An Improved Immunocytochemical Method for ...

0022-1554/87/$3.30 Thejournal Copyright

of Histochemistry and Cytochemistry 1987 by The Histochemical Society,

©

Vol.

OLA and

NILSSON,2 LABS

Institute France

Sweden;

Artide

DAHLSTROM,

MICHEL

GEFFARD,

(ON,

AD),

Laboratoire

Department

ofSurgery

de Neuroimmunologie,

I (HA),

Institut

and Department

de Biochimie

for

publication

May

20,

1986

and

in revised

form

September

Serotothn-like immunoreactivity (5-HT-LI) has been localized at the ultrastructural level in enterochromaffin (EC) cells of rat gastrointestinal tract. Ultra-thin sections of tissues embedded in epoxy resin were incubated with 5-HT antisera and antibody binding sites were visualized with protein A-gold. Three different antisera were compared and were shown to require different fixation regimens for optimal preservation of5-HT-LI. For one antiserum, tissues fixed in glutaraldehyde and osmium tetroxide could be used to demonstrate 5-HT-LI in EC cells. Immunocytochemical localization of 5-HT can thus be performed with good ultrastructural preservation of tissues. Quantitative evaluation of the intracellular distribution of 5-HT-LI was performed on EC cells from antrum, duodenum, and

localize

terochromaffin

5-HT

(EC)

amounts

storage

cells

of 5-HT

munocytochemically

of

antisera

study

of 5-HT-like

immunoreactivity

trointestinal tract. ized over the dense

(23),

we

(5-HT) antibodies it has become sites

in

tissue

gastrointestinal

(3,4,11),

with

In a previous

have

reported

recently

however,

ervation dehyde

been

which

of the fixation

difficult

subcellular

(5-HT-LI)

to determine

ultrastructure

of Goteborg, Bordeaux,

and

omission

of osmium

September

15,

1986

(6A0758).

(J

Cytochem KEY

35:319, Rats;

WORDS:

1987) Enterochromaffin

Immunocytochemistry;

aim

ofthe

preservation

(EC)

Protein

present

oftissues

stration of 5-HT rat gastrointestinal

and

A-gold;

study

cells;

Serotonin

Electron

was to improve

processed

(5-HT);

microscopy.

the ultrastructural

for immunocytochemical

demon-

to re-evaluate, in well-preserved tract, the subcebbubar localization

EC cells of of 5-HT-LI.

store

studied

im-

Materials Tissue

localization

in EC cells

of rat gas-

of the 5-HT-LI was localgranules, but a significant

because

resulting

En-

accepted

and

Methods

from

the dense in EC cells

of non-optimal

the

need

pres-

for formal-

tuna,

Sweden),

Thirty

weight

Supported by grants from the Swedish Medical Research 2207, 5220), the Medical Faculty of Goteborg University,

Council Dr. TAA

Amundson’s Ikundation, OE and E Johansson’s lkundation, and G#{246}teborg Medical Society. 2 Correspondence to: Ola Nibsson, Institute of Neurobiology, University of Goteborg, POB 330 31, 5-400 33 Goteborg, Sweden.

male

100-150

Sprague-Dawley

g, were

used

rats (ALAB;

for the

Soblen-

experiments.

The

in-

structions for care and use of laboratory animals given by the local Ethical Committee for Laboratory Animals (Goteborg) were followed. Rats were fasted overnight with free access to water before they were anesthetized with sodium pentobarbitab (50 mg/kg IP) and fixed by perfusion via the ascending aorta. After brief pre-perfusion with warm (37’C) isotonic saline containing lidocaine (0.1%), rats were perfused for 4 mm with 200-300 The

tetroxide.

Preparation.

ml of freshly

following

prepared

fixatives

from

M sodium

grade; +

GA

cacodybate, periment, mm

and

used

(cf.

paraformaldehyde saline

at room Table

1):

temperature

(Riedeb-.de

Haen

containing

0.02

(PBS)

(18-20’C).

formaldehyde

4%

AG;

(FA),

Hanover,

FRG),

M phosphate

and

0.15

pH 7.4; 4% FA #{247} 0.5% glutarabdehyde (GA; practical AG, Switzerland) in PBS; 4% FA + 1% GA in PBS; 3% FA

chloride,

Fluka

2%

fixative

were

in phosphate-buffered I

CNRS,

colon, fixed in glutaraldehyde only. In all three the majority of the gold partides (90%) in EC cells were localized over the dense core ofthe secretory granules, while a minor fraction (10%) were localized in parts of the cytoplasm devoid of granules. In EC cells fixed in glutaraldehyde and post-fixed in osmium tetroxide, 5-HTLI was reduced by about 85%, although intracellular distribution was essentially the same as in cells fixed in glutaraldehyde alone. The results indicate that 5-HT in EC cells is stored mainly in secretory granules, with a small fraction of 5-HT being localized outside the granules. Histochem

prepared

(537,

University

du

(10,12,17-19,22,23,26,33).

on

We found that 60% core of the secretory

sections.

tract,

to 5-1-IT

in impossible

fraction of the 5-HT-LI (40%) was localized outside cores. The exact intracellular distribution of 5-HT-LI was,

(LEE),

et Neurochimie

proximal locations,

The

With the introduction of serotonin munocytochemistry (6,7,20,24,31,32,34),

large

AHLMAN,

ofAnatomy

Cellulaire

9, 1986;

Introduction

directly

HAKAN

(MG).

Received

to

1987 USA.

ERICSON

ofNeurobiology

Goteborg,

319-326,

Immunocytochemical Method for Subcellular of Serotonin in Rat Enterochromaffin Cells1 ANNICA

E.

3, pp.

Printedin

Original

An Improved Localization

35, No.

Inc.

in PBS;

2%

FA

+

pH

and

5%

GA

rats then

7.2;

were with

perfused a mixture

3%

in PBS;

in 0.05

in two ofFA

and

2.5%

GA

M sodium

steps,

first

in 0.05

cacodylate. with

GA for another

4%

M sodium In one

FA in PBS

4 mm.

After

cxfor 4

perfu-

319

Downloaded from jhc.sagepub.com by guest on September 12, 2016

320

NILSSON,

Table

1 . Summary

localization

offixatives

ofserotonin

testedjor

in

Primary

EC

Post-fixation

A. 4% FA

and

4%

FA

+

0.5%

4%

FA

+

1%

3%

FA

+

2%

GA GA

2%

FA

+

3%

GA

2.5%

GA

GA

0.1-1%

±

by 4% FA

0.5%

+

acetate

FA

+

1%

3%

FA

+

2%

GA GA

2%

FA

+

3%

GA

(21);

(b) etching

0.1 M hydrochloric

distilled

water

60 mm,

followed

sections

were

A-gold.

by extensive

incubated

ERICSON

AHLMAN,

saturated

for

sodium-M-periodate

10 mm,

followed

with saturated

rinsing

the

diluted

water

antisera

rabbit/rat

showed

no specific

sections

in

sodium-M-periodate

in distilled

or normal

for

by washings (5).

absorbed

serum

etched

borohydride controls, with

followed

labeling

for

Some

were treated with 1% sodium incubation. Rr immunocytochemical with

sulphate

Control

with acid

and (c) etching

(9);

HI-creatinine

GA

4%

water

and

and un-etched sections (NaBH4 for S mm before

OsO,

1 % uranyl

B. 4% FA followed

distilled

10 mm

5 % GA

GEFFARD,

water. Finally, sections were contrasted with uranyl acetate and lead citrate. Some sections were etched before incubation with 5-HI antiserum. Three different procedures were tested: (a) etching with 0.5% potassium hydroxide in absolute ethanol for 3 mm, followed by washings in 96% ethanol

mm unocytochemical

cells

fixative

DAHLSTROM,

excess

5-

by protein

EC cells.

over

Electron Microscopy and Quantitative Evaluation. Quantitative evaluof S-HT-U in EC cells was carried out on sections incubated with antiserum A 24. Different incubation times were tested for the S-HI anation

in one step (A) or

Rats were fixed

two steps (B) by perfusion via the ascending fixative. Some tissue pieces were post-fixed in osby uranyl acetate for 1 hr.

with 200-300 ml of primary tetroxide for 1 hr followed

aorta

mium

tissue pieces from the antrum, duodenum, and proximal colon were dissected out and immersed in the fixative for 1-2 hr. Some tissue pieces were post-fixed in osmium tetroxide (OsO) in concentrations ranging from 0.1% to 1% for 1 hr, with or without subsequent staining en bloc

sion,

in 1% uranyb idly

dehydrated

acetate

in distilled

in ethanol

followed

for

1 hr. All tissue

by propylene

resin (TAAB 812; TAAB Laboratories UK). Polymerization was carried

in expoxy

Berkshire, 60C for 48 hr. Ultra-thin nickel

water

sections

were

oxide,

pieces

were

and

embedded

Equipment

LID,

out at 45’C

cut

and

Production were

Characterization

and used:

(a) a rabbit

of Antisera. anti-5-HT

tein

no

at

5-HI

SER

is the

chemical

form

of 5-HI

in tissues

fixed

in FA (25,29);

A-gold

(b)

A-gold

complex

(0.25-2

hr).

Maximal

All sections

with S-HI

complex

for

used

for quantitation

(A 24)for

antiserum

1 hr. Etching

of sections

4001

magnification

and 200-1000 tion a

electron

microscope.

From

was not

each

were

1 hr, followed

level

by pro-

performed,

but

borohydride was carried

beout

of the

gastrointesti-

nal tract (antrum, duodenum, and proximal colon), 12 EC cells (four cells from each of three rats) fixed in 2.5% GA alone were evaluated. In addition, 15 duodenal EC cells (five cells from each ofthree rats) fixed in 2.5% GA and 1% OsO were evaluated. Counting ofgold particles over different cytopbasmic compartments of EC cells was performed on micrographs at a final

7-7)

raised against 5-HI, coupled to bovine serum albumin (BSA)with FA (31). The antiserum reacts primarily with 6-hydroxy-tetrahydro-3-carbobine, which

protein

labeling.

incubated

in a Philips

Reading,

different

(code

the

sections from osmicated tissues were treated with sodium fore incubation. Examination and photography ofsections

on unsupported

Three

antiserum

hr) and

in background

therefore

grids.

antisera

(0.25-6

increase

rap-

for 12 hr and

placed

tiserum

immunobabebing was already obtained after 1-hr incubation with S-HI antiserum and 1-hr incubation with protein A-gold complex. Prolongation ofincubation times did not increase immunolabeling but caused a gradual

areas

point

of

x 85,000.

gold particles of the

counting

different using

Approximately

were counted cytoplasmic

a multi-purpose

55-70

granule

per cell profile. compartments

were

testline

(35)

system

profiles

The relative

sec-

estimated with

by

approxi-

rat monoclonal anti-S-HI antibody (Mas 055 clone YC 5/45 HKL; Sera Lab lid, Crawley Down, Sussex, UK) raised against 5-HT, coupled to BSA with FA (7). The antibody has a specificity similar to that ofSER 7-7 (7); and (c) a rabbit anti-S-HI antiserum (A 24) raised against 5-HI coupled to BSA or human serum albumin (HSA) with GA (S-HI-GA-BSA and 5-HT--GA-HSA). In ELISA tests the antiserum reacts primarily with 5-HI-GA-polybysine (PL) and only to a small extent with 5-methoxytryptamine-GA-PL, S-hydroxytryptophane-.GA-PL, or tryptamine-GAPL. These latter conjugates were 42, SO, and 538 times less recognized by the 5-HI antiserum than was 5-HT-GA-PL (13).

mately 85 test points per tm2 section area. Nonspecific labeling of sections (background labeling) was estimated by calculating the density of gold particles over epithebial non-EC cells. Background labeling was expressed as the relative density ofgold particles (in percent) over epithebial non-EC cells as compared to the density of gold particles over EC cell cytoplasm.

Preparation ofProtein by reducing tetrachboroauric

and duodenum),

Gold

particles

with

A-Gold. Monodisperse gold sobs were prepared acid with sodium citrate and tannic acid (30).

an average

diameter

of 10 nm

were

adsorbed

grids the

carrying various

scheme 1% BSA

antiserum followed

ultra-thin

plastic

incubation

was essentially

solutions according

in PBS for S mm,

sections

sections placed

were on

to Roth

(27).

were

incubated

were carried Unsupported

immersed

in small

Parafllm. Alter

The pre-incubation

with

diluted

measurements

on

EC cells

were

corrected

for

background

labeling.

Statistics. The intracellular distributions ofgobd particles in GA-fixed EC cells from antrum, duodenum, and proximal colon were compared by performing a one-way analysis of variance (2). In the design of the study,

it was

assumed

that

the

direction

of the

difference

if there

were any, would

characteristic

electron-dense

(e.g.,

between

antrum

be the same for all animals.

to protein

A (Pharmacia Fine Chemicals; Uppsala, Sweden) at pH 6.9 and the compInt was then stabilized with polyethylene glycol (PEG 20,000)(16,28). Alter three centrifugations at 40,000 x g for 30 mm, the protein A-gold complex was re-suspended and stored in PBS containing 0.02% PEG and 0.1% sodium azide. bmmunocytochemical Procedures. All incubations room temperature (18-20C) in a moist chamber.

All

out at nickel drops

of

incubation with anti-S-HI

(SER 7.7 1:400; YC 5/45 HKL 1800; A 24 110#{174})for 1 hr, by extensive rinsing in PBS. Sections were transferred to the protemn A-gold solution for 1 hr. followed by washing in PBS and distilled

Results EC cells with

in mucosal

observed

three

levels

studied.

was obtained

after

1%

uranyl

the

EC cells

a limiting

tion

The

best

After

were

shown

membrane space fixation

of the secretory

secretory

granules

of the gastrointestinal ultrastructural

GA fixation

acetate.

electron-lucent the other

epithelium

(2.5%)

such

preservation followed

of tissues

by 1% OSO4 and granules

of

of a dense

core surrounded

by

separated

from

dense

procedures granules

20-50 were

the

at all

to consist

of some

fixation,

were

tract

nm used,

secretory

the (Figure

core

8). When

ultrastructural

was less satisfactory.

Tissues

by

an

any of preservafixed

in

GA were generally much better preserved than tissues fixed in FA (cf. Figures 1, 2, 3, and 5-7). Reduction ofthe 0504 concentration


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Figures 1 and 2. EC cells from rat duodenum fixed in 4% FA and incubated with antiserum SEA 7-7 (Figure 1) or YC 5/45 HKL (Figure 2) and protein A-gold to visualize 5-HT-Iike immunoreactivity. Gold particles are concentrated over dense-core profiles but are also present over extra-granular parts of the cytoplasm, as well as over cell nuclei (N). Fixation quality is inferior to that after fixation in GA (ct. Figures 3 and 5-7). Bars - 0.2 tm.

below 0. 5 % resulted in loss of the limiting membrane of the secretory granules although the dense cores were still clearly visible. As

used

a consequence, a sharp delineation of the granular was difficult to make. Demonstration of 5-HT-U in EC cells could

fixed

performed

with

pendent tamed

on with

all antisera

the

fixation

the three

used,

but

the

procedure

different

success

employed.

5-HT

antisera

compartment

ing

(2-3% could

was highly The

results

are described

in EC Cells as Detected

Antisera

SER

The

obtained

results

7-7

and with

and are therefore described 5-HT coupled to a protein ing ofEC

cells after

YC

de-

perfusion

ob-

procedure,

below.

by

the two antisera

together. These antisera, with FA, gave maximal

fixation

of tissues

in 4%

to be identical raised against immunobabel-

FA alone.

Gold

parti-

des were specifically localized to EC cells, with bow background labeling over the cytoplasm ofother epithebial non-EC cells. In EC cells, a majority of the gold particles were concentrated over the dense core of the secretory granules (Figures 1 and 2). Cell nuclei of both EC cells and non-EC cells were moderately labeled. Fixaiion

of tissues

babebing

with

a mixture

of EC cell granules

of FA and when

high

GA

markedly

concentrations

reduced of GA were

alone EC

was used

cells.

labeling

(Figure

4).

4%

background Osmication

GA

labeling

and fixed

of 0.1%

bow concentration

was

of 0504

labeling

of

EC

hydroxide,

No

could

0.5%-1%

labeling,

be

0504.

strated

This

in secretory

5-HT-LI

this

A

included

in the

tissues

fixation

improved,

over cell nuclei

was re-

labeling

appreciable

in

that with

with potassium borohydride.

after

demonstrated

Cells

still

of EC

0504. The deleterious effect of Ofl immunobabebing could be par-

even

meant

granules

in EC

Antiserum With

cells.

could

in the second

in FA decreased

tialy overcome by etching the sections followed by treatment with sodium other two etching procedures had any ing

granules

of

were

by a mixture

GA was used

preservation

of tissues

tissues

FA followed

3%

no label-

when

of secretory

When

ultrastructural

as fixative,

However,

with

in EC cells when

at a concentration

this

appeared

excellent

step

whereas cells

HKL

5/45

GA,

GA over

perfusion

be demonstrated

duced.

5-HT-LI

When

detected

by sequential

of FA and

be successfully

GA). be

5-HT-LI

with

cells could

preserved

as Detected

effect

etching

EC

hydroxide, None of the on

label-

potassium

post-fixed not

be demon-

limiting

membranes.

coupled

to a protein

by

24

antiserum,


raised

against

5-HT

in

NILSSON,

322

DAHLSTROM,

GEFFARD,

ERICSON

AHLMAN,

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IMMUNOCYIXCHEMICAL

Table 2. Distribution ofgoldparticles processedfor immunocytochemical

323

OF SERONIN

LOCALIZATION

in EC cells ofrat localization ofserotonin

gastrointestinal tract fixed using antiserum A 24

Cytoplasm

Cytoplasm without granules

Relative

number

gold

particles

(%,

mean

SEM)

±

Relative

(%,

of

section

mean

Density

area SEM)

±

of gold

in glutaraldehyde

Total

alone

with

and

granules

Dense

core

Non-dense

core

A:

9.6

±

1.3

90.4

±

1.3

7.4

±

0.8

83.0

±

1.7

D:

5.0

±

0.8

95.0

±

0.8

5.1

±

0.3

89.9

±

0.9

0.9”

C:

5.8

±

0.6a

94.2

±

O.6a

6.0

±

0.4’

88.2

±

A:

80.2

±

1.8

19.8

±

1.8

2.8

±

0.3

17.0

±

1.5

D:

63.4

±

2.8

36.6

±

2.8

4.2

±

0.3

32.4

±

2.6

C: 67.8

±

3.0”

32.2

±

3.0”

4.0

±

0.3”

28.2

±

2.7”

9.7

particles

A:

1

51.7

±

9.0

27.9

±

5.4

55.6

±

(in relation to that of

D:

1

52.4

±

14.8

22.8

±

5.3

56.8

±

16.5

cytoplasm

C:

1

41.0

±

5.7

21.0

±

3.5

43.9

±

6.0

mean

without

granules;

n.s.’

n.s.’

From

each level of the gastrointestinal tract 12 EC cells were analyzed. Average number of gold particles per dense-core profile of EC cells from different levels of the gastrointestinal tract was performed by one-way analysis of variance. The test variable of freedom. Background labeling was 1.0%-2.8%. A, antrum; D, duodenum; C, colon.

was

Comparison

a