0022-1554/90/$3.30 The Journal of HistOCheIniStry and Cytochemistry Copyright © 1990 by The Histochemical Society,

Vol. 38, No. 8, pp.

Immunogold-Silver Staining Method for Light Electron Microscopic Detection of Lymphocyte Antigens with Monoclonal Antibodies’

Department

Osaka

Histology

SchoolService,

TbeJohn

Received

for

March

due

publication

to loss of manuscript

S. MAGARI,

MAXWELL,

L. E.

ofAnatomy,

Medical Curtin i6,

in transit

College,

Takatsuki,

and Osaka

SchoolofMedicalResearcb,

1989

the immunogold-silver staining method (IGSS) for of lymphocyte cell surface antigens with monodonal antibodies in light and electron microscopy and cornthis procedure with the immunogold staining method. Two different sizes ofcolloidal gold partides (5 nm and 15

-

were

used

in

this

study.

Immunolabeling

on

cell

rut-

faces was visualized as Ime granules only by IGSS in light miaoscopy. The labeling density (silver-gold complexes/cell) and diameters of silver-enhanced gold partides on cell stirfaces were examined by electron microscopy. Labeling density was influenced not by the enhancement time ofthe physiral developer but by the size ofthe gold partides. However, the development ofshells ofsilver-enhanced gold partides correlated with the enhancement time of the physical de-

monoclonal have been

antibodies produced

(MAb) to lymphocyte for various species and

cell surface anhave been used

for demonstration of lymphocyte subpopulations by means of the immunogold staining method (3), immunofluorescence and flow microfluorometry (i2), and immunoenzyme staining methods (i5). The

immunogold

many

staining

investigators,

since

the recent

who

antigen

immunoenzyme been reported background were usually ing

method.

has attracted

used

development

The method utilizing has several advantages, beled

method have

it for

ofsilver

colloidal including

the

the attention

detection

enhancement

ability

of

of antigens

techniques

gold labeled anti-immunoglobulin the probable identification

sites and the greater

(YO,SM,HK),

i3, i989;

accepted

E/ectmn

6,

1990.

KEY

Acceptance

WORDS:

to quantify

labeling

(iO).

(IGSS)

than

has

lymphocytes

scopic

level.

resin-embedded microscopic

sections level.

could

complexes/cell)

for detection

The

with

be measured, could

be

Medical

College,

2-7,

Daigaku-cho,

Animals.

and

Th.katsuki,

Osaka

of Anatomy, 569,

Japan.

intensity

done

study

and

of labeling

at the electron

was to develop

microa method

gold particles on the could be visualized blue

the density

determined

staining

cell surin i-tm

at the light

complexes

oflabeling

at the

of

in Epon-

on

cell

(silver-gold

electron

microscopic

Methods

Ten Merino efferent cages

sheep

were used as the source

lymphatics. and were

cannulated by the method collected at intervals through

MD, Department

of the

in smears

(17),

of silver-gold

and

Osaka with

Preparation

ofCdlls.

a model

Fn Coulter

The

fed oats.

were Foundation.

of antigens tissue

toluidine

diameters

Light micros-

level.

or metabolic

stain-

used

staining;

Lymph;

in paraffin-embedded

by which silver-enhanced colloidal faces of sheep lymph cell suspensions

pens

Naito

(LEM).

Immunogold-silver

antigens;

The aim of the present

in popliteal

immunogold-silver

been

(5),

tions

to: Y. Otsuki,

Australia

delayed

embedded tissue (1). Detailed studies by the IGSS method have not yet been

surfaces

of Ia-

Immunocytochemistry;

Immunogold staining; Cell surface copy; Electron microscopy; Sheep.

At the light

the

and

ACT26OJ,

veloper rather than the size of the colloidal gold partides. Five-nm gold partides enhanced with the physical developer for 3 mm were considered optimal for this IGSS method because of reduced background staining and high specific staining in the cell suspensions in sheep lymph. Moreover, this method may make it possible to show the ultrastructure ofidentical positive cells detected in 1-pim sections counterstained with toluidine blue by electron miaoscopy, in addition to the percentage of positive cells by light microscopy. (J Hiscochem Cytochem 38:1215-1221)

Materials

I Supported by 2 Correspondence

Micmscopy

Canberra,

April

staining methods provide (1,7). Moreover, it has by Holgate et al. (9) that both specific staining and staining by the indirect immunoperoxidase method inferior to those obtained by the immunogold stainmicroscopiclevel,

and

Nationa/University,

ing

Introduction Many tigens

Japan,

(9T1650).

We used detection

nm)

and Cell Surface

H. KUBO

569,

Australian

form July

in revised

and

1990

USA.

Note

Technical

Y. OTSUKI,2

1215-1221, Pri,,tedin

Inc.

ofLascelles

these

sheep

The

The cell concentration Counter

(Coulter

popliteal

and

cannulae

were

for cell populahoused

indoors

efferent

lymphatics

Morris(li),

in conscious in lymph Electronics;

and

in

lymph

was

sheep. was determined Dunstable,

UK).

1215

Downloaded from jhc.sagepub.com at SEIR on January 27, 2015

OTSUKI,

1216

Suspensions of 1.5 x 106 cells/mI were made in 0.1 M cacodylate buffer, pH 7.4, containing 1% BSA (cacodylate/BSA). The cells were washed with cacodylate/BSA and centrifuged at 200 x g three times for 5 mm. One ml of the cell suspension containing approximately 106 cells was transferred to an EM embedding capsule and centrifuged at 200 x g for 5 mm, and the wash buffer was pipetted off. Monoclonal in this

Antibodies.

study

were

SBU-T1,

SBU-T4,

SBU-T8,

by and

purchased

from

developed

and SBU-lI Dr.

M.B.

used

Brandon

(Embryology Research Unit, School ofVeterinary Science, University of Melbourne, Melbourne, Australia). These monoclonal antibodies (MAb) were raised against cell surface antigens of sheep lymphocytes and are reported to be the sheep homologues of human , T4 , T8 , and HLA-DR, respectively

(13,14,16).

Institute

for

E53, an MAb Immunology

Colloidal Gold-labeled (whole molecule) antiserum obtained

for sheep

(Basel,

fromJanssen

B-cells,

was supplied

by the

Basel

pH

8.2,

Secondary conjugated

Life Science

supplemented

Antibodies. to 5- and

Products

Goat 15-nm

(Beerse,

anti-mouse gold

IgG

particles

Belgium).

was

The

solution

to shield

the

with

were

mixed

0.1%

BSA

immediately

silver

enhancement

ture was insensitive

to light.

Staining

Procedure.

ing procedures

were

and

0.02

cells were incubated dilutions

from

saline

(pH

M sodium

use.

from

The immunogold

azide.

at 4C

in 100 .tl ofeach

a buffer

supplemented

with

was no need

because

the

mix-

composed 1%

BSA

(4,7).

stain-

The

glutaraldehyde

room

in 0.1

temperature.

tilled

water,

tilled

water

After

M cacodylate

further

primary

of0.02

MAb

in

The

cells

were

at room

7.2)

for 30 mm

two centrifugations

and

with

(pH

2%

temperature.

osmium

The

with

tetroxide

cells

at dis-

distilled

water,

they

were

were

sections

post-fixed

(1 p.m thick)

were stained

in 1% toluidine

thin

sections

(60-80

with

with

lead

4 mm,

citrate

and

the same

any nonspecific particles

with

on

MAb

fixation cell

uranyl

from

fixative.

After

brief

ofcolloidal

sections. used

SBU-T4, Cells

with

H 7000 gold

by light

microscopy.

block

electron from

block

were and

lead

particles

stained

citrate

microscope. the procedure

Ultra-

triple

for

As a negato quantify

or silver-enhanced

gold

surfaces.

for determination

(SBU-T1,

Spurr’s-embedded

for 8 mm,

was omitted

More than 400 cells in the sheep med

the

the same

acetate

a Hitachi

the primary

from

and examined

made

for 4 mm,

examined

tive control,

made

blue

nm thick)

of the SBU-T8, SBU-T1

for quantitative

efferent

percentages

SBU-II, and

Cell

popliteal

Concentration

efferent

lymph

was generally

concentration of the lymph was 1.49 a range of 0.97 x iO to i.93 x i0

and

E53)

silver-gold

evaluation complexes

ofthe

popliteal ofcells

lymph

labeled

in 1-pm

complexes labeling

at the

were each

examMAb

Spurr’s-embedded on their

intensity

electron

with

(the

microscopic

Reactivity

ofAnti-Sheep

Light

clear

0.62 cells/mi.

yellow.

(i07

±

The

cell

cells/ml),

with

Lymphocyte

Antibody

Microscopic

dal gold visualized

Findings.

la). Silver-enhanced

munolabeling

gold

particles

on cell surfaces

were

clearly

Both

5-nm

and

iS-nm

ob-

method no im-

gold

parti-

developer for 3 mm gave extremely staining was undetectable, and there staining between the two particle sizes.

in specific

staining physical

was slightly

developer

cells enhanced

and

for

mm.

for iO

SBU-II,

5i.4%, centage

or colloi-

in the slides treated with the IGSS for immunogold staining showed

on cell surfaces.

no difference

the

gold

on cell surfaces were of x 200 (Figure

with the physical when background

Background

T8,

No silver-enhanced

particles (5 nm or i5 nm in diameter) in the control slides at a magnification

served as crenated shapes (Figure ib). Slides used

E53

most

lymphoid

enhanced

intense ,

in the

SBU-T4,

cells

SBU-

73.4%,

was

12.2%, respectively (Table i). The cells was highest and the percentage

cells was greater The

than

combined

cells

was

of SBU-Ti

sheep

in all instances.

SBU-Tg-labeled

in the cells

and

reactivity

against

9.5%, 24.4%, and of SBU-T1 -labeled

increased

mm

5

The

SBU-T4-labeled

surfaces number level.

Electron

fur-

washing and two more centrifugations with distilled water, the cells for the immunogold staining and IGSS were stained en bloc with 1% aqueous uranyl acetate for 10 mm and then routinely dehydrated and embedded in Spurr’s. Semi-thin

and

did

that

of SBU-T8-labeled

percentages

not

exceed

ofstained

that

perof cells

SBU-T4-

and

of SBU-T-labeled

cells.

in dis-

for IGSS

ther incubated in 1 ml ofthe physical developer for 3, 5, or 10 mm at room temperature before post-fixation. After washing and two centrifugations with

The

with

M Tris-buffered

(Tris/BSA).

buffer

washing

the cells were post-fixed for 20 mm

Lymph

was

suspended

washed twice with Tris/BSA and further incubated with 100 tm of colloidii gold particles (5 or 15 nm in diameter) conjugated with goat anti-mouse IgG in dilutions from 120 to 1:40 with Tris/BSA for 30 mm at 4C. After washing and two centrifugations with Tris, the cells were fixed with 2 ml of 2%

Results

des enhanced good results

and immunogold-silver elsewhere

for 30 mm

There

daylight

as described

1:10 to 1:20 with

8.2)

before

mixture

performed

KUBO

x 30,000 in six groups: 5-nm gold particles enhanced with the physical developer for 3 mm, 5 mm, and 10 mm, and 15-nm gold particles enhanced for 3 mm, 5 mm, and 10 mm. The density oflabeling (the number of silver-gold complexes/cell) was calculated in 30 SBU-T1-labeled cells of each group at the same magnification.

Monoclonal

opti-

Physical Developer. The physical developer(lntenSE Il)was purchased fromJanssen and contained silver lactate, hydroquinone, trisodium citrate, and citric acid. The silver lactate and hydroquinone solutions and the remiming

MAGARI,

Switzerland).

cal densities of the stock solution at 520 nm were 2.5 for the 5-nm size and 3.5 for the 15-nm size. They were suspended in 0.02 M Tris-buffered saline,

MAXWELL,

were and

diameter)

of silver-gold

The

diameters T1-labeled

ofmore than 500 silver-gold complexes on the surfaces of SBUcells were measured on montages at a final magnification of

Findings.

Microscopic

Most

cells in the control

had no patches of gold particles or silver-enhanced on their cell surfaces, but a few particles were Background with

the

staining physical

study. Lymphocytes distributed in the

was increased developer

with

patches

immunogold

in the cell suspensions

for

reacting

in small

staining

sections

gold particles occasionally seen.

mm

10

the

as in the

MAb

or clusters study

had

on their

(Figure

2).

light

many

enhanced microscopic

gold

particles

surface

membranes

SBU-II

was

positive

on the cell surfaces of all macrophages and some lymphocytes (Figure 3). Lymphocytes possessed round nuclei with peripheral chromatin, many free ribosomes, and mitochondria. There were no morphological E53-labeled in the

and ules.

differences among SBU-T1-, cells. However, large granular

lymph

were

E53-labeled Only

distinguished

cells by their

a few plasma

all the specimens. The IGSS method or 5 mm showed evenly distributed

cells treated

from

SBU-T4-, SBU-T8-, lymphocytes rarely SBU-Ti

intracytoplasmic and

some

with

macrophages

physical

that patches ofsilver-enhanced on the cell surface, having

Downloaded from jhc.sagepub.com at SEIR on January 27, 2015

-, SBU-T4-, electron-dense

developer gold a round

and seen

SBU-T8-, gran-

were for

seen

in

3 mm

particles were or polygonal

IMMUNOGOLD-SILVER

STAINING

FOR

CELL

SURFACE

1217

ANTIGENS

i,l’

\LF

“LI

.,t.!l., S

,.j,

-,p

I.

.

‘&.v

) la

#{149} ‘:

Figure 1. (a) Light micrograph of 1-pm Spurr’s-embedded sections of cells in lymph counterstained with toluidine blue. These cells, exposed to no primary MAb, were incubated in goat anti-mouse lgG conjugated to 15-nm gold particles foir 30 mm and further enhanced with physical developer for 3 mm. There is no specific staining, and only a few tiny grains of background silver are present. (b) Light micrograph of 1-pm Spurr’s-embedded sections of cells in lymph. These cells were exposed to SBU-T, for 30 mm, incubated in goat anti-mouse lgG conjugated to 15-nm gold particles for 30 mm, further enhanced with physical doveloper for 3 mm, and counterstained with toluidine blue. Silver-enhanced gold partides are clearly visualized on the cell surfaces as crenated shapes (arrows). Original magnification x 200. Bars = 50 pm. Figure 2. Electron micrograph of a lymphocyte incubated with SBU-T4 for 30 mm and goat anti-mouse lgG conjugated to 5-nm gold particlesfor 30 mm. Colloidal gold partides are evenly distributed only on the cell surface. Original magnification x 10,000. Bar = 1 pm.

shape

and

not cially

3

ofvarious

sizes

to be encapsulated in the

mm

specimens

(Figure

Table

5).

(Figure

enhanced

However,

1 . Reactivity

4).

in growing

A few

gold

shells

of metallic

with

gold

particles

ofanti-sheep

lymphocyte

SBU-T1

MAb Mean

valuS’

the

73.4

±

particles

physical (5 nm

appeared

silver,

espe-

developer and

MAb

en-

in popliteal

SBU-T4 8.1

51.4

±

with

to form

crenated

the physical

for iO mm

on cell surfaces to assess

the

diameter

on cell surfaces

in these

became

in some and

groups.

aggregated places

the

(Figure

number

The

of

density

lymph

SBU-T8 6.0

developer

structures

6), and it was difficult silver-gold complexes

for

15 nm)

hanced

9.5

±

2.1

SBU-II 24.4

±

E53 7.3

12.2

±

Minimal

value

63.8

45.2

6.3

15.1

6.0

Maximal

value

83.4

58.1

12.7

35.6

19.8

a

Abbreviation: represent

b Data

MAb. mean

monoclonal ±

antibody.

SD.

Downloaded from jhc.sagepub.com at SEIR on January 27, 2015

4.6

1218

OTSUKI,

MAXWELL,

MAGARI,

KUBO

Figure a Electron micrograph of a lymphocyte and macrophage labeled with SBU-ll and the immunogold method. Five-nm gold particles are present on the cell surfaces of the lymphocyte (L) and macrophage (M), and on tube-like structures (arrows) invaginating from the cell surface ofthe macrophage. Original magnification x 12,000. Bar = 1 pm.

‘,k

Figure 4. A lymphocyte labeled with SBUT, and the IGSS method. This cell is one of the SBU-T,-labeled cells seen in Figure 2. Fifteen-nm gold particles enhanced with metallic silver for 3 mm are evenly distributed and clearly visualized only on the cell surface. The size ofthe gold-silver complexes is variable. Original magnification x 8000. Bar = 1 pm.

4.

“-.4,-’

.-.

I

1 -

:

3

__

p

‘..

,

I

‘

..,

-.)

‘5

,

.

i

I

S

4

.

:

=

oflabeling using

for 3

(silver-gold 5-nm

mm

gold

(the

complexes/cell)

particles

5 nm

and 5 mm group, 26.8 ± 4.1 in the

and

25.7 15 nm

was 65.0

enhanced

±

3

mm

with

group),

4.6 in the and

5

mm

the

67.5

15 nm and group.

The

±

3.9 in the

physical 7.3

±

3

mm

density

group

developer in

the

group,

5 nm

and

of labeling

.

in the

groups

using

5-nm

gold

15-nm particles. The range ofdiameters faces

is shown

in Figure

most

frequent

in the

Downloaded from jhc.sagepub.com at SEIR on January 27, 2015

particles

was

superior

ofthe

silver-gold

7. The

151-180-nm

sized

5 mm

the

5 nm

and

and

to that

complexes

on cell sur-

complexes

is

using

nm

and

were 5 mm

IMMUNOGOLD-SILVER

STAINING

FOR

CELL

SURFACE

Figure 5. Electron micrograph of lymphocytes labeled with SBU-T and the lOSS method. Silver-gold complexes vary in size and form. One 15-nm gold particle (arrow) treated with physicaldeveloperfor3 mm appears not to be encapsulated in a growing shell of metallic silver. Original magnification x 15,000. Bar = 0.1 pm.

ANTIGENS

1219

9’

J’-. .‘.‘.t,

:

‘

,

e

#{149}

Figure 6. Electron micrograph ofa lymphocyte labeled with SBU-T, and silver-gold complex. Five-nm gold particlestreated with physical developer for 10 mm tend to cluster on the cell surface and no longer mdicate the probable localization ofthe antigen site. Original magnification x 15,000. Bar = 1pm.

..

.

.‘-,

V

‘ t)’_

.

:‘

I

,‘-

*.

.. ,‘,‘

groups,

whereas

the

size

3 mm and the iS nm and 3 diameter of gold-silver and 3 mm, 5 nm and 128.3

nm,

i30.4

nm,

of most

mm

complexes

groups

was

in the 121-150

complexes in the 5 nm 5 mm, and is nm and i56.8

nm,

and

i55.6

nm,

nm.

S nm

and

The mean

and 3 mm, iS nm 5 mm groups was respectively.

Discussion A few studies croscopic clonal particles

have

described

detection antibodies

(2,4).

was achieved

Downloaded from jhc.sagepub.com at SEIR on January 27, 2015

immunogold

of leukocyte However, only

with

cell surface adequate the

staining

for light

antigens

with

visualization

use of 40-nm

gold

mi-

monoof gold particles

OTSUKI,

1220

NUMBER

0

OF

SILVER-GOLD

35

COMPLEXES

105

70

than was

140

50

w

:

0 81-

120

121-

150

151-

180

nJ..w

C

i,i.#{241}.W)i1i!)l,#{241}.#{189}

.-.-.,.-.-

.WJfliiii/,Y,5JfrDflhiiJJ/.’)5)))#{228}

I

w U) L

data

181-210

211-250

Figure 7. Effect of silver-gold complex sizes on gold particle size and enhancemont time of physical developer. The 151-180-nm sized complexes were most frequent in the groups using 5 nm and 15 nm gold particles enhanced with the physical developer for 5 mm (5 nm and 15 mm, 15 nm and 5 mm), where the complex size in the groups using 5 nm and 15 nm gold particles enhanced with developer for 3 mm (5 nm and 3 mm, is nm and 3 mm) was 121-150 nm. The diameter of silver-gold complexes correlates with the enhancement time of the physical developer rather than the gold particle size.

at a final magnification ofmore than by Yokota (i8) that labeling density

with

nification be counted

of

idine

staining.

x

low background

200,

easily

blue

x 1000. It has been reported was inversely proportional to

gold particles, and that large with problems of background 5- and 15-nm gold particles

on cell surfaces

and

each

in i-tm The

gold particles (40 nm) staining. In our study, were clearly observed

staining

at a primary

lymphocyte

obtained

Spurr’s-embedded percentage

by the IGSS

method.

T-cell

faces by the immunoperoxidase in the sections stained with

lymph

node in and flow

In all specimens,

the

sum

of

which

is SBU-T4

negative

staining method toluidine blue. This

by electron

microscopy

the

and

SBU-

(8).

This IGSS gold partisections with on cell sur-

was barely advantage

visible makes

ultrastructure

of

identical positive cells detected by light microscopy. In our electron microscopic observations, cells with a few gold particles were rarely seen in the control sections, although i% BSA added

considered agent

to prevent

nonspecific

to be owing

on some

of the

cells

in the

staining.

to fixation cells,

by Dc Wade et al. (3). The labeling density ofpositive

that

silver-gold

probably

using

phenomenon

of the goat

anti-mouse

on Fe receptors,

of silver-gold

groups

This

particles.

complexes 5-nm

gold

gold

on the

particles

was

KUBO

This

staining

the density

well with than the

the mean

finding

at the

of labeling

the enhancement size of the gold

diameter

light

of silver-

time particles.

of silver-gold

enhanced

with

greater

we show

were

complexes

the physical

that

not uniform

5-nm

gold

suspended

cells.

Moreover,

developer

the enhancein this study

but varied

in size

enhanced

with

particles

this

of the Our

method

result in IGSS specific stainmakes

evaluation of immunolabeling electron microscopic level.

possible

in the

posi-

Cited Light microscopic tissue.J Histochem

visualization of colloiCytochem 31:1394, 1983

2. Dc Mey J, Hacker GW, Dc Wade M, Springall DR: Gold probes light microscopy. In PolakJM, Van Noorden 5, eds. Immunocytochemistry. Bristol, J Wright and Sons, 1986, 71 3.

in

Dc Wade M, Dc MeyJ, Moeremans M, Dc Brabander M, Van Camp B: Immunogold staining method for the light microscopic detection of leukocyte Histochem

cell surface antigens Cylochem 31:376, 1983

Dc Wade M, Dc MeyJ, B: An immunogold-silver

surface antigens 5. Dc Wade

with

monoclonal

Renmans W, Labeur staining method

in light microscopy.J

M, Renmans

tive detection epipolarization

J

antibodies.

C, Reynaert Ph, Van Camp for the detection of cell

Histochem

W, Segers E,Jochmans

Cytochem

34:935,

K, Van Camp

1986

B: Sensi-

of immunogold-silver staining with darkfield and microscopy. J Histochem Cytochem 36:679, 1988

6. Ellis 10, BellJ, BancroftJD: An investigation ofoptimal particle size for immunohistological immunogold and immunogold-silver staining to be viewed by polarized incident light (EPI polarization) microscopy. J Histochem Cytochem 36:121, 1988 7. Geoghegan

WD, ScillianJJ,

B-lymphocytes

by both

dal gold-labeled

Ackerman

light

and

electron

antiimmunoglobulin.

8. Grossi C: Immunology. involved in the immune Publishing, 1985, 2.8

In Roitt response.

GA: The detection microscopy

Immunol IM, BrostoffJ, London, New

of human

utilizing

Commun Male York,

colloi7:1,

1978

DK, eds. Cells Gower Medical

9. Holgate C, Jackson P, Pollard K, Lunny D, Cohn C: Effect of fixation on T and B lymphocyte surface membrane antigen: demonstration in paraffin processed tissue. J Pathol 149:293, 1986 10.

Holgate

C,Jackson

P. Cowen

P. Bird

new method ofimmunostaining Cytochem 31:938, 1983

re-

surfaces

complexes

1. Danscher G, NorgaardJOR: dal gold on resin-embedded

was

as reported

in the

Literature

for SBU-T4, and identical with our

the

was

Moreover,

in the groups

the semiquantitative tive cells at the

4.

and SBU-T8-labeled cells did not cxcells. These data indicate the presence

subpopulation

to observe

tolu-

subpopu-

T8 negative (12), or of large granular lymphocytes method revealed both cell features and silver-enhanced des on the cell surfaces in i-tm Spurr’s-embedded toluidine blue staining, although the immunolabeling

it possible

with

lymphocyte

SBU-T1, 49% were almost

ing

could

sections

ofeach

(12): 74% for These percentages

the percentage of SBU-T4ceed that of SBU-T1-labeled of a third

gold

the physical developer for 3 mm yield the optimal because of reduced background staining and high

mag-

subpopulation

in the efferent lymph from the prescapular has already been assessed by immunofluorescence

microfluorometry 18% for SBU-T8. data

shown

and form. In our study,

25 1

lation sheep

have

was that

0

the size ofthe were associated silver-enhanced

15-nm

by immunogold

for 5 mm was larger than that in the groups in which ment time was 3 mm. One of the problems noted

,,,,bU,L’J

-,.-‘,

US LU

a

level (6,9).

on cell surfaces

0

cr

using

obtained

gold particles correlated physical developer rather

0

(5

to that

MAGARI,

enhanced gold particles was influenced not by the enhancement time ofthe physical developer but by the size ofthe gold particles. On the other hand, the development of shells of silver-enhanced

15,,,,,&5,,,

51-80

a U

in the groups

microscopic

_

w -J 0.

that similar

MAXWELL,

11.

12.

CC:

Immunogold-silver

with enhanced

sensitivity.J

Lascelles AK, Morris B: Surgical techniques for the collection from unanesthetized sheep. Q J Exp Physiol 46:199, 1961 Mackay

of sheep

CR.

MaddoxJF,

T lymphocytes.

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MR:

Eur J Immunol

Three

staining:

Histochem of lymph

distinct subpopulations 16:19, 1986

IMMUNOGOLD-SILVER

13.

Mackay ization

15.

Immunology

MaddoxJF, SBU-T8 bodies. Otsuki venules electron

FOR

CELL

SURFACE

Mackay

55:729,

16.

Pun NK, Mackay CR, major histocompatibility 1985

17.

RothJ:

1985

CR, Brandon

of sheep T lymphocyte Immunology 55:739,

1221

ANTIGENS

CR, MaddoxJF, Gogolin-Ewens KJ, Brandon MR: Characterof two sheep lymphocyte differentiation antigens. SBU-T4 and

SBU-T6. 14.

STAINING

MR: Surface

subsets 1985

Y, Magari S: Lymphocyte and lymphatic capillaries microscopic study. Acta

defined

antigens,

SBU-T4 and

by monoclonal

subpopulations in high endothelial in rat Peyer’s patches: an immunoHistochem Cytochem 21:3, 1988

Applications

tion ofprotein

anti-

cation tions. 18.

Brandon MR: Sheep lymphocyte antigen complex class molecules. Immunology

ofimmunocolloids

A-silver

for localization

J Histochem

and protein ofsingle

Cytochem

Yokota 5: Effect ofparticle tein

A-gold

and multiple 30:691, 1982

size on labeling

immunocytochemistry.J

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in light

A-gold

Histochem

microscopy.

complexes

Prepara-

and their appli-

antigens

density

(SLA) 56:725,

in paraffin

for catalase

Cytochem

36:107,

sec-

in pro1988