THE JOURNAL OF HISTOCHEMISTRY AND Copyright © 1977 by The Histochemical

ULTRASTRUCTURAL COMPONENTS

Vol.

CYTOCHEMISTRY

Society,

VISUALIZATION OF MEANS OF LECTINS

BY

METHACRYLATE D. GROS,

25, No.

2, pp. 104-114, 1977 Printed in U.S.A.

Inc.

A. OBRENOVITCH,

C.E.

CELLULAR CARBOHYDRATE ON ULTRATHIN GLYCOL

SECTIONS’ CHALLICE,

M. MONSIGNY

J.

AND

SCHREVEL

Laboratoire de Zoologie et de Biologie Cellulaire, U.E.R. Sciences Fondamentales et Appliqu#{233}es, 40, avenue du RecteurPineau 86022 Poitiers C#{233}dex, France (D. G., J. S.); Centre de Biophysique Mol#{233}culaire du CNRS, 45045 Orleans C#{233}dex,France (A. 0., M. M.) and Physics Department, University of Calgary, Calgary, Alberta, T2N iN4, Canada (C. E. C.) Received

for publication

April

20, 1976,

and

in revised

form

July

6, 1976

A method for the visualization of cellular carbohydrate components by both light and electron microscopy using lectins on glycol methacrylate sections is proposed. This method, which is an application of the lectin-peroxidase affinity technique, solves the problem of limited penetration when it is attempted to demonstrate lectins receptors within the tissue block. Following partial dissolution of glycol methacrylate from thin sections using alcohol, they are incubated successively with lectin (Concanavalin A or wheat germ agglutinin), horseradish peroxidase (Sigma, type II), 3-3’ diaminobenzidine and H202 and then with 0s04. Different kinds of tissues and cells have been used to test the method: mouse myocardium, rat epididymis, a protozoon Gregarina blaberae and the bacterium Escherichia coli. The localization of carbohydrate residues demonstrated by this method within the different tissues and cells is consistent with the findings from other published studies. Controls have been performed (i.e., omission of the lectin, lectin and its inhibitor) and these demonstrate the specificity of the method. The specific

lectin Concanavalin for a-r-glucopyranosyl,

anosyl

and

was

employed

try

by

A (Con A), which a-D-mannopyr-

f3-D-fructofuranosyl for

Bernhard

the

residues,

first

and

time

in

is

sections proved

(4).

Wheat

germ agglutinin (WGA) specific for N-acetyl glucosamine residues (7), was first used in ultrastructural research by Fran#{231}ois et al. (9) and Huet and Garrido (18). Several different techniques have thin tissue

been used to visualize sections by transmission

microscopy: e.g., horseradish (3) and microperoxidase (32), (17), ferritin labeling (25, complex (21) and mannan-iron

the

nique

ing

of the factor

istry cellular

chemicals in the

has

effective

of both

tissue

sites.

The

proved

to be the of the

cell

surfaces

use

of

the

Samples

work

was

Grant

744229.03,

7570790, Council

and a grant of Canada.

partly

DGRST from

supported

Grants the

National

by

of the

present

paper

lectins

are

thereby avoiding into tissue blocks

AND

parasite

extracted from were fixed for solution

internal

buffer.

to the

METHODS

buffered After

protozoa

Gregarina

blaberae

the intestine of Blabera craniifer, 2 hr at 4#{176}C in 6% glutaraldehyde at pH 7.4 with 0.1 M phosphate several

rinses

in

buffer

they

were

on 2

‘This

in the

MATERIAL

limit-

techniques

imGuil-

Fixation: Small pieces of mouse ventricular myocardium and rat epididymis2 were immersed for 1 hr at 4#{176}C in 5% glutaraldehyde solution buffered at pH 7.4 with 0.1 M cacodylate buffer, washed in cacodylate buffer (CB) and embedded in glycol methacrylate (GMA) according to Leduc and Bernhard (19).

cytochem-

and

greatly and

and allowing the chemicals direct access interand intracellular receptor sites.

lectins in electron

peroxidase (HRP) mercury labeling 30), iron-dextran complex (29).

study

described

used on ultrathin sections, the problem of penetration

All these techniques have been used on cell suspensions or in tissue blocks. In the latter case, the reactions have tended to be limited to the periphery of the tissue block. Poor penetration

has however (6). Feldmann

louzo (8) reported an intracellular localization of Con A binding sites in rat hepatocytes but the cells were damaged by freezing. In the tech-

(12)

cytochemis-

Avrameas

of 30-40 jim the penetration

7470791

Rat

epididymis

and

E.

coli

are

generous

gifts

from, respectively, Doctor A. Malassin#{233} (Laboratory of Physiology, University of Poitiers, France) and Doctor W. Costerton (Biology Department, University of Calgary, Canada).

INSERM and

Research

104

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on May 29, 2015

OF CELLULAR

VISUALIZATION

embedded in GMA. 280, serotype 08:K Brain-Heart

Escherichia 27(A):H)

infusion

agar

coli 082 (Strain were maintained

plates

ferred at 1 month intervals. the production of alkaline

CARBOHYDRATE

(Difco)

and

trans-

induced for phosphatase by growth in a medium described by Garen and Levinthal (10). Broth cultures were incubated at 35#{176}C on a New Brunswick G-25 Controlled Environment Incubator shaker

at

vested

and

150

rpm.

After

1 ml

cacodylate

Cells

D

on

8.5

of 5%

buffer

were

hr,

the

cells

(pH

6.7)

was

added

After 20 mm the cells were 12,500 x g for 10 mm and resuspended glutaraldehyde in 0.067 M cacodylate aliquot.

for

1.5

hr.

for

10 mm

The

cells

and

the

were

were

M

to

a

ml at

in 10 ml

buffer at

“enrobed”

10

of 5%

(pH 6.7) x g

12,500

in 4% agar.

After

five 10 mm washes in cacodylate buffer the gelled agar-cell suspensions were embedded in GMA. Use of lectins on ultrathin sections: technique for electron microscopy: Ultrathin sections (white or

yellow

sections)

(20)

and

The

water

which

floated was

was

ethanol was

carefully

scope

double-distilled replaced

2 ml

controlled

sections

were

the

the

absolute

GMA This

(from process

binocular

and with

then the

drop,

ethanol).

under

alcohol

well-controlling

by

rings

(DDW). ethanol to

water

dissolving of 30%

plastic

30%

by

ultramicrotome,

of the

on

drop

partially

for

of the

dilution

on

slowly,

thereby 6 drops

collected

then

added,

3 to

The

were

was

micro-

arrested

by

double-distilled

rinsed

water.

in DDW.

dissolving

of

(A way

GMA

is

to

DDW (from 1 to 24 hr) the sections were stained in a 1% 0504 solution for 15 mm, then rinsed in DDW. Several controls were used: (a) omission of the lectin; (b) lectin and its inhibitor (a-methyi-D-mannoside, 0.2 M for Con A and N-acetyi-D-glucosamine, 0.2 M for WGA); (c) omission of HRP. Sections

of use

were

uncoated in

0.067

centrifuged

centrifuged

pellet

har-

in

glutaraldehyde

105

COMPONENTS

a

grids

collected and

Siemens

on

observed

coated

(Parlodion)

without

Elmiskop

la

or

other

or

staining

Hitachi

HU

11

Cs

electron microscope. Use of lectins on thick sections: technique for light microscopy: Thick sections (0.5-1 ji in thickness) were used for light microscopy. These were placed

on

a glass

slide

and

the

embedding

alone (from 15 to 30 the specimens were but, between each

step,

the

only

with

DDW.

sections

were

This

with

medium

(GMA) was partially removed (from 15 to 30 mm) and absolute 30 mm) or by absolute ethanol mm). After rinsing with DDW treated as for electron microscopy

ethanol

washed

whole

100%

process

xyiene

(from

twice

takes

for

only

15 to

5 mm

a few

hours.

Periodic acid-thiocarbohydrazide-silver proteinate technique: Protozoa G. blaberae were fixed for 1 hr at 4#{176}C in 6% glutaraldehyde solution buffered at pH 7.4 with 0.1 M phosphate buffer, rinsed carefully in buffer, then postfixed in 1% osmium tetroxide solution in phosphate buffer (pH 7.4; 0.1 M). Following fixation the specimens were dehydrated in graded acetone solutions and embedded in Araldite. Ultrathin

sections,

collected

on

gold

grids,

were

processed as described by Thiery (33) and floated 24 hr in thiocarbohydrazide solution. Two kinds of controls were performed: omission of periodic acid oxitissue free-GMA allow to estimate the partially disdation and omission of exposure to thiocarbohydrasolution of the section). zide solution. The Con A was purified according to Agrawal and Goldstein (1) and the WGA (Purchased from IndusImmunodiffusion: Reactions of Con A and WGA trie Biologique Fran#{231}aise, 16 Boulevard du G#{233}n#{233}ralwith two kinds of peroxidase HRP type II (Sigma) Leclerc 92115 CLICHY, France) according to Bouand HRP type VI (Sigma)were tested by a diffusion-precipitation reaction in a 2% gel plate of Agar chard et al. (5). Uectin solutions were prepared immediately before use at a concentration of 50 j.ig/ml, Noble (Difco) in veronal buffer, pH 8.6, (I = 0.1). Gels were stained with Coomassie brillant blue R. 250 j.ig/ml or 500 jig/ml in 0.1 M cacodylate buffer pH 7.4 containing 1 mM of each of MgCl2, MnCl2 and sections containing tissue free-GMA.

CaC12

both tissue and a small part The change in color of the part

of of

(28).

RESULTS

Sections were floated for 20 to 30 mm on the lectin solutions maintained at room temperature and constantly agitated. Following intensive rinses in stirred DDW (from 15 to 24 hr) the sections were placed on a 250 j.tg/ml or 500 jig/mi peroxidase solution (horseradish peroxidase, Sigma, Type II) for 15 mm,

these

tions

were

rinsed

then

treated

with

dine

tetrahydrochioride

ide

tion.

from

also

being

10 to

(pH 7.4) peroxide

DAB

agitated.

the

in

15

3.3’

per 10 ml.

and

mm, again with filtered on 0.22

(Sigma)

to which

stirred

substrate (DAB)

The substrate,

50 jig/ml

constantly overnight

was After

0.1

extensive

ml

secand

diaminobenzihydrogen

perox-

constant ji

agita-

Millipore,

in 0.2 M Tris

solution added

The DDW

of 1% rinsing

was

buffer

Immunodiffusion:

Gel

experiments have demonstrated capable of precipitating with and tive

HRP with

type HRP

does not react gives, at the

immunodiffusion both

that Con A is HRP type II

VI. The results are more type II (Fig. 1A and B). with highest

HRP type VI concentration,

(Fig.

sensiWGA

1D) but precipita-

tion bands with HRP type II (Fig. 1C). Con A: For light microscopy, partial removal of GMA is an indispensable and essential step. Dissolution with absolute alcohol alone seems

hydrogen

to give

in stirred

lene

more followed

consistent with

results absolute

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on May 29, 2015

than alcohol.

with In

xythick

106

GROS

ET

AL.

ConA

WGA

HRPIi

(

lB.

HRPvi

r .,1

IC

I

1D

a-

FIG. 1. Gel immunodiffusion. A, wells a, b and c, Con A (2.5; and 10 mg/mi); central well, HRP type II (2.5 mg/mi). B, wells a, b and c, WGA (2.5; 5 and 10 mg/mi); central well, HRP type 11(2.5 mg/mi). C, wells a, b and c, Con A (2.5; 5 and 10 mg/mi); central well, HRP type VI (2.5 mg/ml). D, wells a, band c, WGA (2.5; 5 and 10 mg/mi); central well, HRP type VI (2.5 mg/ml).

sections ing to

of mouse myocardiumn the technique described

treated above,

accordall the

celis are shown to be enclosed within an opaque deposit (Fig. 2), whereas control sections (no Con A or Con A and its inhibitor, a-methyl-Dmannoside, 0.2 M) are free of deposit. Ultrathin sections of G. blaberae treated electron microscopy tion of the embedding sites opaque

of

reaction deposit

following plastic with

is present

Con on the

partial exhibit A.

An folds

ver

proteinate

the

carbohydrate

which

residues

c). No deposit is detected (Fig. 3b). In the rat epididymis smooth

for

technique

whereas

muscle the

cells basal

in the

lamina,

and the columnar cells, reacts (Fig. 4a). An electron-opaque

electron

observed

cell

surface, on subcortical vesicles and on amylopectin (paraglycogen) granules (Fig. 3a). These localizations are similar to those demonstrated using the periodic acid-thiocarbohydrazide-sil-

area

on of the

large columnar

granules cells

surfaces

between

for

3a and sections

a weak

of the reaction

these

cells

more intensively deposit is also in the (Fig.

spermatozoa flagellar periphery columnar cell surfaces are free 4a). Control sections demonstrate ity of the reactions (Fig. 4b and

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on May 29, 2015

Fig.

control

sections, present

dissoluseveral of the

is specific

(compare

4a)

perinuclear and

at the

(Fig. 5a). The of deposit (Fig. the specific5b). After treat-

OF CELLULAR

VISUALIZATION

-,

CARBOHYDRATE

107

COMPONENTS

.-

4

=‘

‘

/

,

:

I

*

..‘#

51.L

2 FIG.

Light

2.

jig/ml), myocardial

HRP

ment of the is increased In E. the

coli

external

microscopy.

Thick section (iji) of mouse myocardium, successively DAB (50 jig/ml) and H92(0.01%). An opaque deposit

(250 j.ig/ml), cell. x645.

sections the contrast (Fig. 4a and 4b).

of some

sections

the

reaction

surface

of the

cell

nuclei

is present wall

and

used for the enrobing of the bacteria mouse myocardial muscle the reaction

HRP on

on agar

(Fig. 6). In sites are

at the cell surfaces, the T-tubules (their trances as well as their lumenal surfaces) the glycogen particles (Fig. 7). In both case coli. and myocardium) control sections are

enand (E. free

of deposit.

WGA: In ultrathin sections of mouse myocardium, incubated with WGA and treated with peroxidase type II as previously described, the muscle cells surfaces react positively (Fig. 8a). In

the

capillaries

present

an

the

irregular cells

contiguous

continuous reaction

red

blood

deposit, where

cell

surfaces

except

the

deposit

line (Fig. 9). In control is observed (Fig. 8b).

between is

a thin

sections

no

used

have

type

noticed that not exhibit

II for all this

WGA

is weak

the batches property.

and

it must

be

of HRP type II do On the other hand,

(Sigma)

the

is not

ultrastructural

(9). partial

suitable

were plastic

for

of GMA

of

is an

success of the any dissolution

the

affinity of already been this

impor-

technique dothe results

always negative. Partial removal of the embedding from ultrathin sections by

absolute 1 mm)

alcohol is much than when xylene

hol has dissolve

the further the plastic

sections. incubation the tion

A (250 each

detection

dissolution

tant factor in the scribed. Without

faster (often is used (23),

advantage that rings used to

Agitation with the

of the lectin,

to a minimum. precipitates

concentration

must

clean sections. The presence

and

the

bohydrate components sues that we chose are been studied previously ray et al. blaberae,

(221,

The

is directly of DAB. be intensive and

for

Nicolson

E.

less and

it does handle

sections, with HRP

mixture DAB-H202 minimizes of nonspecific precipitates and

background

washes

shown that HRP type II (Sigma) can be directly bound to WGA without using the glutaraldehyde coupling method (2). However, affinity of HRP

for

lectin The

the

experiments

VI

by Con surrounds

detection of WGA. The spontaneous WGA for certain kinds of HRP has

specific

DISCUSSION

Gel-immunodiffusion

type

treated (arrows)

than alconot the

following and with the formareduces the

density

of non-

proportional to After each step lengthy to obtain

distribution

of the

car-

in the cells and the tisknown since they have by other methods: Mur-

coli, Schr#{233}vel (31) for G. and Yanagimachi (26) for

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on May 29, 2015

108

GROS

ET

AL.

.

sy.

.1

#{149}

#{149}..

#{149}

S.,.

-

.

a’

#{149} -

q

‘p

#{149}

.4

.

.

So

.

vsv

a

9. S

.,

#{149}

S

.#{149}J

#{149}

#{149}

S

.

9 #{149}

#{149} #{149}#{149}-

.

-

#{149}0

#{149}

#{149} .#{149}#{149}

..

S.

35

3c FIG.

jig/mi;

‘

Electron microscopy DAB, 50 j.tg/ml; H2O2,

3.

d’

of G. blaberae. a, ultrathin section treated with: Con A, 50 j.tg/ml; HRP, 50 0.01%. The reaction product is present on surface membrane folds (black

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on May 29, 2015

VISUALIZATION

R

OF

CARBOHYDRATE

109

COMPONENTS

.:.

#{149}‘‘#{149}

.hT1

CELLULAR

#{149}.#{149}/‘:

bS_._

.S

..._

r.

1;

‘1k.

..

.14

w.. .:-

0(

‘,.‘.

#{149}e‘

Mr

-

#{149}

‘‘:

-#{149}

S

.

.,

v _.2.:#{149}.*

.-#{149},

.5-,

I.

‘

S

#{149}%4.

\(,,

#{149}

‘fr

-...

4

#{149}-.#{149}

J

-

..1:f:

-

N

:r?:

#{149};:

.Q :-:.

. .fp

aL 4b

FIG. 4. Rat epididymis. a, ultrathin section treated with Con A, 200 jig/mi; HRP, 200 jig/mi; DAB, 50 jig/ ml; H2O2, 0.01%. The reaction is especially visible between muscle cells (M) and columnar cells (C) i.e., at the level of the basal lamina (opposed black arrows). Muscle cell surfaces react weakly whereas intercellular spaces between columnar cells are free of deposit (opposed black head arrows). Within columnar cells, large granules (black arrows) in the perinuclear area react strongly. N, nucleus. x 10,000. b, control section (Con A, 200 jig/mi and a-D-methylmannoside, 0.2 M; HRP, 200 jig/mi; DAB, 50 jig/mi; H2O2, 0.01%). Muscle cells (M) and columnar cells (C). Control sections do not exhibit any deposit. Note the contrast of the nuclei. N, nucleus.

x 10,000

arrows), subcortical vesicles (sv) and amylopectin (paragiycogen) granules (PG). U, lipid extracted during the embedding process. x7,500. b, control section (no Con A, HRP, 50 jig/mi; DAB, 50 jtg/ml; H2O2 0.01%). Section free of deposit. Black arrows indicate the surface of the membrane folds. x 15,000. c, demonstration of glycoi groups with periodic acid-thiocarbohydrazide-silver proteinate technique (periodic oxidation, 30 mm; thiocarbohydrazide, 24 hr; silver proteinate, 30 mm). Compare Fig. 3a and c. In both cases the reaction products (sv) and

are localized paraglycogen

on the granules

same structure: (PG). x 15,000.

membrane

folds

surface

(black

arrows),

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on May 29, 2015

subcorticai

vesicies

110

GROSETAL.

-

#{149}

-

r’a . #{149}5b

FIG. 5. Rat epididymis. Transverse section of spermatozoa in the lumen. a, section treaed with Con A, 200 jig/mi; HRP, 200 jig/mi; DAB, 50 jig/mi: H2O2, 0.01%. Opposed black arrows indicate the electron opaque deposit on surface of spermatozoa. x 10,000. b, control section (Con A, 200 jig/mi and a-n-methylmannoside, 0.2 M; HRP, 200 jig/mi; DAB, 50 jig/mi; H202, 0.01%). Control sections are free of deposit. xlO,000. FIG. 6. E. coli. Uitrathin section treated with Con A, 250 jig/mi; HRP, 250 jig/mi; DAB, 50 jig/mi; H2O2, 0.01%. Reaction product on the external surface of the cell wali (opposed black and white arrows) and on the

agar

(black

arrows)

used

for

enrobing.

x 15,000.

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on May 29, 2015

VISUALIZATION

OF

CELLULAR

CARBOHYDRATE

111

COMPONENTS

r

*

Cl

15

p

m

#{149} .

-

-

-

mf

*

.

4

RBC5

.:

.#{149} .

#{149}.

.-.

I

.. . (-

-

#{149}1.

.

#{149} ..

.

#{149}

ml;

FIG. 7. Mouse H202, 0.01%.

myocardial Section

muscle. Sections treated with passes through three myocardial

Con A, 250 jig/mi; HRP, 250 jig/mi; cells (Cl, C2, C3) and a capillary arrow), on glycogen particles (arrows),

deposit is localized on the external cells surface (opposed (asterisks) and on lumenal surfaces (double arrows). m, mitochondria; cell.

mf,

myofilaments;

DAB, 50 jig/ (Ca). Opaque in T-tubuies

RBC,

red

blood

xlO,000.

spermatozoa, and Gros

and

Parmley Challice

for

et al.

(27)

(13)

for the

blood mouse

cells myo-

A

receptor

their

sites

staining

cardium. The present results are in agreement with those of the earlier studies. In the myocar-

tion

dium, endogene peroxidase activity, present in the peroxisomes, was not (14, 15) and the usual pseudo-peroxidase

carbohydrates. to the receptor high dissociation

usually observed activ-

on the

essarily

constant (or used throughout

specific surface

for glycoproteins, of columnar cells

membranes

in their

perinuclear

3Malassin#{233}

A:

areas.:; Personal

with large

the cell granules

The

presence

of Con

communication.

eliminated

these

PTA.

columnar

imply

ity of hemoglobin in the red blood cells has not been detected (6). In the rat epididymis, phosphotungstic acid (PTA) at low pH, which is

reacts and the

in

by

The cell

the

granules

confirms

absence

of a reac-

surfaces

absence

does

of

Con

not

nec-

A-specific

The binding of Con A molecules sites, in this case, could have constant or a low association both) the

the

after

a

and the intensive rinses, experiments, could have

A molecules incubation with

Con

bound to the the lectin. A

possible extraction of certain Con A binding sites by the embedding and/or dissolution procedures cannot be discared, also. Both, Con A and

WGA

receptors

have

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on May 29, 2015

been

localized

on liver

U

#{149} #{149}-#{149} ___

#{149} #{149}

S.

Ql #{149}

-

I

.\

;

‘.1

,,

1

:-

#{149}:‘(

-

#{149}‘51

.SS#{149}#{149},.

.

.4

H

‘t5”_’” -

.‘

I

L

#{149}

I

#%Ie%I?

&

,..

“4,j

.[

#{149}

-

/,

..v I

#{149}

1

k

c

,S_1I7[!:

S

#{149}

f

#{149}

.1

#{149}15#{149},’

S

-

t

:

4

#{149}.‘

#{149}

S-#{149},

‘p

p ‘5

.

‘I.. .#{149}.

-

-

.‘#{149}

/

:v:.

S

.I.

...:

#{149}

#{149}

‘

S.,

_,

C3

.“

-

#{149}

.#{149} -,

4

-

5frP

.

S

I

#{149}1’. #{149}

rnf

#{149}

-

#{149}:.. .G4’

f#{149} .4.

#{149}#{149} ,#{149}#{149}

S .-

#{149} -

.

p

#{149} .

#{149}. -

.

-.-,S;

I.,,

..‘-t.

.:../;

#{149}5.

-

‘I

.,

C2

S

rn

-. . #{149} I.

/

8b

4

-N-

8. Mouse myocardial muscle, a, section treated with WGA, 500 j.ig/ml; HRP type II, 500 jig/ml; DAB, 50 jig/mi; H2O2, 0.01%. Section passes through three myocardial cells (Cl, C2, C3). Opaque deposit is present in the extraceiiular space between the cells (opposed arrows). m, mitochondria; mf, myofilaments. x 10,000. b, controi section (WGA, 500 jig/mi and N-acetyl glucosamine, 0.2 M; HRP type II, 500 jig/ml; FIG.

DAB, 50 jig/mI; (Ca). Intercellular xlO,000.

H2O2, 0.01%). Section passes through spaces (opposed head arrows) are

four free

myocardial of deposit.

cells (Cl, C2, m, mitochondria;

112

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on May 29, 2015

C3,

C4) mf,

and a capillary myofilaments.

VISUALIZATION

OF

________

CELLULAR

CARBOHYDRATE

113

COMPONENTS

#{149}

#{149} f

-;

-:

-,

-#{149}

-

-

W

-

#{149} #{149}S4

S

#{149} 511

.‘

‘‘-4

H

$

p’s

-

9

M

FIG.

jig/mi; (arrows)

9.

Red

blood

cells

in a myocardial

capillary.

cell

nuclei

on

membranes

mic

and

reticulum

mitochondria of

(11, 24) as well rough endoplas-

and

smooth

of myeloma

used in our localizations.

cells

experiments

we

(16).

did

In

not

The extensive control experiments of the lectin, lectin and its inhibitor, of HRP) demonstrate the specificity nique. This

method

thin sections, inates widely tins

Section

DAB, 50 ji/ml; H202, 0.01%. The reaction on free surfaces, and as a continuous line

and

using

lectins

enzyme

through

cells

such

and

advantages. of penetration

markers

the

find

4.

in

blocks. In addition, serially sectioned material may be used, and sugar residues specific for Con A (for instance) may section and sugar residues the following section.

be localized on one specific for WGA on

The

authors

are

grateful

to

Dr.

J.

W.

Cos-

terton for helpful suggestions during the progress of the study. They are also indebted to Miss Kieda, Mrs. Caigneaux and Mrs. Besse for their valuable photographic the

help, to Mr. work and Miss

preparation

of the LITERATURE

1. Agrawal

drate

2.

BBL, interaction.

Blanchard Decourt

manuscript. CITED

Goldstein IJ: IV. Isolation

A by specific adsorption gels. Biochim Biophys Avrameas 5: Coupling

for the for help in

Protein

carbohy-

of Concanavalin on cross linked dextran Acta 147:262, 1967 of enzymes to proteins

500

jig/mi;

glutaraldehyde.

HRP

type

II, 500

cell surfaces as patches head arrows). x 25,000. Use

of the

conjugates

for

of cellular

carbohydrates

components

by means of Concanavalin A. Expti Cell Res 64:232, 1971 5. Bouchard P, Moroux Y, Tixier R, Privat JP, Monsigny M: Optimum purification of wheat germ agglutinin (lectin) by affinity chromatography. 6.

Bretton, tion of

Biochimie,

in

R. Bariety Concanavalin

press J;

Ultrastructural A in normal

rat

localizakidney

glomeruli and arterioles. J Ultrastruct 48:396, 1974 7. Burger MM, Goldberg AR: Identification

8. ACKNOWLEDGMENTS

WGA,

on red blood cells (opposed

the detection of antigens and antibodies. Immunochemistry 6:987, 1969 Avrameas 5: Emploi de la Concanavaline A pour i’isolement, la detection et la mesure des glycoproteines et glucides extra ou endocellulaires. CR Acad Sci Paris 270:2205, 1970 Bernhard W, Avrameas 5: Ultrastructural visualization

ultra-

tissues

with

3.

It elimof lec-

the

with

is present contiguous

as

(omission omission of the tech-

on thick

has several the problem

treated

product between

Res of a

tumor-specific determinant on neoplastic cell surfaces. Proc Nat Acad Sci USA 57:359, 1967 Feldmann G, Guillouzo A: Localisation intracellulaire des sites r#{233}cepteurs de la Concanavaline A dans les h#{233}patocytes de Rat. CR Acad Sci

Paris 282:191, 1976 9. Fran#{231}oisD, Van Tuyen F: Etude tion de

au microscope lectines marquees

des

V, Lebvre electronique a la

Raifort

sur

maines sarcome

transform#{233}es in vitro de Rous (RSV), souche

H, Haguenau de la peroxydase

fixade

cellules embryonnaires hupar le virus du Bryan.

CR

Acad

Sci Paris 274:1981, 1972 10. Garen A, Levinthal C: A fine structure genetic and chemical study of the enzyme alkaline phosphatase ofE. coli. I. Purification and characterization of alkaline phosphatase. Biochim Biophys Acta 38:470, 1960 11. Glew RH, Kayman SC, Kuhlenschmidt MS: Studies on the binding of Concanavalin A to rat liver mitochondria. J Biol Chem 248:3137, 1973

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on May 29, 2015

114

GROS

12. Goldstein IH, So LU, Young Y, Collies QC: Protein carbohydrate interaction. XX. The interaction of Concanavalin A with Ig M and the glycoprotein phytohemagglutinins of the waxbean and the soybean. J Immunolo 103:695, 1969 13. Gros D, Challice CE: The coating of mouse myocardiai cells. A cytochemical electron microscopical study. J Histochem Cytochem 23:727, 1975 14. Hand AR: Peroxisomes (microbodies) in striated muscle cells. J Histochem Cytochem 22:207,

Singer SJ: Distribution on membrane fragments homogenate: its implication

of saccharide resifrom a myeloma for membrane

biogenesis. Proc Nat Acad Sci USA 17.

Horisberger

M,

Bauer

H,

iabeiled Concanavalin microscopy localization wails.

FEBS

Uett

Bush

A as a marker of mannan

18:311,

Uitrastruct

Res

Marinozzi

21.

nucleoie-RNA et iaires. J Ultrastruct Martin BJ, Spicer

technique

substances.

23.

V:

19:196,

20.

22.

24.

27.

28.

in eiectron in yeast cell

Nicoison GU, plant agglutinin

du

A-iron dexsurface muco22:206,

saccharides Exptl

Cell

of bovine nuRes 21:468,

Singer SJ: Ferritin-conjugated as specific saccharide stains

for

electron microscopy: application to saccharide bound to cell membranes. Proc Nat Acad Sci USA 68:942, 1971 Nicolson GU, Yanagimachi R: Terminal saccharides on sperm plasma membranes: identification by specific aggiutins. Science 177:276, 1972 Parmley RT, Martin BJ, Spicer SS: Staining of

blood ceil surfaces with a lectin-horseradish peroxidase method. J Histochem Cytochem 21:912, 1973 Pratt RM, Gibson WA: An immunofluorescent technique for observing the binding of ConcanaA to

frozen

tissue

sections.

J

Histochem

29.

30.

41:365, 1975 Roth J, Thoss K, Wagner M, Meyer HW: Electron microscopic demonstration of cell surface

tion

ultrastructurale

Cytochem

terminal mitochondria.

21:229, 1973 Roth J, Franz H: Uitrastructurai detection of lectin receptors by cytochemical affinity reacMannan-iron

by

means

complex.

Histochemie

of peroxidase

tin compiexes of the Lens culinaris chemie 43:275, 1975 31. Schrevel J: Les polysaccharides surface

ceiiulaire des gr#{233}garines parasites). I. Ultrastructure et

and

lectin.

ferri-

Histo-

associ#{233}s a la (protozoaires cytochimie.

J

Microsc (Paris) 15: 21, 1972 32. Temmink JHM, Collard JG, Spits H, Roos E: A comparative tion methods

1974

Murray RGE, Steed P, Elson HE: The localization of the mucopeptide in sections of the cell wall of Escherichia coli and other gram-negative bacteria. Can J Microbioi 11:547, 1965 Nakane PK: Application of peroxidase-iabelled

using

carbohydrates

des prot#{233}ines intranuci#{233}oRes 10:433, 1964

J Histochem

membrane clei and

Cytochem

1971

SS: Concanavaiin for staining cell

to the intracellular localization of Acta Endocrinoi 67:190, 1971 GU, Lacorbiere M, Delmonte P: Outer

valin

Mercury-

1967

Cytochimie

antibodies hormones. Nicolson

1971 25.

69:2945, 1972 DA:

18. Huet C, Garrido J: Uitrastructural visualization of celi coat components by means of wheat germ gglutinin. Exptl Ceil Res 75:523, 1972 19. Ueduc EH, Bernhard W: Recent modifications of the glycol methacrylate embedding procedure. J

tran

AL.

26.

1974

15. Herzog V, Fahimi HD: Microbodies (peroxisomes) containing catalase in myocardium: morphological and biochemical evidence. Science 185:271, 1974 16. Hirano H, Parkhouse B, Nicolson GU, Lennox ES; dues ceil

ET

33.

study of four of Concanavalin

the cell membrane. Exptl Thiery JP: Mise en evidence sur coupes en microscopie crosc (Paris) 6:987, 1967

Downloaded from jhc.sagepub.com at WESTERN OREGON UNIVERSITY on May 29, 2015

cytochemical A binding

detecsites on

Cell Res 92:307, 1975 des polysaccharides electronique. J Mi-