierre etMa rie Curie, 7, q uai Saint Bernard, 75252 Paris Cedex 05, France.

Received for publication May 3, 1996; accepted August 15,1996 (6A3954).

KEY WORDS

Freeze-fixation

Freeze-substitution

Compact connective t issues

Immunogold electron

microscopy

Cuticular collagen

Type I collagen


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120

N icolas, Gaill , Zylberberg

has provided invaluable data about the molecular to-pology of the collagen fibrils (Fleischmajer et al.,1990; Birk et a l., 1988), but these immunoelectron mi-croscopic techniques have been applied after disrup-tive treatments that may induce disorganization o f thetissue architecture. Another strategy for concomi-

tantly preserving ultrastructure and antigenicity of tis-sues involves cryotechniques (Nicolas, 1991; Nicolaset al. 1989), such as ultra-rapid freeze-fixation orhigh-pressure freezing followed by freeze-substitution,which improve the quality of the ultrastructural pres-ervation and preserve the antigenicity of connectivetissues (Young et al., 1995; Nicolas et al., 1994; Hun-ziker, 1993; Keene and McDonald, 1993; Hunzikerand H errmann, 1987).

In the present study, immunological labeling wasperformed on tissues prepared by two procedures: (a)fresh material was treated either by ultra-rapid freez-ing or high-pressure freezing, freeze-substitution, and

resin embedding; and (b) chemically fixed materialw as submitt ed to freeze-fixa tion, freeze-substitution,and embedding in the same way as the freshly isolatedmaterial. We tested the latter procedure because, dueto the way the worms were collected during oceanicmissions, the majority of the worm tissues were acces-sible only after glutaraldehyde fixation. This proce-dure w as a lso applied on f ish scales to compare the in-fluence of the glutaraldehyde prefixation on theimmunolabeling of two types of fibrillar collagens: (a)the unbanded fibrillar collagen of the worm cuticle, anextremely long molecule with a globular domain(Gaill et al., 1994,1995; Mann et al., 1992; Murray

and Tanzer, 1985), which has no counterpart in verte-brates (Har-El and Tanzer, 1993), and (b) the ubiqui-tous vertebrate Type I collagen, which is the maincomponent of fish scales (Zylberberg et al., 1992a;Kimura et al., 1991).

Materials and Methods

Animals

I nvertebrates.

Different types of marine worms were used,

including the usual coastal annelid species and deep-sea hy-drothermal vent worms. The coastal annelids, H armothoe

lunulata

and Arenicola lunulata

, were collected at the

Roscoff marine station, w hereas t he vent vestimentifera spe-cies Riftia pachyptila

was collected at a depth of 2600meters in the Pacific Ocean during a FrenchAmerican cruise

(HERO 92). The cuticle of A. marina

and of R. pachypti la

was examined on small pieces removed from the body wall.

In the shallow seawater scale worm H armothoe lunulata

,each ring presents tw o b ioluminescent elytra covered w ith a

cuticle. These elytra were used in the present study.

Vertebrates.

G oldfish (

Carassius aur atus

) of 6080 mm SL(standard length; from snout to end of the caudal peduncle)

were obtained commercially and kept in an aquarium with

tapwater at about 20

C. Scales w ere removed fro m the mid-

dorsal area below the dorsal fin to exclude structural varia-

tions that could be related to their localization.

Fixations

Chemical fix ation.

Immunolabeling using a pre-embedding

procedure was carried out on chemically fixed worm cuticleand goldfish scales. Only the results obtained with the fishscale are mentioned in the present study, t hose obta ined f or

the cuticle being published elsewhere (Hamraoui, 1994). To

apply pre-embedding labeling techniques, some scales of the

goldfish were fixed in 2% paraformaldehyde in 0.1 M so-dium cacodylate buffer, pH 7.4. They were washed in PBS

containing 0.1 M lysine. The scales were submitted either to

mechanical disruptive treatments (freezing in liquid nitro-

gen, tearing) or to chemical treatments (acetic acid a ction orenzymatic digestion using hyaluronidase or proteinases such

as pronase or proteinase K) to facilitate penetration of the

antibodygold complexes. The scales were labeled according

to the process described for the sections. Then the scales

were fixed in 2.5% glutaraldehyde in PBS and postfixed in1% osmium tetroxide in PBS. After dehydration in ethanol,

the scales were embedded in Epon. The thin sections were

double stained with uranyl acetate and lead citrate.Postembedding immunolabeling after cryofixation and

cryosubstitution of prefixed material was tested on speci-

mens treated by gluta raldehyde. Pieces of the body w all from

the vent worm R. pachypti la

were fixed immediately afterrecovery on board ship by injecting a 3% glutaraldehyde,

0.1 M sodium cacodylate-buffered solution (pH 7.4; 1 hr at

4

C). They were stored in sodium cacodylate buffer until

further treatment in the laboratory. Both solutions werebrought to sea osmolarity by addition of NaCl as previously

described (G aill et al., 1991).

Scales of the goldfish w ere fixed in 2% glutara ldehyde in

0.1 M sodium cacodylate [pH 7.4; 1 hr at room temperature(RT)] and stored in sodium cacodylate buffer before perfor-

mance of cryotechniques.

The samples were immersed fo r 1 hr in a so lution o f 20%

glycerol in buffer and then cryofixed by the slam-freezingmethod as described below; for high-pressure freezing no

glycerol w as a dded in the buffer.

Cryofixations.

The tissues were fixed by those of the fol-

low ing procedures tha t w ere opportune.

Cryofi xati on by Plunge-freezing.

Small pieces of bodywall of the vent worm R. pachyptila

were cryofixed on

boa rd ship by plunging into liquid propa ne.

Cryofi xati on by Slam-fr eezing.

The cuticle of the shallowseawater annelids H . lunulata

and A. marina

and the scalesof the goldfish w ere rapid-frozen by slamming aga inst a cop-

per block cooled w ith liquid helium at

260

C, using a C ry-

oblock ReichertJung Leica (Escaig, 1982) as previously de-

scribed (Nicolas, 1991).

H igh-pressure Freezing.

This procedure w as a pplied to the

elytra of the annelid H . lunulata

. Small pieces of elytra

moistened with the freezing medium (1-hexadecene) (Ho-henberg et al., 1994; Studer et al., 1989) w ere load ed into in-

terlocking specimen holders (Craig et al., 1987) and frozen


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Imm unoelectro n M icroscopy of Connective Tissues

121

w ith liquid nitrogen under 2100 bars using the Balzers HP M

010 High Pressure Freezer (Bal-Tec Products; Selles sur

Cher, France) (Moor, 1987). The same procedure was ap-

plied to some scales of the goldfish.

Freeze-substitution

The frozen samples, stored in liquid nitrogen, were thentransferred into the freeze-substitution medium at

90

C for

3 days as described previously (Nicolas, 1991), either in pure

acetone or in acetone containing 2.5% osmium tetroxide. A

molecula r sieve (type 4A 1/16; Unio n Ca rbide, R ungis,France) was added to the medium, which was gradually re-

w armed from

90

C to

30

C over a period o f 3 hr and left

a t

30

C for 2 hr. Some samples were kept in acetone at

20

C and embedded in LR White at

20

C. After a finalrewa rming to reach RT with a step of 30 min at this temper-

ature, the ot her samples w ere w ashed in the pure solvent and

embedded either in LR White polymerized at 50

C or in

Epon.

Postembedding Immunogold Labeling

All steps of the labeling procedure, except the primary anti-

body incubation overnight, w ere performed at RT.

Ultrathin sections were collected on gold grids. They

were washed for 15 min in a blocking solution of PBS con-taining 0.5% BSA (bovine serum albumin) and 0.05%

Tween 20. The sections were then incubated overnight at

4

C in 20-

l drops of the blocking PBS containing the pri-

mary antibodies at the appropriate dilution. After severalwashes in pure PBS and one more in the PBSBSATween 20

for 15 min, the sections were incubated in the secondary

gold-conjugated a ntibody. The sections w ere then w ashed in

PBS. The immunoreaction was stabilized by floating thegrids on 0.2% glutaraldehyde in PBS for 2 min. After several

rinses in PBS and in distilled water, they were contrasted inaqueous saturated uranyl acetate solution.

Primary A ntibodi es.

A polyclonal antibody raised against

the native purified cuticular collagen of Arenicola

following

the standard protocols (Timpl, 1982) was used as primaryantibody, at a concentration of 1:200 in the PBSBSA

Tween 20 solution. This antibody was known to crossreact

with cuticle collagen of annelid and vestimentiferan species

(Ga ill et al., 1994; Ha mraoui, 1994).The Type I collagen was identified in the goldfish scales

using as primary antibody a polyclonal antibody raised

against the dermal pepsin-digested Type I collagen of the

goldfish (provided by Dr. D. J. Hartmann, Institut Pasteur,

Lyon). This antibody was the same as that previously tested(Zylberberg et al., 1992a). It was used at a concentration of

1:60 in the PBSBSATween 20 solution.

Secondary Anti bodi es.

The sections of the worm cuticle

w ere incubated w ith either protein Agold complex usingcolloidal gold particles of 10 nm (Biocell; Cardiff, UK) at a

concentration of 1:100 in the PBSBSATween solution or

goa t a nti-rabbit IgG coupled w ith 5-nm gold pa rticles (IgG

5

)(Amersham International; Poole, UK) at a concentration of

1:50 in the same solution. The sections of the fish scales

w ere incubated w ith protein Agold only.

To control the specificity of the labeling, the primary an-

tibodies were substituted with appropriate dilutions of non-

immune rabbit serum or PBSBSATween solution.

Results

G oldfish scales and w orm cuticles were submitted to

the same cryot echniques before postembedding immu-nolabeling.

Fish Scale Colla g en

The basal plate of the goldfish is composed of thickcollagen fibrils (about 100

m in diameter) organizedin superimposed layers parallel to the scale surfaceand arranged in a plywood-like structure. In eachlayer, the fibrils are parallel to each other and their di-rection varies from one layer to the other. In the gold-fish basal plate, thin collagen fibrils called TC fibers(Onozato and Watabe, 1979) perpendicular to thescale surface cross the plywood-like structure. Thick

Figure 1 Goldfish scale. Pre-embedding immunolabeling afterproteinase K disruptive trea tment . Low er part o f the basal plate.

Note tha t only the collag en f ibrils of t he disrupted pa rt are labe led

by the g old particles, which do not penetrat e w ithin the compact

tissue. Original magnification 50,000. Bar 200 nm.


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122


Figures 28 Goldfish scale. Postembedding immunolabeling after freeze-substitution in OsO 4acet one , LR White . Type I collag en a nt i-bod yprot ein Ago ld (10 nm). Figures 24Slam-freez ing, LR White , 50C. Gold pa rticles are distribut ed over the b asa l plat e (bp) on t he thickcollag en f ibrils forming t he plyw ood -like structure a nd on the thin TC fibrils crossing t his structure. Figure 3Detail of the ba sal plate. Figure4 Collagen f ibrils decorated w ith go ld particles form the innermost layer o f t he ba sal plat e (bp) in contact w ith a hyposquama l scleroblast(hs) containing flat RER saccules (er). m, mitochondrion. Figure 5Slam-freezing, LR White, 20C. Figures 6 and 7High-pressure f reezing.Figure 6Innermost layer of the basal plate (bp) in contact with the hyposquamal scleroblasts (hs). Figure 7Detail of the ba sal plate. Figure8Slam -freezing , LR White. Control. Section incuba ted w ith nonimmu ne serum. Origina l mag nificat ions: Figures 2 and 830,000; Figures 3,4, and 760,000; Figures 5 and 640,000. Bars 200 nm.


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123

and thin collagen fibrils of the basal plate are synthe-sized by the hyposquamal scleroblasts that form acontinuous lay er lining the basal surface of the scales.

Immunogold labeling using pre-embedding proce-dures was obtained only after disruptive treatments.These treatments disorganized the collagenous net-

work, at least at the surface of the specimens. Onlyisolated collagen fibrils were decorated with gold par-ticles when antibody against Type I collagen was used(Figure 1). No labeling was observed on the fibrilswithin the compact collagenous network. The scalesthat were cryofixed by slam-freezing, cryosubstituted

Figures 9 and 10 Harmothoe lunulata. Cuticle underlined by epidermal cells (ec) in the elytrum. Slam-freezing, freeze-substitution inOsO 4aceton e. co, collag en; m i, microvilli. Figure 9Epon, cuticular collagen a ntibody, g oa t a nti-rabbit secondary ant ibody conjuga ted t o5-nm go ld. Figure 10Embe dding in LR White, cuticular collag en a ntibo dyprotein Ago ld (10 nm). The collage n net w ork is labe led by a bun-dant gold particles in both cases. Original magnifications: Figure 960,000; Figure 1030,000. Bars 200 nm.


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w ith acetone supplemented w ith osmium tetroxide,an d embedded in Epon show ed improved preserva tionof the cells and of the collagen fibrils, as described

elsewhere (Zylberberg and Nicolas, 1982). However,the immunolabeling of these specimens was not spe-cific, even when osmium tetroxide was omitted duringcryosubstitution. Specific labeling was obtained onlyin cryofixed samples cryosubstituted in acetone eitherwith or without osmium tetroxide and embedded inLR White at both

20

C a nd 50

C (Figures 2, 3, and5). The gold particles were distributed over the basalplate on the thick collagen fibrils forming the ply-wood-like structure and on the thin TC fibers (Figure2). No labeling was observed in the hyposquamal scle-

robla sts, w hich exhibit fla t R ER sa ccules (Figure 4).The labeling was not improved in specimens embed-ded in LR White at low tempera ture (Figure 5).

Scales submitted to high-pressure freezing and thento freeze-substitution in acetone containing osmiumtetroxide and embedded in LR White showed a well-

preserved collagenous network, the labeling of whichdid not appear as intense as that obtained after slam-freezing (Figures 6 and 7).

No specific labeling was obtained with sections ofgoldfish scales that were chemically fixed before cryo-fixation by slam-freezing, cryosubstitution in acetonewith or without osmium tetroxide, and embedding inEpon or in LR White.

No labeling was obtained on control sections sub-mitted to nonimmune rabbit serum (Figure 8) or w henthe primary antibody was omitted.

Cuticular Colla g en

The w orm cuticle secreted by the epidermal cells iscomposed of unbanded fibrils organized in superim-posed layers, forming a plywood-like structure that isnot as dense as that of the fish scale. The cuticle iscrossed by microvilli that arise from the epidermal cellsand reach the body surface.

For studying the coasta l annelids, all labo rato ry fa -cilities were available. Therefore, slam-freezing andcryosubstitution, which were known to preserve theultrastructure a nd t he antigenicity of t he tissues (Nico-las, 1991), were applied on both species, H . lunulata

and A. marina

. Elytra of H . lunulata

w ere freeze-sub-stituted with acetoneosmium tetroxide and embed-

ded in either LR White or Epon. H igh specific labelingof the collagenous network was obtained with bothresins (Figures 9 and 10). Low-temperature embed-ding did not improve the intensity of the labeling.Moreover, the labeling of the cuticular collagen wasobtained only after a cryosubstitution with osmiumtetroxide added to the medium; no labeling was ob-served after the use of pure acetone. The same resultsw ere obtained w ith A. marina

embedded in Epon.Immunolabeling of the cuticular collagen after high-

pressure freezing followed by cryosubstitution in ace-toneosmium tetroxide and embedding in Epon wastested on the elytra of H . lunulata

(Figure 11). With

the use of high-pressure freezing, extractions were ob-served in the ground cytoplasm and the vesicles wereswollen, whereas the preservation of the cuticle ultra-structure was improved. However, the immunolabel-ing did not appear as intense as that obtained withslam-freezing (Figure 9).

The specimens of R. pachyptil a

were cryofixed onshipboard by plunging in liquid propane. Few resultswere obtained because of the difficulty of reproducinggood quality freeze-fixations without specialized de-vices, and only small, shallow areas appeared to be

Figure 11 Harmothoe lunulata. Cuticle covering the elytrum.High-pressure freezing, freeze-substitution in OsO4acetone, em-

bedding in Epon, cuticular collagen antibodyprotein Agold (10

nm). The cut icular structure is very w ell preserved a nd spe cifica lly

labeled. Original ma gnif icat ion 30,000. Bar = 200 nm.


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125

Figures 1214 Rift ia p achypt i la,bo dy cuticle. Figure 12Propa ne-plunging , freeze -substitution in OsO4acetone , embed ding in Epon, cutic-ular collag en a ntibo dyprotein Ago ld (10 nm). The collage n fibrils (co) labeled w ith g old pa rticles form a n etw ork aroun d t he cross-sec-

tioned microvilli (mi). Figures 13 and 14Chemically fixed and cryoprotected specimens. Figure 13Slam-freezing . Figure 14 High pressurefreez ing. In bot h cases, the g old pa rticles are localized on the collag en f ibrils (co), forming a net w ork crossed by t he microvilli (mi). ec, epi-

derma l cell. Original mag nificat ions: Figure 1260,000; Figure 1350,000; Figure 1440,000. Ba rs 200 nm.


9/11

126 N icolas, Gaill , Zylberberg

w ell preserved. In these areas the collagen fibrils orga-nized in an orthogonal plywood-like structure werewell preserved and were labeled with gold particles(Figure 12).

M ost o f the tissues collected d uring the cruise wereaccessible after fixation by aldehydes only, mostly glu-tara ldehyde, w hich is know n to inhibit immunolabelingreactions. N evertheless, w e carried out immunolabel-ing techniques after freeze-fixation of these chemically

fixed tissues. Slam-freezing (Figure 13) and high-pres-sure freezing (Figure 14) w ere used. The samples werefreeze-substituted in a medium containing osmiumtetroxide and embedded in Epon. The freeze-fixationof chemically pre-fixed and cryoprotected specimensensures the preservation of a thicker area (about 200m with slam-freezing) from the freezing front thanthat obtained with fresh material without cryoprotec-tion (about 1015 m). The high-pressure freezingproduced some swelling of the cytoplasmic vesicles,similar to tha t o bserved in non-pre-fixed ma terial. Thecollagenous network was well preserved and abun-dant gold particles were specifically distributed on the

collagen fibrils (Figure 14).Control sections submitted to the nonimmune rab-

bit serum or to PBSBSATw een solution w ere not la-beled.

The results are summarized in Table 1. After theslam-freezing method, we tested two cryosubstitutionmedia, pure acetone or acetoneosmium, and the tworesins, Epon or LR White. This latt er w as po lymerizedat 20C or 50C. The use of LR White polymerizedat low temperature did not improve the immunolabel-ing compared to that obtained at 50C . Therefore, forthe other experiments the polymerization was achievedat 50C. It appeared that the best compromise for

characterization o f Type I collagen w as o bta ined a ftercryosubstitution with acetoneosmium and embed-ding in LR White, whereas for cuticular collagen itw as o bta ined aft er cryosubstitution in acetoneosmiumand embedding either in Epon or LR White.

Discussion

The present study has show n tha t immunoelectron mi-croscopy of fibrillar collagens in compact tissues isachieved using postembedding labeling after freeze-

fixation and freeze-substitution, which also yieldsgood preservation of the spatial architecture of thecollagenous network in the worm cuticle and in thefish scale. Previous studies using the same antibodieshave shown that postembedding immunolabeling hasfailed after chemical fixation in worm cuticle (Ham-raoui, 1994) and in fish scales (unpublished results).These results are consistent w ith the fa ct genera lly ad -mitted that conventional techniqueschemical fixa-

tions, dehydration, and embeddingproduce alter-ations tha t lead to masking of t he antigen.In vertebrates, collagen epitopes are highly sensitive

to aldehydes contained in the fixatives and particu-larly to glutaraldehyde, the most successful aldehydeused for electron microscopy (Young et al., 1995;Hunziker, 1993), these aldehydes showing a high af-finity for -helices (Hayat, 1981). The formation ofintermolecular crosslinks is shown by changes in thestaining patterns of the fixed fibrillar collagens (Bair-ati et al., 1968). Cryotechniques offer advantages be-cause they minimize modifications of the morphologi-cal fea tures and preserve antigenicity (Hunziker, 1993).

Slam-freezing of fresh worm cuticle and fish scale hassucceeded in pro viding w ell-preserved structures w ithinthe cells as w ell as in the compact extr acellular ma trix.High-pressure freezing of fresh tissues has improvedthe structural preservation of the compact extracellu-lar matrix but not its immunoreactivity, as observed inanother extracellular matrix (Nanci et al., 1994). Ithas induced some artifacts in the cell structures, suchas sw elling of the vesicles and extraction of t he groundcytoplasm. Such artifacts were believed to occur dur-ing specimen mounting before exposure to high-pres-sure freezing (Kiss and Staehelin, 1995).

Fine preservation of the structures as w ell a s intense

immunolabeling w as o bta ined w hen osmium tetroxidew as a dded to acetone, the freeze-substitution medium,although osmium tetroxide is known to destroy anti-genicity by proteinprotein crosslinking (Newman andJasani, 1984; Nielson and G riffith, 1979). Mo reover,osmium tetroxide must be added to the freeze-substi-tution medium to preserve the antigenicity of thew orm cuticular collagen. In o ur study, w e have foundthat the antigenicity of the compact collagenous net-w ork of w orm cuticle w as a lso preserved in chemicallyfixed specimens subsequently freeze-fixed either by

Table 1 Results obt ained a fter t he dif ferent procedures used a

Cryo f ixa t io n Pro pa ne Slam HP Pref ix. cryofix.

Resins Epon Epon LR W LRW20C Epon LR W Epon LR W

Type I co lla g en Acet one ND ND

Aceto neosmium ND

Worm cut icula r co lla g en Acet one ND ND ND ND

Acetoneosmium ND NDaLR W, LR w hite; ND, not do ne.


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Imm unoelectro n M icroscopy of Connective Tissues 127

slam-freezing or by high-pressure freezing and freeze-substitution. These positive results suggest that themain cause of labeling loss of the worm cuticular col-lagen is not due to the aldehyde fixation but to theother procedural steps. The dehydration at room tem-perature might be a disturbing step, as it induces os-

motic changes and shrinkage (Oprins et al., 1994;H ay at , 1970). Freeze-substitution, w hich consists ofthe withdrawal of water in a solid state, results in avery slow and gentle dehydration, which reduces thepossibility of ultrastructural changes to a minimum(Chan et al., 1993; Yamashita a nd Ya suda, 1992).

In contrast, the immunoreactivity of the Type I col-lagen of the fish scale was not preserved in samplesfixed before freeze-fixation and freeze-substitution.On the other hand, unlike the worm cuticular col-lagen, w hich is immunolabeled aft er embedding in LRWhite and in Epon, the Type I collagen of fish scale isimmunoreactive only in specimens embedded in LR

White, a hydrophilic acrylic resin that provides goodaccess to epitopes (Brorson et a l., 1994; G riffiths,1993; Newman and Hobot, 1993). No labeling wasobt ained in sa mples embedded in Epo n. This might beexplained by the fact that this resin could co-polymer-ize with the tissue, forming covalent bonds (Kellen-berger et a l., 1987) and ha mpering immunolab eling.

The failure of labeling of the collagenous netw orkin the fish scales while the worm cuticle remains im-munoreactive can be relat ed to t he presence of noncol-lagenous components such as those involved in themineralization of the fish scales (Zylberberg et al.,1992b; Zylberberg and Nicolas, 1982), which prevent

access to Type I collagen epitopes. Immunolabelingmight also be hampered by the higher compactness ofthe plyw ood -like structure in the fish scales tha n in thecuticle. Alternatively, it is possible that epitopes of thecuticular collagen are less sensitive to aldehydes thanthose of Type I collagen of vertebra tes. Epitopes of t hecuticular collagen are not destroyed by chemical fixa-tion and become accessible to antibodies after freeze-fixation and freeze-substitution.

Fixation is considered to be the most important ofthe many steps involved in the processing of specimensfor immunoelectron microscopy (reviewed by Will-iams and Faulkner, 1993). Nevertheless, freeze-substi-

tution a nd embedding appeared to be importa nt a lso,since the substitution medium a nd t he resin could a lsoprevent the labeling of antigen as observed in thepresent study .

Our results are consistent with data reporting thatcryotechniques are of value for postembedding immu-nogold labeling in compact connective tissues. How-ever, they are not a standa rd a pplication (Young et al.,1995; Hunziker, 1993). A compromise between thepreservation of ultrastructure and immunoreactivitymust be found for each tissue.

Acknowledgments

This work was supported by grants from the Centre Na-tional de la Recherche Scientifique (CNRS) URA 1488, UPR9042, URA 1137.

We are indebted to Dr. Aries Kovoor (Universit Paris 7and CN RS) for the English correction of t he manuscript. Wealso acknowledge the excellent service of the photographic

department of the CIME.

Literature Cited

Baira ti A, Petruccioli MG , Torri Tarelli L (1968) Strutt ura periodicadelle fibrille collagene e technique di prepar azio ne. - I. Fissazionealdeidica. Boll Soc Ital Biol Sper 44:12571260

Birk DE, Fitch JM, Barbiaz JP, Linsenmayer TF (1988) Collagentype I and V are present in the same fibril in the avian cornealstroma. J Cell Biol 106:9991008

Birk DE, Linsenmayer TF (1994) Collagen fibril assembly deposi-tion and organization into tissue specific matrices. In YurchenkoPD, Birk DE, Mecham RP, eds. Extracellular Matrix Assemblyand Struct ure. New Yo rk, Academic Press, 91128

Brorson SH, Roos N, Skjrten F (1994) Antibody penetration into

LR White sections. Micron 25:453460Chan FL, Inoue S, Leblond CP (1993) The basement membranes ofcryofixed or aldehyde-fixed freeze-substituted tissues are com-posed of a dense lamina and do not contain a lamina lucida. CellTissue Res 273:4152

Cra ig S, G ilkey JC , Sta ehlin JA (1987) Improved specimen cups andauxiliary devices for Balzers high pressure apparatus. J Microsc48:103106

Escaig J (1982) New instruments w hich facilitat e rapid freezing at83K and 6K. J Microsc 126:221229

Fleischmajer R, MacDonald ED, Perlish JS, Burgeson RE, Fisher L(1990) Dermal collagen fibrils are hybrids of type I and type IIIcollagen molecules. J Struct Biol 105:162169

Gaill F, Hamraoui L, Sicot FX, Timpl R (1994) Immunologicalproperties and tissue localization o f tw o d ifferent collagen typesin annelid and vestimentifera species. Eur J Cell Biol 65:392401

G aill F, Ma nn K, Wiedeman n H , Engel J, Timpl R (1995) Structural

comparison of cuticle and interstitial collagens from annelids liv-ing in shallow sea water and deep sea hydrothermal vents. J MolBiol 246:284294

G aill F, Wiedemann H , M ann K , Khn K, Timpl R, Engel J (1991)Molecular characterization of cuticle and interstitial collagensfrom w orms collected at deep-sea hydrothermal vents. J M ol Biol221:209223

Giraud MM, Castanet J, Meunier FJ, Bouligand Y (1978) The fi-brous structure of coelacanth: a twisted plywood. Tissue Cell10:671686

G riffiths G (1993) Fine Structure Immunocyto chemistry. Berlin,Springer-Verlag

Hamraoui L (1994) In situ and in vitro assembly of cuticle collagensfrom ma rine worms. C ell Mo l Biol 40:495506

Har-El R, Tanzer ML (1993) Extracellular matrix. 3: Evolution ofthe extracellular matrix in invertebrates. FASEB J 7:11151123

Hayat MA (1981) Fixation for Electron Microscopy. New York,Academic PressHayat MA (1970) Principles and Techniques of Electron Micros-

copy. Vol 1. Biological Applications. New York, Van NostrandReinhold

Hohenberg H, Mannweiler K, Mller M (1994) High-pressurefreezing of cell suspensions in cellulose capillary tubes. J Microsc175:3443

Hunziker EB (1993) Application of cryotechniques in cartilage tis-sue preservation and immunoelectron microscopy: potentials andproblems. M icrosc Res Tech 24:457464

Hunziker EB, Hermann W (1987) In situ localization of extracellu-lar ma trix components by electron microscopy a fter cryotechni-cal tissue processing. J Histochem Cytochem 35:647655


11/11

128 N icolas, Gaill , Zylberberg

Keene DR, McDonald K (1993) The ultrastructure of the connectivetissue matrix of skin and cartilage after high-pressure freezingand freeze-substitution. J Histochem Cytochem 41:11411153

Kellenberger E, Durrenberger M, Villiger W, Carlemalm E, WurtzM (1987) The efficiency of immunola bel on Low icryl sectionscompared to theoretical predictions. J Histochem Cytochem35:959969

Kellenberger E, Hayat MA (1991) Some basic concepts for the

choice of methods. In Ha yat M A, ed. Co lloidal Go ld. Vol 3. SanDiego, Academic Press, 230Kielty CM, Hopkinson I, Grant ME (1993) Collagen: the collagen

family structure, assembly and organization in the extracellularmatrix. In Royce PM, Steinmann B, eds. Connective Tissue andIts Heritable Disorders. Vol 3. New York, WileyLiss, 103130

Kimura S, Miyauchi Y, Uchida N (1991) Scale and bone type-I col-lagen of carp (Cyprinius carpio). Comp Biochem Physiol 99B:473476

Kiss JZ, Staehelin LA (1995) High pressure freezing. In Severs NJ,Shotton DM, eds. Rapid Freezing, Freeze Fracture and DeepEtching. New York, WileyLiss, 89104

M ann K , G aill F, Timpl R (1992) Amino a cid sequence and cell ad -hesion activity of a fibril-forming collagen from the tube wormRiftia pachyptila living at deep sea hydrothermal vents. Eur JBiochem 210:839847

Mayne R, Burgeson RE (1987) Structure and Function of the Col-

lagen Types. New York, Academic PressMoor H (1987) Theory and practice of high pressure freezing. In

Steinbrecht RA, Zierold K, eds. Cryotechniques in BiologicalElectron Microscopy. Berlin, Springer-Verlag, 175191

Murray LW, Tanzer LM (1985) The collagen of the annelida. InBairati A, Garonne R, eds. Biology of Invertebrate and LowerVertebrate Collagens. New York, Plenum Press, 243258

Nanci A, Kawaguchi H, Kogaya Y (1994) Ultrastructural studiesand immunolocalization of enamel proteins in rodent secretorystage ameloblasts processed by various cryofixation methods.Anat Rec 238:425436

Newman GR, Hobot JA (1993) Resin Microscopy and On-sectionImmunocytochemistry. Berlin, Springer-Verlag

Newman GR, Jasani B (1984) Post-embedding immunoenzymetechniques. In Polak JM, Varndell IM, eds. Immunolabeling forElectron Microscopy. Amsterdam, Elsevier, 5370

Nicolas G (1991) Advantages of fast-freeze-fixation followed byfreeze-substitution for the preservation of cell integrity. J Elec-tron Microsc Tech 18:395405

Nicolas M T, Ba ssot JM , N icolas G (1989) Immunogold labelling ofthe luminous bacterium Vibrio harveyi after fast-freeze-fixation

and different freeze-substitution and embedding procedures. JHistochem C ytochem 37:663674

Nicolas G , G aill F, Z ylberberg L (1994) The use of cryofixa tion incompact connective tissue immunoelectron microscopy. In Elec-tron Microscopy, 1994. Proc 13th Int Congr Electron Microsc.Applicatio n in Bio logical Sciences. Vol. 3A:133134

Nielson AJ, G riffith WP (1979) Tissue fixa tion b y osmium tetrox -idea possible role for proteins. J Histochem Cytochem 27:997

999Onozato H, Watabe N (1979) Studies on fish scale formation andresorption: III. Fine structure and calcification of the fibrillaryplate of the scales in Carassius auratus (Cypriniformes: Cyprin-idae). Cell Tissue Res 201:409422

Oprins A, G euze HJ, Slot JW (1994) Cryosubstitution dehydrat ionof a ldehyde-fixed tissue: a favo rable approach to qua ntitative im-munocytochemistry. J H istochem Cyto chem 42:497503

Studer D, Michel M, Mller M (1989) High pressure freezing comesof age. Scan Microsc(suppl 3):253269

Timpl R (1982) Antibodies to collagens and procollagens. MethodsEnzymol 82:472478

Van der Rest M, G arrone R (1991) Collagen family of prot eins.FASEB J 5:28142823

Williams G V, Faulkner P (1993) Detection of vira l and cellularstructures by post-embedding immunocytochemistry. In HayatAD, ed. Immunogold EM in Virus Diagno sis and Research. Bo ca

Ra ton, FL, C RC Press, 177230Yamashita S, Yasuda K (1992) Freeze-substitution fixation for im-

munohistochemistry at the light microscopic level: effects of sol-vent and chemical fixatives. Acta Histochem Cyt ochem 25:641650

Young RD, Lawrence PA, Duance VC, Aigner T, Monaghan P(1995) Immunolocalization of type III collagen in human articu-lar cartilage prepared by high pressure cryofixation, freeze-sub-stitution, and low temperature embedding. J Histochem Cy-tochem 43:421427

Zylberberg L, Bonaventure J, CohenSolal L, Hartmann DJ, Bere-iterHahn J (1992a) Organization and characterization of fibril-lar collagen in fish scales in situ and in vitro. J Cell Sci 103:273285

Z ylberberg L, G raudie J, M eunier FJ, Sire JY (1992b) Biomineral-ization in the integumental skeleton of the living lower verte-

brates. In Hall BK, ed. Bone. Vol 4. Boca Raton, FL, CRC Press,171224

Z ylberberg L, Nicolas G (1982) Ultra structure of scales in a teleost(Carassius auratus L.) after use of rapid freeze-fixation andfreeze-substitution. Cell Tissue Res 223:349367

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