0022-1554/90/13.30

The Journal of Histochemistry and Cytochemistry Copyright © 1990 by The Histochemical Society.

Vol. 38,

No.

3,

Inc.

Rapid

pp. 307-313, 1990 Printed in USA.

Communication

A Reliable Method for Simultaneous Demonstration Two Antigens Using a Novel Combination of Immunogold-Silver Staining and Immunoenzymatic Labeling’ REINHARD The

Walter

GILLITZER,2 and

E/:za

Ha/I

Department

of Dermatology

University

of Erlangen-Nurnberg

Received

for publication

RUDOLF

Institute

ofMedica/

I, University Medical

November

BERGER, Research,

of Vienna School,

and

Melbourne,

Medical Federal

15, 1989; accepted

School,

Republic

December

We have developed a reliable and sensitive immunohistochemical staining technique which allows the simultaneous demonstration of two different antigens expressed in or on the same cell (referred to as mixed labeling), together with the evaluation of the general histopathological appearance of the tissue. The staining procedure combines a three-step (streptavidin-biotin) immunogold-silver staining (IGSS) with a three-step immunoenzymatic labeling. For this purpose, we investigated the compatibility ofIGSS with various substrates ofperoxidase or alkaline phosphatase (AP). Highly reliable and discernible mixed labeling was achieved only after initial labeling with IGSS followed by AP labeling using the substrates naphthol AS-MX phosphate/Fast Blue or naphthol AS-BI phosphate/New Fuchsin, respectively. To

Introduction Double immunoenzymatic labeling (IEL) of cryostat sections has become a widely applied technique in immunohistology since its first description (23). Its major advantage is based on the possibility of studying both labels simultaneously. This is in contrast to double immunofluorescence (IF) procedures (17,30), in which the use of different filters and the invisibility of the general histology during fluorescence microscopy constitute considerable disadvantages. In addition, the fading ofcolor signals does not allow longterm documentation and retrospective evaluation of IF-stained tissue samples. On the other hand, the use of double IEL methods for identification of two different surface or cytoplasmic antigens of the very same cell or of other tissue components (referred to as mixed labeling) is associated with fundamental difficulties that have not yet been

overcome

(4,19).

Thus

far, all the enzymatic

color

reac-

HEIDRUN

Victoria

3050,

Austria

(RG,

of Germany

I

Supported

by the Deutsche

Forschungsgemeinschaft,

Federal

Repub-

MOLL Australth RB);

(RG,

and

HM);

institute

for

Clinical

Microbiology,

(HM).

1, 1989 (9C1846).

ensure utmost specificity, we applied Flit-conjugated mouse monoclonal antibodies and rabbit anti-Flit immunoglobulins visualized by AP-labeled immunoglobulins and the respective substrate in a final step. This novel approach provides an excellent means for demonstration of immunocompetent cells and unequivocal determination of the percentage of specific cell subsets in infiltrated tissue. The advantages of this method, as compared with double immunofluorescence or double immunoenzymatic labeling, were investigated and are discussed. (JHisrochem Cytochem 38:307-313, KEY

1990) Immunohistochemistry;

WORDS:

Flit-anti-Flit

system;

Immunogold-silver

Leucocyte

subpopulations;

staining;

Two-color

staining.

tions that have been combined either interfered with each other or resulted in markedly decreased sensitivity (24). These are the major reasons for the more frequent use ofdouble IF staining procedures despite the disadvantages mentioned above. In a variety of diseases, analysis of immunocompetent cells infiltrating the affected tissue is important for understanding the pathogenetic mechanisms (25,26,28). In addition to the identification ofvarious lymphocyte populations (B-cells, CD4 and CD8 T-cells), the demonstration of lymphocyte subsets expressing memory or activation antigens (interleukin-2 receptor, HLA-DR antigen,

CD44,

CD45RO,

transferrin

Our

interests

focus

on the analysis

disorders orders)

(acute

and chronic

to evaluate

their

further insight come effective. mixed

tic of Germany. 2 Correspondence to: R. Gillitzer, Department of Dermatology I, University of Vienna Medical School, Alserstr. 4, A-1090 Vienna, Austria.

of

labeling

role

receptor)

is ofutmost

of these

skin inflammation, in the course

we investigated

significance. in various

autoimmune of disease

into the immunopathological In view of the importance system,

subsets

and

skin

disto gain

mechanisms that befor these analyses of a the

pssibility

of a novel

staining protocol combining an IGSS (9,15) with an immunoenzymatic method. We evaluated the various combinations of IGSS and either peroxidase (POX) or alkaline phosphatase (AP) labeling, using different substrates for the enzymatic reactions. Our 307

Downloaded from jhc.sagepub.com at SUNY HEALTH SCIENCE CENTER on March 20, 2015

308

GILLITZER,

results

demonstrate

that

lowed

by AP staining

phate

(NAMP)/Fast

three-step

monoclonal ulins the

IGSS

sensitivity

discernible. tioned

This

obtained

frozen thawed

to block

nonspecific

taming

5% receptors,

ature

(RT) Mouse

MAb jugates

milk

MAb,

(Miles

the aforemen-

from

from

OCT

in a solution

and

0.1%

Tween

20 in PBS,

sheep

Polyclonal

in Table

mm

pH

7.4.

used

sources

and

(Sigma

(AEC)

( 7)

and

For

IGSS

Bronckers

incubated

with

hr (overnight) three

times

to remain

Table

(Sigma)

et al. (6) was used

at 4C. and

in all experiments.

antibodies

Thereafter,

Antibodies

for

and

(for

the slides

the biotin-conjugated sections

according

second-stage

1 hr at RT

The

dilution

were After

to Coggi

thoroughly antibodies a brief

wash

were

and

hum

(NBT)

(Sigma)

for

0.5 mg/mI

the

DAB,

reaction,

AP

0.01%

Anti-HLA-DR FITC-conjugated (UCHL-1)

Anti-CD45RO

(UCHL.1),

Flit-conjugated

(Sigma;

The

cobalt

The

NAMP/Fast

Red and

in Tris buffer(0.1

and

of0.5

0.5%),

New

(stock

pH

9.0).

The

dissolved in 70%

1 mg/mI

Blue

NAPB

The

(Sigma), Darm-

(12,16)

con-

imidazole

6 mM sodium solution

BCIP/NBT

nitrite,

diluted

solution

substrates

contained

onto

0.38

and

and

5-20

M,

(50 mg/mI

mg/mi color

(w/v)

(0.2 dissolved

(100 mM,

and

after

4%

buffer

M BCIP

saline

the slides

2 mm in the case ofDAB

after were

(22) (final

in Tris

0.4 M NBT(75

filtered

respec-

solution

levamisole,

in Tris-buffered

were

2%),

TR salt,

Fuchsin

1 mM

in N,N-dimethylformamide)and solutions

0.2 mg/mI

concentration

in 2 M HC1)

(20)

N,N-dimethylformamide)

substrate

contained

in N,N-dimethylformamide

pH 9.5).

development

mm

when

other

used.

were

in Blotto allowed

in Blotto,

Combination

ing with to give

all

RT);

ofIGSS

IGSS the

mouse

and

best MAb

With

IEL,

results:

the Blotto

for first

POX/AP

following

Labeling. sequence

containing

(IGSS)

For the

mixed

of treatments

20%

labeling

sheep

label-

was shown

serum

(overnight,

(20-30

4’C);

mm,

biotinylated

Source

1/30-1/100

Becton-Dickinson

1/60-1/200

Becton-Dickinson

1/30-1/60 1 /60-1 / 100

Dakopatts

(Copenhagen,

Becton-Dickinson Becton-Dickinson

Anti-Leu-4

(CD3),

FITC-conjugated

1/30-1/100

FITC-conjugated

1/30-1/

1 / 500-1/

Becton-Dickinson

1000

East

Amersham

anti-rabbit

1g. POX-conjugated

1/200

Goat

anti-rabbit

1g. AP-conjugated

1/200

Accurate

1/200

Jackson

Chemicals

Accurate

Chemicals

gold

Streptavidin-conjugated

Texas

1/40-1/50 Red

1 / 50

Downloaded from jhc.sagepub.com at SUNY HEALTH SCIENCE CENTER on March 20, 2015

Janssen Amersham

UK) (Southbridge, (San

Chemicals Immuno

(Avondale, Streptavidin-conjugated

Denmark)

(Amersham,

Acres

Goat

FITC-conjugated

CA)

Becton-Dickinson 100

1/ 100-1/200

Ig

(Sunnyvale,

Dakopatts

100

Ig,

and

tetrazo-

solution

NABP/New

dissolved

100

anti-rabbit

Red or Merck,

(final

1/60-1/

Donkey

Tris rinsing

6.8 mg/ml

solutions

1/30-1/

anti-FITC

Blotto,

with

Fast Red TR salt or Fast Blue

M, pH 8.2).

mg/mi

concentration Fuchsin

NAMP/Fast

in N,N-dimethylformamide

FITC-conjugated

Rabbit

DAB

chloride,

(HLe)

1g. biotin-conjugated

or AP. Between with

(BCIP)/nitroblue

(HLe),

Anti-mouse

rabbit

diaminobenzidine

NAMP/Fast

Anti-CD45

(CD8),

an-

with

3-amino-9-ethylcarbazole

and

Anti-CD45 Anti-Leu-2a

un(10).

and 0.03% (v/v) H202 in PBS (pH 7.4), and the AEC solution (13) contained 0.2 mg/mI AEC, previously dissolved in N,N-dimethylformamide (final concentration 5%), 0.015% H202 in acetate buffer (50 mM, pH 5).

Dilution

Anti-CD45RO

POX

Fuchsin

reaction. (w/v)

labeled

the substrates and

was

FIlE-conjugated

of substrates,

MO)

Belgium)

illumination

rinsed

bromochloro-indolyl-phosphate

tively,

are

addition

buffer enhance-

was monitored

with were

silver

Olen,

with

sections

NABP/New

7.6) gold

Conjugates

Antibodies/complexes

Anti-HLA-DR,

Life Science;

with

M, pH

in Tris rinsing prepared

Ig conjugated the

POX

FRG),

1) for 10-14

washed

the

stadt,

consisted

et al.

sections

see Table

for

levamisole,

1. procedure

(0.05

were subsequently

St Louis,

Blue (Sigma),

The

a three-step

the first-step

on the

1.

(8),

washes freshly

incubated

was visualized

1 mM

con-

were

before

Chemicals;

NAMP/Fast

was stopped IGSS.

step,

con-

All and

three

at 4’C and anti-rabbit

washing

dissolved

temper-

all reagents

of antibodies

final

NAMP

Blotto

Conjugates.

buffer

MOLL

Streptavidin-conjugated

water,

II Uanssen

sections

sheep

and

For blocking

at room

and

are commonly and

slides

serum.

Antibodies,

dilutions

San Diego,

stated.

for 20-30

20%

study

otherwise

after

step (RT, 1 hr),

The labeling

(29),

(Blotto)

Ig and

buffer.

IL) using were

The

(DAB)

tamed

compound

rinsed

unless

atopic

Gelatin-coated

SE TM

1)ovemight

each incubation

was snap(4 jim)

Chemicals;

10 mm). slides

inflamed

Naperville, sections

were

pre-incubated

summarized

suffering

of the material

they

were

The

patients

(Accurate

To clear

containing

in the present

of

Scientific;

(4’C,

steps

Conjugated

specimens

(0.2%).

washes in distilled

IEL.

anti-FIlE in the

IF.

biopsy

in Tris rinsing BSA

1 hr at RT, and

Intense

POX/AP

are clearly

double

Cryostat

Histostik

in acetone

powder

available.

are

and

Some

nitrogen.

washing

sections

applied

from

fungoides.

for IGSS.

in Blotto

commercially

histology

with

fixed

in all further

ofFc

human

reactions,

skim

IEL

for

mixture

performed

20(O.1%)and

by three

tibodies(lble

irnrnunocornpe-

combines

overlaid

were

applied for 5-12 mm. The silver enhancement process der the microscope using brightfield or epipolarization

Ig ensured

As a result,

Fresh

in liquid

and

recommended

was used

double

OCTcompound

onto slides coated

are not

label.

Tween

ment

irnmunoglob-

anti-rabbit

washes

followed

of FIlE-conjugated

anti-FIlE

therefore

mucosis

pre-cooled

air-dried,

and rabbit

Sections.

and

in Tissue-Tek

CA),

application

following containing was

(NABP)/

for one or for two antigens

both

folphos-

mixed labeling. All other cornsatisfactory results. In addition

second

for routine

eczema,

isopentane

IGSS,

AS-MX

Methods

O(Skin

dermatitis,

with

phosphate

by AP-labeled

method

of

Preparation were

(MAb)

novel

and

Materials

AS-B!

the

are positive

advantages

skin

(6,7),

of the

cells that

staining naphthol

or naphthol

detected

(14,31,32)

high

tent

initial

substrates

in excellent not produce

antibodies

(Ig)

the

the

Blue

New Fuchsin, resulted binations tested did to the

only

with

BERGER,

(Olen,

Research PA) Belgium)

Diego,

MA) CA)

PAIRED

IGSS-IMMUNOENZYMATIC

Figure

(A) Dermal

1.

HLA-DR

antigen

vessels

expression

DOUBLE

stained

for

by a three-step

Bar

incident =

Figure

polarized

light

2.

(A) Skin

-

.

J

I

1A

reflection.

lB

section

with

mycosis

fun-

for CD3

with IGSS and for HLA-DR with AP-NAMP/Fast Blue. The figure shows mutually exclusive expression of the two antigens, with only a few cells being doubly positive (arrowhead). Brightfield illumination. (B) NAMP/Fast Blue labeling does not show incident polarized light reflection and therefore does not interfere with IGSS. Bar = 25 m. stained

brightfield

and(D)

illumination.

Bars:

under A

: :: T:.

.

.‘

.

4:

,

‘;;I

.Tc

t’jE’....

sm.

,,

‘

IC

16

=

i,

,;

epipolariza-

25 rim; C

=

l

I

Figure 3. Double staining with IGSS (CD8) and AP-NAPB/New Fuchsin (HLA-DR) in a skin lesion of atopic dermatitis (A) under brightfield illumination. (B) Only a few cells co-express both antigens and are therefore IGSS positive (epipolarization illumination). (C) The same tissue stained with AP-NAMP! New Fuchsin (CD45) and IGSS (HLA-DR) under

,*

25 Hm.

goides

tion

.

.ka

:vft

EL with a final AP-conjugated Ab and NAMP/Fast Red as substrate; hematoxylin counterstaining. (B) The same area as in A under epipolarization illumination shows an intrinsic shining ofthe NAMP/Fast Red color product similar to IGSS. (C) Dermal vessel stainedfor HLA-DR antigen as in A, but with NABP/New Fuchsin as substrate; hematoxylin counterstaining. (D) Same area as in C under epipolarization illumination shows no intrinsic

309

LABELING

1:1’

f

2A

sheep anti-mouse (1 hr. RT); blocking conjugated

MAb

RT);

anti-Flit

rabbit

Ig (1 hr. RT), strate

(2-20

tion,

should Double

for the

IGSS mm).

Ig antibodies with 10% second

labeling

Ig (1 hr. RT); enhancement The

IGSS

be monitored IF.

(1 hr. RT); mouse serum

For double

enhancement, time

(5-12

ofstreptavidin-conjugated

gold

donkey

anti-rabbit

(Table

Ig antibodies

(overnight,

4’C goat

mm,

RT);

RT);

(see Table 1) instead

under

gold Flit-

or 2 hr at

POX/AP

2B

Counterstaining

sub-

and

counterstained mount; equipped

anti-rabbit

as well as the AP-substrate to time

IF, streptavidin-conjugated

instead antibodies.

(IEL)

POX/AP-conjugated

mixture from

streptavidin-conjugated in Blotto (20 mm,

.

4:

with

BDH,

Poole,

UK).

for polarized

Oberkochen,

FRG)

Microscopic

hematoxylin,

light

The sections

ofSections.

indicated

For evaluation

incident was

Analysis where

and

a Zeiss

mounted Axiophot

(epipolarization)

(Gurr

were Aqua-

microscope

microscopy

(Zeiss;

used.

reac-

a microscope. Texas

1), and

Red was used

Flit-conjugated

of enzyme-conjugated

Results Single

IGSS

For one-color

and staining

Single with

Downloaded from jhc.sagepub.com at SUNY HEALTH SCIENCE CENTER on March 20, 2015

IEL either

IGSS

or IEL,

our

results

confirmed

GILLITZER,

310

previous

reports

cedure

used

IEL

very specific

MAb.

we found

could

Epipolarization

compensate is comparable in terms

three-step

prosystem

beneficial

high

to three-step

for the signal-

primary

MAb

to fivefold

although

lowering

for IGSS,

of the

three-step

streptavidin-biotin

as substrates

well in terms

de-

which

methods

of sensitivity.

incubation”).

sequently,

Combined

When, staining

after establishing the conditions for IGSS and IEL, both protocols were combined for double labeling, the sequence

of labeling

was found

Performance nals ofcells

of IEL before already labeled

not shown). on the color cific color before

the more

signals

ofthe

without

to be critical

This can substrates enzyme

ble mixed enhancement

tion.

The

was obtained LEL. The

antigen

latter

resulted

products.

In contrast,

ofIGSS in double

the IGSS specific

For these

procedure

sections.

and stronger IGSS

reasons,

by IEL, whereby

the sil-

before the AP/POX-substrate with POX labeling, using

reacDAB as

strated

the high

specificity

By this

technique,

with

substrate

poorly AEC

discernible

yields

color

product and therefore appeared more suitable to be combined IGSS. However, the POX reaction with AEC almost completely

with neu-

the IGSS and resulted in insufficient staining. In summary, the two most widely used POX substrates are not compatible with

Blue

we applied (“reversed

expression

distribution

ofthe and

This

presented doubly

was traced

yellow

of single-

result

clearly

mixed positive

epipolarization

cells displayed

bright

grains,

lesions

(Figures

by staining

5A, and

and the relatively

high proportion

CD45RO, label.

is unambiguously

which sensitivity

for CD3

SB). CD34

of mixed

seen

cells addition-

with

primary

higher

Ab,

the second antigen, two to three times

however, higher.

the

CD45RO by IGSS,

by the additional

was compared

the sensitivity

for tracing

and

cells are co-expressing

for which AP and gold conjugates were replaced Red conjugates, respectively (data not shown). of the

NAMP/Fast

tool to define Using this techT-lymphocytes

or CD8

ofactivated

labeling

first

blue

AP

to double

IF,

by FIlE and Texas As judged by the

of IF was similar antigen.

the sensitivity

be

single-

a shift ofthe bright (Figures 4C, 4D, 5A,

cells are visualized

seen

even

IGSS

In summary, the mixed labeling is a powerful singly and doubly positive cells in frozen sections. nique, we can analyze the distribution of activated in inflammatory

method.

could

whereas

Blue)showed blueish color

and

demon-

labeling cells

illumination.

and SB). This beneficial effect was primarily Blue, which was therefore used preferentially.

dilution

equally

out whether this mixed enough to detect anti-

HLA-DR

not shown).

singly under

to two times

a red reaction

performed ofNAMP/Fast

To find sensitive

an identical

(data

ally labeled with AP(NAMP/Fast yellow color towards a bright

The

for mixed

NABP/New

on immunocompetent cells, in two reciprocal combinations

We found cells

expression

dou-

staining

the POX

precipitation a highly spe-

out first in all further

followed

performed

combination

yields

was carried

experiments,

substrate,

using

to be detected.

enhancement was

nonspecific IGSS sigofPOX or AP(results

be partly explained by silver of the preceding IEL. However,

sensitive

labeling

ver

and sensitivity.

for specificity

IGSS produced with the substrates

reaction

second

silver

JElL

and

substrates

the preparation

For example,

double-labeled

labeled

IGSS

Both

but

Blue

MOLL

and CD45 expression with AP, as well as vice versa. Suball the singly and doubly positive cells were counted in

IGSS

both

AP.

consuming. is specific and

procedure

distinguished

Vith

for

ofsensitivity,

to be utilized

NAMP/Fast

and less time

is simpler labeling

with

ofMAb

using

order

the

POX/AP

Fuchsin

combinations

examined

with

not fully IEL,

various

were

used higher

could

Therefore, labeling

gens that are co-expressed the corresponding MAb

stain-

for IEL as compared

antigens

sensitivity

buffer

background

in a three-

microscopy,

the

the

sensitivitiy be used

showed ratio

as rinsing

For all the

ofgold-labeled

for

of Blotto

reduced

procedures. higher

threshold

The

FITC-anti-FITC

particularly

a significantly

tection

an

use

it significantly

For IEL, the MAb

dilution.

on

The

in preparation)was

to-noise ratio, because ing for both labeling IGSS.

(6,7,10,15).

based

the fluorochrorne as a hapten, and regardless of the fluorochrome/protein

fluoresceinated

(manuscript

methods

was

employing staining

(14,31,32)

of the

on these for

BERGER,

or up

For detection

of mixed

labeling

of was

tralized

IGSS for double mixed labeling. For this purpose, in the next series

ofexperiments,

ing protocol

determinant

which

would

commonly

for the second be suitable used

NABP/New

for combination

substrates,

Fuchsin,

perfectly visualized could be expected,

and

with

NAMP/Fast BCIP/NBT,

Red, were

the antigenic determinant the dark purple color

four

most Blue,

applied.

All substrates

(e.g., product

HLA-DR). As of BCIP/NBT

only

before

NAMP/Fast

IEL Blue

Among

many

AP-specific

and NABP/New

Fuchsin

substrates revealed

sig-

color

to IGSS. Under epipolarization sin (Figure 1D) and NAMP/Fast any intrinsic polarized well with IGSS under

microscopic

is performed

tested,

brown

incident

light

IGSS

is althat

detected by IGSS and supports the The designed method does not require

however,

under

tion of antigens co-expressed on the same cell. This technique referred to as mixed labeling. The staining protocol developed lows highly specific and sensitive double labeling, provided

brightfield illumination, (Figure 1A) and could polarized

IGSS

The

NAMP/Fast

a novel combination of IGSS and unambiguous demonstra-

Under visible

ofIGSS;

from

IGSS.

In the present study we demonstrated with IEL which allows simultaneous

visualization and therefore is not an appropriate substrate for mixed labeling. NAMP/Fast Red was not suitable for this purpose either.

uct reflected

to discriminate

an AP stainwas established

that did not interfere with IGSS under either brightfield or epipolarization illumination. The use of FIlE-conjugated primary MAb for IEL (31,32) prevents any crossreactivity with the antigen

was

difficult

antigenic

Discussion

nals

its red reaction product was clearly be expected to contrast well with the the NAMP/Fast light

(Figure

Red reaction

illumination, only NABP/New Blue (Figure 2B) did not

light reflection and brightfield illumination

prod-

1B) in a way similar

nevertheless (Figures

Richexhibit

contrasted 2, 3,

and

4).

with risk

haptens ofloss

such

as arsanilic

ofantibody

activity

acid

specificity conjugation (11),

or inefficient

which

of the procedure. ofprimary MAb bears

coupling.

the

inherent

The applica-

bility and practicability is facilitated by the utilization of commonly and commerically available substances. For this reason, it can easily be applied As compared

for

routine work and diagnostic with double IF techniques (24),

Downloaded from jhc.sagepub.com at SUNY HEALTH SCIENCE CENTER on March 20, 2015

purposes. this

new method

PAIRED

IGSS-IMMUNOENZYMATIC

Figure 4. Double staining and HLA-DR(AP-NAMPIFast section

DOUBLE

311

LABELING

for CD45 (IGSS) Blue) in a skin

ofmycosisfungoides.(A)A

high

per-

centage ofcells are doubly positivefor CD45 and HLA-DR under brightfield illumination and (B) under epipolarization illumination. (C, D) The same section with higher magnification. Single IGSS-positive cells show yellow

grains,

whereas

double-labeled

are light blue. Bars: A

25 tm; C

=

cells

16 tm.

=

Figure 5. Double staining for CD3 (IGSS) and CD45RO (AP-NAMP/Fast Blue). Detail with two CD3 lymphocytes. One lymphocyte is activated (CD45RO ), demonstrated by the additional blue label (A), which appears light blue under epipolarization illumination (B). Bar = 16 tm.

2k*:t: (

‘.4.

,-‘e,

4C./’:a:,. -

#{149}::,‘

.*

‘t

:

Ji

%i;

5A

offers the

several

important

labeling

mixed

although

switching

nation tine

details

IEL do not fade

and

re-examination

three-step

has

allow

section

second

was two

a slightly been

of IGSS

a mixed

lower

in several

The

IEL should

mixed

to

stain

labeling

the

less

achieved

and

and

to IF, IGSS

documentation

IEL.

stained

cells

is hardly

an appropriate In our hands, continuous

change

general,

mixed

using

FIlE-

plication

and

has been

a simi-

(4,17,19).

The

problems

as compared (1),

combination

however,

with

whereas

IF.

others

for IGSS, especially when intracel(6,15). The difference in sensitivbe taken is being

used. antigen

brown

into

account For by

IGSS

this

when reason,

IEL.

and

well-

in the

present

to be most

with

only

study,

the

enzyme

mutual

it is more

laborious,

tion ofglucoxidase

time

reliable

above

one

can

AP,

find

IEL (2),

consuming, not seem

and

much

by several

In ap-

authors using

the

than IEL and

POX,

appears

IGSS,

because

POX activity. The basic labels offer the advantage

This would and

Downloaded from jhc.sagepub.com at SUNY HEALTH SCIENCE CENTER on March 20, 2015

to

IEL. As described

rather with

any endogenous between both

(21)does

espenot alsingle-

unfeasible.

be avoided

appropriate

labeling

interference.

of autoradiography

it is difficult

assessment

as not

and

bination

of better

does from

IEL has not gained

mentioned

it does not interfere with chemophysical differences

of avoiding

making double

described

for mixed

and

to evaluate,

antigen that distinction

In addition,

of IGSS suitable

visible

is difficult

enzyme combination for double IEL. of blue and red dyes often resulted

of hue,

labeling

than Ag,

is clearly

latter

possible.

labeling first

The

substrate and the combination

sensitive

of IGSS

with

or red AP labeling

mixed

more

of the

abundant

than

cially in the case of a low-density second low strong labeling. As a result, unequivocal

in

date.

approaches

therefore protocol

blue

quality

the discrimiuse of rou-

as opposed

contrasting

mixed

staining

sensitivity

double-labeling

it is advisable

times

of

increases

long-term

antigen, For

encountered

and

products

(brightfield),

irregularities

at a later

to three

revealed a fairly high sensitivity lular antigens had been labeled ity

Third,

therefore

IF procedure.

we found This

ofthe

MAb

pathological

be revealed.

and

of the

For detection conjugated

color

lighting

illumination

Therefore,

can easily

and

both

the same

Second, this method enables morphology with concomitant

stains.

histological

notable

First,

under

to epipolarization

the IGSS signal. ofcell and tissue

clarifies

lar

advantages.

are visible

also apply but

hazardous.

appropriate

for the corn-

in contrast

to IGSS

The

applica-

for mixed

labeling

312

either,

because

label

of adjacent

ble

its diffuse

color

cells which,

localization

may

as a result,

may

interfere

with

be mistaken

method

the

as dou-

nm),

do not exhibit

tion

illumination.

cells

and

steric This

even

allows

We could

levels

of HLA-DR

high

density

that have previously which, unlike larger hindrance,

the

antigen

of shining

under

the detection

semiquantitative CD4Y

epipolariza-

assessment

grains.

The

illumination)and

different

contrast

IGSS

be used for computerized automated image analysis. both mixed labeling and double IF allow documentation and-white photographs, documentation.

whereas

double

IEL

It may be beneficial, for time reduction, labeling for use with a novel capillary action cently developed been demonstrated of

two

time

can

reagents The

also

(e.g.

present

be

achieved

by

streptavidin-gold

,

technique,

the demonstration tigens

hormone

production ofviral

in situ hybridization

taneous present

with

in situ detection communication

need

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able

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Literature 1. Allen

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