THE

JOURNAL

Copyright

OF HISTOCHEMISTRY

© 1976

Vol.

AND CYTOcHEMISTRY

by The

Histochemical

ACROLEIN

AS

Society,

of Morphology, Received

FOR SAITO

Institute

for publication

12, pp.

1258-1269, Printed

A FIXATIVE TAKUMA

Department

24, No.

Inc.

ENZYME

February

27,

CYTOCHEMISTRY

HIROMI

AND

for Developmental 1976,

1976

in U.S.A.

KEINO

Research, and

Kasugai,

in revised

form

Aichi July

480-03,

Japan

12, 1976

Since acrolein can penetrate more quickly and deeply into tissue blocks than glutaraldehyde, the possibility of the use of this aldehyde as a prefixative in enzyme cytochemistry was reinvestigated. At low concentrations, acrolein preserves the activities of the enzymes investigated, including those of glucose-6-phosphatase, which is known as one of the most vulnerable to aldehyde fixation; thus, acrolein is usable in enzyme ultracytochemistry. Enzyme activities are also preserved in tissues fixed with acrolein and glutaraldehyde combined. The rapid penetration of acrolein enables fixation in larger tissue blocks and provides greater freedom in specimen selection, especially important advantages when encountering heterogeneous materials as in pathology. Since Sabatini et al. effects of glutaraldehyde, other aldehydes in search to electron

microscopic

cytochemistry,

of glutaraldehyde as a prefixative tetroxide as a postfixative has practice

in ultracytochemical

tural studies. But as restrictions

anesthesia,

and osmium enjoyed wide ultrastruc-

been

encountered

fixation with

hyde, because its slow results in an unavoidable

in

rate of penetration fixation gradient from

fixation

methods

have

(10,

solid.

been

acrolein

increasingly applied to fine structural studies (1, 26, 27) as well as to prefixations in enzyme cytochemistry

(5, 9, 25, 32) to avoid the fixation

as

surgical

biopsy

tissues (23, 34), mammalian stages (31) and amphibian fertilized

embryos

The primary to achieve a

(16,

the been

penetration

capable

was enzyme

of deep

and

into tissue blocks. Therefore,

was focused

introduced

at early eggs and

17, 35).

and

penetration

attention

(8), plant

embryos unfertilized

intent of this investigation fixative suitable for

ultracytochemistry rapid

samples

as

causing MATERIALS

on acrolein,

a potent

fixative

little shrinkage AND

which

The

tissue

was

acrolein,

containing

0.1%

hydroquinone

plus

peak absorption obtained from

differences in the tissue blocks. However, there are some materials that are hard to perfuse, such

the

removed

and

cut

under

a

Chemicals: Acrolein, propenal, is a monoaldehyde of molecular weight 56.06, and of specific gravity 0.84. It has an irritating smell and is a strong lacrimatory stimulant. It is rather unstable and has a tendency to polymerize under light forming disacryl, a plastic

glutaralde-

the surface to the center of the tissue blocks perfusion

throughout the experiment. Immediately animals were sacrificed under light ether

few drops of fixative into thin strips, varying in length of about 1 mm2 across the end. To compare the penetration rates of acrolein and glutaraldehyde, the whole liver lobes were dissected, immediately immersed into the fixatives, and processed for cyto-

the use

and

used the

chemistry.

have

the use of immersion

15),

was after

(29, 30) studied the acrolein and many of a fixative of benefit

has

of rapid (20).

METHODS

Animals: Male Sprague-Dawley rats of 150 to 300 g body weight, fed ad libitum on a normal laboratory diet, Oriental NMF, were used as experimental animals. For the sake of convenience, only the liver tissue

spectrum the Tokyo

a minimum as stabilizer,

of

98% had

a

of about 210 mi; it was Kasei Kogyo Co., Ltd. Glutaraldehyde (25%) (molecular weight 100.12, specific gravity 1.08) was purchased commercially (Union Carbide, Lot V4N857) through Nakarai Chemical Co., Ltd., Kyoto, Japan, and paraformaldehyde powder was purchased through the Katayama Chemical Industries Co., Ltd., Osaka, Japan. Sodium $-glycerophosphate was obtained from E. Merck, Darmstadt, Germany. Thiamine pyrophosphate (Cocarboxylase), glucose-6-phosphate dipotassium salt, and sodium cacodylate were obtained from Sigma Chemical Co., St. Louis, Missouri. All other reagents were of analytical grade. Distilled and deionized water was used throughout the experiments. Fixation: The following fixation procedures were applied: (a) Immersion fixation in 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% (v/v) acrolein in 0.1 M cacodylate buffer, pH 7.2, for 5, 10, 15, 20, 30, 60, 90 and 120 mm at 0#{176}C;tissues were occasionally fixed at room temperature too. (b) Immersion fixation in a mixture of 2% acrolein and 1% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 5, 10, 15, 20, 30, 60,

1258

Downloaded from jhc.sagepub.com at GEORGIAN COURT UNIV on April 18, 2015

ACROLEIN

and

AS

A FIXATIVE

FOR

mm at 0#{176}C and at room temperature, (c) Immersion fixation in a mixture of 4% acrolein and glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, for 5, 10, 15, 20, 30, 60, 90 and 120 mm 90

120

respectively.

at

0#{176}C and

at

room

temperature,

respectively.

Cytoehemistry and electron microscopy: Mter fixation, the materials were washed overnight in a 0.1 M cacodylate buffer, pH 7.2, containing 6-8% sucrose. The fixed tissues were cut into 40 frozen or nonfrozen sections (Vibratome, Oxford, California) (2, 36). The free sections were incubated in the reaction mixture for 15 mm at 37#{176}C in a water bath with continuous ferred

shaking. directly

The

into

a

1%

sections

were

then

osmium

tetroxide

transsolution

(6), to stop the reaction and to postfix the tissue for 60 mm at 0#{176}C. Then, the specimens were dehydrated through graded alcohols and propylene oxide, and were

embedded

tion,

ultrathin

in

Epon

sections

812

(21).

were

After

made

polymeriza-

on LKB

Ultrotome

III using a glass or diamond knife (du Pont, de Nemours, & Co.) and collected on grids without coating. The sections were double stained with uranyl acetate (38) and lead citrate (28) and were examined with

a JEOL

100-B

electron

microscope.

Epon

sec-

) were also made, and stained with 1% toluidine blue. Incubation media: Acid phosphatase: Incubation was carried out at pH 5.0 in a medium containing 10 mM fi-glycerophosphate, 0.05 M acetate buffer, 3 mM Pb(NO3)2 and 8% sucrose (14). For the controls the same medium without substrate or with 10 mM NaF added, was used. Thiamine pyrophosphatase: Incubation was carried out at pH 7.2 in a medium containing 2 mM thiamine pyrophosphate, 0.08 M Tris-maleate buffer, 3.6 mM Pb(NO3)2, 5 mM MnCl2 and 6-8% sucrose (24). For tions

the

(1

controls,

a substrate

free

medium

was

used.

Light

microscopy:

sections

15 mm

of fixed

in distilled

ammonium they were

sulfide, mounted

Immediately tissue

were

water,

then

after washed

rinsed

incubation, for

for

more

2 mm

than in

1%

and then rinsed in water before on slides with glycerine jelly. RESULTS

Cytochemical experiments copy: The preliminary data enzyme

activities

could

on light showed

survive

after

microsthat the acrolein

fixation under diluted conditions. Figure 1 shows the acid phosphatase activity in rat liver, after a 4% The

acrolein fixation at 0#{176}C for 120 mm.

lysosomes

were

clearly seen

and

the

CYTOCHEMISTRY

phosphatase bile tion portal the

acid

1259

activity

was

canaliculi. No was observed areas.

and

glucose-6-phosphatase,

from

the

after shows

the

fixation 10.0%

activity higher

time

activity; up

could

be

of the

lead

to

concentrations mm.

120

Thiamine active

Table activity

.

from

enzyme 1.5%

permit-

without

activity

pyrophosphatase in

tissue

remained

fixed

in

concentra-

below 4% at 0#{176}C, and was localized canaliculi (Table II). Even though

cose-6-phosphatase sensitive remained

is

0.5 With fix-

decreased below

fixation

I

phosphatase

positively demonstrable. of acrolein, increased

mm

positively

sulfide sections

Acid

concentrations 30

judged

microscopical

5 to

decrease. tions bile

color

light

activities,

pyrophosphatase,

proportionately but

of

acrolein

enzyme

at various for

was still concentrations

ation ted

three

brown

in

one

4%

acrolein fixation (Tables I-Ill) the degrees of acid phosphatase

after to

after

thiamine

dark

precipitate

to see that

even 2).

of

phosphatase,

the

in distribucentral and

glucose-6-phosphatase,

retainment

acid

around

change the

surprising

enzymes,

also appeared positive fixation for 90 mm (Fig. The

positive

lobular between

It was

weakest

regarded

as

to aldehyde fixation, positive in the cytoplasm

on the glu-

extremely its

activity after fixa-

tions up to 2 hr with 4% acrolein concentrations at 0#{176}C (Table III). The enzyme activity tended towards gradual weakening after fixation at higher

concentrations

with

longer

times; this was especially evident thiamine

Glucose-6-phosphatase: The sections were incubated at pH 6.2 in a medium containing 1.5 mM glucose-6-phosphate dipotassium salt, 0.08 M Trismaleate buffer, 3.6 mM Pb(NO3)2, and 6-8% sucrose (37). For the controls, a substrate free medium was used. the

ENZYME

pyrophosphatase

phatase. ties after

On the other short fixations

trations,

showed

Electron enzyme

and

the same fixation.

glucose-6-phos-

hand, the enzyme with very low

weaker

activiconcen-

staining.

microscopy: activities

fixation

in the cases of

The

after

localization

acrolein

as that described after The acid phosphatase

observed as electron the lysosomal matrix tivesicular body (Fig.

of

fixation

were

glutaraldehyde activity

was

dense lead phosphate in as well as on the mul3). Thiamine pyrophos-

phatase

reaction

cisternae

of the

of reaction first and

products were heaviest within the second Golgi cisternae along the con-

cave,

trans,

products Golgi

surface

often completely Similarly, although cipitates

were

also

were

complex

of the

seen within (Fig.

Golgi

the

4). Deposits

stacks,

and

it

filled the innermost layer. less extensively, fine preseen

over

near the Golgi apparatus,

Downloaded from jhc.sagepub.com at GEORGIAN COURT UNIV on April 18, 2015

the

small

and sometimes

vesicles within

SAITO

1260

AND

KEINO

‘,.

k4

..

-.

1. Light micrograph of a section of rat liver, fixed in 4% acrolein for 120 mm. Acid phosphatase activity is seen positive in the lysosomes around the bile canaliculi. x 1,200. FIG. 2. Light micrograph of a section of rat liver, fixed in 4% acrolein for 90 mm at 0#{176}C. The glucose-6-phosphatase activity is weakly positive in the cytoplasm of the hepatic parenchymal cells. x 1,200. FIG.

smooth

vesicles

inner

Golgi

located

in

cisternae.

the

vicinity

Thiamine

tase activity was found in the reticulum (ER) in only extremely was

surprising

hyde, The the

after

a diluted

acrolein

It

localization comparable glutaralde-

fixation

(Fig.

5).

deposits were localized exclusively within cisternal spaces of both the rough and

smooth were

activity, fixed with

ERs

(Figs.

5 and

also observed

inner and outer nuclear reaction

product

6). Reaction

in the spaces was

(Fig.

membranes evident

membrane, in the elements plex, or over other intracellular Controls: In the substrate

products

between

along

the 5). No

the plasma

of the Golgi components. free controls,

comno

reaction product for any of the phosphatase activities studied was found. Nor was any reaction

product

found

when

added to the complete medium phosphatase preparations. Morphologic

preservation:

0.1 M

NaF

was

in the

acid

Figures

show liver

some tissue

the

endoplasmic rare cases.

to find an excellent

of glucose-6-phosphatase to that seen in material

of the

pyrophospha-

3 to 6

typical hepatocytes fixed by acrolein

cytochemical

medium.

The

liver

maintained retained

of the cells of the very fine morphology normal other tissues.

as

and

smooth

ERs

recognizable appearing

as ER

tubules. nuclear small the The

their Both

were

well

(Figs.

5

long

appearing

the

cisternae

as a network

myelin

figures

were

of smooth

glycogen

dark, probably had not been

regions

ER

to

each fixed intracellumorand easily

rough and

ER the

of the ERs, although

occasionally or Golgi

appeared

same same

of connected

There was no vacuolation envelope, or mitochondria,

vicinity

the the

and

6),

parallel

in

accepted the rough

preserved and

adult

ultrastructure

topographical relationships those seen in glutaraldehyde The Golgi complex and other

lar components retained phological appearances.

smooth

seen in incubated

and

light,

seen

in

membranes. instead

of

because the glycogen particles well preserved by the diluted

acrolein fixative. Fixation with

Downloaded from jhc.sagepub.com at GEORGIAN COURT UNIV on April 18, 2015

a combination

of acrolein

ACROLEIN

AS

A FIXATIVE

FOR

ENZYME

CYTOCHEMISTRY

1261

++++

++++ ++++++ +

a)a)

+++++++

so. .

.

+ +

++ ++

+

+ +

a)

‘-4

+?

+

a

.

++

+-

0. C.)

50

.0

+ + ++

++

+ + +

+++

+

+

+

o

a

+ ‘-4

+

++++

Cl)

0.. Cl)

.

0 cl3a)

++++

+++

a

+++

++++++

a 0

E

I-

+

+++ +++

a

5a) U_c dna)

a 0

.0 S 44

++

+++ a)

++

++

ca)

+++++

++++

.

+±

C’)

a)

N .

o -

+++

dna

a

+++

+

00+ U

‘q

La

++++

+

±+++

+ +

+++

U-

0+ +++

+++ +++

a o4o -c bl)o. S

+ + ++ +

+

++++t+++++

a)dna

S

++++ + +

a.

-c a S

+

.

‘

+

.

a)

.dl)+

50 cl35

S

.‘

a5o o. -o. 0 .

.s 2U

-co.2 -L’

.

;

...

C

.

..

H.

#{149}

#{163} ; :T1K

-

s’r:

..

4aas”

Downloaded from jhc.sagepub.com at GEORGIAN COURT UNIV on April 18, 2015

#{231},.

.4t,:Ir;:14

ACROLEIN

and

glutaraldehyde:

The

AS

idea

A FIXATIVE

of fixation

FOR

with

be

a mixture of acrolein and glutaraldehyde (18) where one might expect rapid penetration of acrolein

followed

by further

fixation

followed. The mixtures of 2% acrolein with

taraldehyde

4%

and

dehyde.

The

three

acrolein

v ith

enzyme

activities

phosphatase, thiamine glucose-6-phosphatase, positive

after

fixation

at 0#{176}C (Tables activities tested

with

mixed

at room temperature

although

enzyme

some

activities

Activities

After

that

and as

after

positively 0.4 to

Fixati 10

+++

Thiamine

pyrophospha-

the

the

TABLE IV in a Mixture of 2% Acrolein

Fixation

5

Acidphosphatase

from

or during

15

+++ +

+++ +

on Time

1%

to

wash,

and

sec-

only

to a depth

(Fig. 7).

lost

during

the

in

of about

The

cells

buffer

the

reaction

Glutaraldehyde

in

wash me-

at 0#{176}C”

(mm) 60

++ +

rate

contrast-

this material, demonstrated

buffer

surface

30

+++ +

The

was

by attempting

incubation

and

20

the

In tissue fixed in 2% glutaraldethe acid phosphatase activity was

the inner area were

continued

the in

penetration: into tissue

demonstrated

mm

VII).

improvement

fixation, .

and alone,

of rat livers. In activity was

immersion

tioning to 40 hyde for 1 hr,

Fixation retention,

VI

acrolein

of glutaraldehyde

fix whole lobes acid phosphatase

fixatives

temperature. reduced

(Tables with

of acrolein

rate

ed with

IV and V). As all of the enzyme were retained, further fixations

were performed at this elevated

Enzyme

these

The

acid

pyrophosphatase, were demonstrated

observable to fixation

of acrolein penetration

glutaralof

positively

1265

combination afforded an degree of overall preservation.

tested 1% glu-

2%

CYTOCHEMISTRY

Compared

by glutaral-

dehyde, was then were a combination

ENZYME

90

+-.-++

±

+

120

+.-++

±

+

+.-++ ±

±

+

±

tase

Glucose-6-phosphatase “Acid sections

+

phosphatase, fixed

+

thiamine

in a mixture

+

+

+

pyrophosphatase,

of 2%

acrolein

at 0#{176}C. The

degrees

positive

and

strongly

positive

+

Activities

After

Fixation

in

+ +,

+

of colored

and

and

reaction

+

graded

Acrolein

4%

+

as:

activities

negative

durations

±, slightly

trace

-,

were ranging

demonstrated from

positive

in

5 to 120 mm +,

moderately

V

and 2% Glutaraldehyde

10

+++

pyrophospha-

+

+.

5

Thiamine

+

at various

Fixati

Acidphosphatase

+

glucose-6-phosphatase

TABLE

Enzyme

+

1% glutaraldehyde

were +

+

15

+++ +

on Time

20

+

60

+-.++

++ +

Buffer

at 0#{176}C”

(mm)

30

+++

+++ +

in 0.1 M Cacodylate

±

90

120

+-++

+‘++

±

-

±

+

+

±

±

-

tase

Glucose-6-phosphatase

+

+

+

+

+

+

+

+

+

+

+ +

±

“Three enzyme activities, acid phosphatase, thiamine pyrophosphatase, and glucose-6-phosphatase, were demonstrated in sections fixed with 4% acrolein and 2% glutaraldehyde in a 0.1 M cacodylate buffer, pH 7.2, at various durations of ranging from 5 to 120 mm at 0#{176}C. The degrees of enzyme activity were graded as: negative -‘ trace slightly positive +, moderately positive + +, and strongly positive + + +.

±,

FIG. 5. Electron micrograph of a part of the hepatic parenchymal cells fixed in 4% acrolein for 30 mm. End products of glucose-6-phosphatase activity are seen in the cisternal space of the endoplasmic reticulum as well as in the space between the inner and outer nuclear membrane. Glycogen, washed out from the cytoplasmic matrix,

probably

account

for

the

electron

lucent

area.

The

mitochondria

and

ribosomes

are

clearly

defined.

x28,000. FIG. 6. Higher magnification of a part of a hepatic parenchymal cell fixed in 4% acrolein for 120 mm. Glucose-6-phosphatase activity is clearly seen in the cmsternal spaces of the endoplasmic reticulum. The localization of the enzyme is similar to that reported after glutaraldehyde fixation. x 70,000.

Downloaded from jhc.sagepub.com at GEORGIAN COURT UNIV on April 18, 2015

SAITO

1266

Enzyme

Activities

After

Fixation

AND

KEINO

TABLE VI of 2% Acrolein

in a Mixture

and

1% Glutaraldehyde

Fixati on Time

Acid phosphatase Thiamine pyrophosphatase Glucose-6-phosphatase “Three

enzyme

demonstrated

on

10

15

20

+ + +

+ +

+ +

+ +

+

+

+

+

+ +

activities, sections

Activities

After

+

acid fixed

various durations of ranging graded as: negative -, trace

Enzyme

5

+ +

+

+ +

phosphatae,

from

and

5 to 120 mm at room +, moderate +

in a Mixture

+ +

and

5

10

15

+ + +

+ +

+ +

+

+

+

The

+ +

+

±

“Three enzyme activities, acid phosphatase, demonstrated in sections fixed in 4% acrolein various

durations

ranging

from

were graded as: negative

dium

at 37#{176}C,possibly

(Fig.

8); fixation

in to at least

7%

acrolein

preservation the

of the

for

the

1 hr

at

0#{176}Cresulted

than

1.5

mm,

i.e.,

in

One

of the

the

adequate

the

tissues.

found

important

Since the

towards

+ +,

of a good

aldehyde

had to be

widely

as a prefixative in cytochemistry and morphologic fixative in ultrastructural glutaraldehyde

its slow penetration

may

been used

as a purely studies.

preserve

any other known

to

in this

of choice

(29, 30), it has come

better than

is fixative

glutaraldehyde

structure

at Room

Temperature”

60

90

120

+ +

+

+

+

-

-

-

±

-

-

-

The

and

were

±

temperature.

degrees

strongly

of colored

positive

+

+

were at

pH

enzyme

7.2,

activity

+.

bility to small blocks of tissue which must be cut from the source. This problem can be considerably diminished by in vivo perfusion of fixatives through the channels

whereby

pected

to

diffusion 22, 26,

distance 27). The

thus

a

obtaining

ultracytochemistry,

Although

activity

stabilize

especially

the

perfusions tissue

by

from the application useful

system,

and

in

are

fixative of this

with

respect

to the

come

to receive

fields,

the

source method

it has

various

cx-

reducing

(7, is

central wide

including

histo-

chemistry (5, 9, 19, 25, 32). Recently, Glaumann (13) utilized vascular perfusion incubation with

steps

in enzyme application

to be

respect

+

+

±

(mm)

application

lobe.

results

+

enzyme

±

-

were pH 7.2, at

buffer,

of the

-

+ + +.

30

nervous

almost

DISCUSSION

better

Time

vascular

in tissue surface

4% acrolein resulted in 1.2 mm or more; fixation

to more

entire

at room

moderate

+,

glucose-6-phosphatase,

thiamine pyrophosphatase, and glucose-6-phosphatase, 2% glutaraldehyde in a 0.1 M cacodylate buffer,

(Materials and Methods), only a thin surface layer of On the other hand, fixa-

acrolein for 1 hr resulted to about 1.0 mm from

preservation in

minutes

±, slight

as a consequence

continuous shaking the result being that tissue was preserved. tion in 2% preservation

5 to 120

trace

-,

and

on

+

±

±

2% Glutaraldehyde

20

+

+

degrees

positive

+ ±

in a 0.1 M cacodylate

strongly

+

+

±

and

120

-

±

+ +

temperature. +,

90

+

pyrophosphatase,

Fixati

Acid phosphatase Thiamine pyrophosphatase Glucose-6-phosphatase

+ +

+

TABLE VII 4% Acrolein and

of

60

±

1% glutaraldehyde

±, slight

Fixation

+

Temperature”

(mm)

30

+

thiamine

in 2% acrolein

at Room

the fine fixative,

rate often limits its applica-

medium

containing

the

enzyme

glutaraldehyde

perfusion.

fixation, however, ence is needed

considerable for performing

after

cannulation time.

during

Also,

for

rigid temperature required as well fixative

flow

number

of

inapplicable

the

enzyme

rate.

materials such

Downloaded from jhc.sagepub.com at GEORGIAN COURT UNIV on April 18, 2015

With

minimum

as some

experiand

permissible a more

during fixation is regulation of the

Another to

perfusion

skill and dissection

histochemistry,

control as a stricter

substrate

limitation which seeds,

this the

is method pregastru-

the is

ACROLEIN

AS

A FIXATIVE

FOR

ENZYME

CYTOCHEMISTRY

1267

FIG. 7. A thick section of a liver lobe fixed in 2% glutaraldehyde for 60 mm at 0#{176}C. The form of the tissue appears as a ribbon from the surface. The central area has been lost during buffer washing and incubation in an acid phosphatase medium of continuous shaking. The width of the ribbon is less than 0.4 mm. The acid phosphatase activity is positive. x8. FIG. 8. A similar section of a whole liver lobe immersed in 2% acrolein for 60 mm and then processed to demonstrate

acid

distorted be about

phosphatase

ral stage of sected tissues materials

amphibian of surgical

also (23)

during minative

the the

thus

most

studied late stages,

found

that

fix

images.

that

acrolein,

a

for a

to fix with

preserved.

and on

seeds

with

authors

a and very

(16,

amphibian

applied

a

embryos.

17, 35)

combined

glutaraldehyde

in

culture When

solution. Luft

prefixative

for

deeply

other

aldehydes,

and

of

acrolein

a half-strength

(20)

that its superiority more

fixative

(3)

electron lay

acrolein

microscopy,

he

in its ability

react

more

an

advantage

aldehydes

also

completely.

and

acrolein

showed

to penetrate

quickly

than

whereby

most fresh

reagent

is espe-

The tissue

surprisingly

greater

with respect introduced

a

than rapid

able also

great,

than

glutaralde-

are

to the stability (4). The rates

able

to

fix

against

it

the

washing

necessitates

of

proteins care

in in

biological materials. tion of acrolein as

the

application However, an electron

Downloaded from jhc.sagepub.com at GEORGIAN COURT UNIV on April 18, 2015

almost

shown in

effect (12). and

protein, acting

protein is

effective

more

any other aldehydes rate of penetration

bilization

this

Furthermore,

of the which

at

to preserve soluble indicates that rapidly

is much

albumin as

to

of relatively large practical reasons,

was

times

the

as the can be

into smaller pieces. acrolein into the

of

immersion

aldehydes are e. g., albumin,

New-Twitty

introduced

area;

hyde.

collagen cross-links

and

Bluemink

this

in the fixation which, for

two

liver

was calculated

more reliable results, of living conditions

dissected speed

than

of the

The superiority of acrolein and glutaraldehyde has been shown on the model system using

containing

formaldehyde,

furnish locations

1 hr

more

all

of the fixation

Consequently,

during

and micros-

almost

depth

effective of tissue,

dehydrated electron

sulfoxide in a 0.1 M cacodylate buffer, an excellent and uniform preservation

dimethyl provided

can

cannot be penetration

fixative

glutaraldehyde,

cially areas

over

The

and early gerare the periods

formaldehyde, postfixation gives

Several

is positive

fixation.

tissues molecular

prefixation

combined

activity

disThese

fixatives

partially to

of glutaraldehyde, followed by

acceptable

of

are

the

Mollenhauer

several

difficult

enzyme

is due to weak

and samples.

developmental since these

tissues

they

mixture acrolein found

The

to be difficult

by immersion.

Totten

when

embryos, biopsy

happen

glutaraldehyde

copy;

activity.

appearance of the innermost cells 1.0 mm from the surface. x8.

Of the tissues of

that

stabilizing in a buffer course, speedy

this sta-

examined fixation

to

on his introducmicroscopical

1268 prefixative,

Luft

ultrastructure

(20)

indicated

well

except

phologic excellence was reported similar aldehyde

that

dehyde

presented

acrolein

that

et al.

(29).

either

other

and

than

could

be

the

extreme

reactivity

of

for such

an extensive

to account

alone.

is said

destruction enzyme However,

of enzyme activities that histochemistry is severely there has been some evidence possibility under

of the retention acrolein fixation.

showed that some enzymes acceptable level of activity and

similar

by Feustel

and

results

also

(11).

positive.

The

shows

that

alkaline esterase

present

enzyme

been

Sabatini

themselves showed that while tivate most enzyme activities, thepsine C, slight trace cytoplasmic

maintain acrolein

reported

clearly

are

could be acrolein. The reports

here with

dilute acthat even

demonstrated contradiction

in the

literature

is

more apparent than real in some instances. For example, Sabatini et al. (29) did report having successfully applied acrolein in 10% for from 30 to 120 mm; in this instance, in the present

report simply

concentration examined. Acid phosphatase activity strated after fixation in 10% mm,

and

the

excellent. On enzymes phatase, 4% acrolein

fine

can be acrolein are

for more

ultrastructures

than

observed conditions at between

for most

other

90 mm

one

of the

demonfor 120 to

be

weakest

volume per cent Our laboratory

at 0#{176}C, and

were

of acrolein

of formaldehyde. tive, 4% acrolein mm

the

enzymes

enzyme milder

it is stronger

tested

were

still

activities a broad

could

arose as to whether longer concentrations or shorter

range

of acrolein

higher

concentrations

would

enzyme

cytochemistry.

However,

remains

unanswered. be concluded of acrolein with and

rapid

makes

it possible

retain

a tissue

tages

of

than

be

question lower

a uniform

this

fixative,

at at

better

for

this

cial materials which, for some in large

its

4.

is equivalent mM, and the

6.

enzymes.

7.

which they

the

terrible

can suffi-

of enzyme disadvanodor

and

may have on spe-

to perfuse, may have

and to be

blocks. CITED

R: Microperfusion

for ultrastructural 41:176, 1972 2. Ago Y, Ogawa

3.

and the ensures

morphology

that are hard reason or other,

LITERATURE 1. Abrunhosa

question

so that

the purposes Considering

60

sustained

distribution,

cellular

for

after

that acrolein glutaraldehyde

fixative

and

fixamost

fixations fixations

be

to fix tissues

of that

solutions,

powerful lacrimatory stimulus, its use to be limited to enzyme cytochemistry

fixed

that

positive

quite

mixture

activ-

than

within

It might

fixation mm, the

Even in the combined and 2% glutaraldehyde,

fixation. As enzyme

5.

is 108 glutaralde-

after 120

to inactivate

though

glu(216

glucose-6-phos-

survive mM) for

glutaraldehyde,

for fixation should be

of glutaraldehyde standard for

could (600

of

glutaraldehyde

at 0#{176}C.Since,

to be somewhat

acceptable.

suitable 4-10%

One volume per cent of acrolein to a molar concentration of 150

mM.

known

one

of

demonstration

ity appeared

for aldehyde fixation, glucose-6-phoscould be demonstrated positive after

Therefore, the with acrolein applicable

structures

the other hand,

tendency

solutions the data

the range

extend

30 mm activity acrolein

ciently suitable ultracytochemistry.

histochemi-

cally demonstrable in the tissue after rolein fixation. It is important to note

presented

macca-

phosphatase and activities remained investigation

the

is 2%

for

(29)

acrolein did moderate

activities

glucose-6-phosphatase in tissue fixed with

an fixa-

et al.

for

phatase with 4%

of the

its use in restricted. suggestof enzyme Flitney (12)

might after

have

Geyer

glutaralof the

acrolein

ing the activities

(33)

preservation

aldehyde

hand,

mM)

glutar-

Sandborn

of acrolein

a better

with the

for

fixation

cose-6-phosphatase

mor-

fixation

reported

microtubules

obtained

The

KEINO hyde

it preserved

lipid.

after

to

a mixture

cytoplasmic On

that for

seen

by Sabatini

showed

tion

AND

SAITO

fixation

studies. K:

The

J use

of embryos

Ultrastruct of vibratome

Res in

ultracytochemistry. Acta Histochem Cytochem 6:1, 1973 Bluemink JG: The first cleavage of the amphibian egg. An electron microscope study of the onset of cytokinesis in the egg of Ambystoma mexicanum. J Ultrastruct Res 32:142, 1970 Bowes JH, Cater CW: The reaction of glutaraldehyde with proteins and other biological materials. J Roy Micro Soc 85:193, 1966 Casanova 5, Marchetti M, Bovina C, Laschi R: A study of the effects of fixation on liver glucose-6phosphatase activity for electron microscope cytochemistry. J Submicr Cytol 4:261, 1972 Caulfield JB: Effects of varying the vehicle for osmium tetroxide in tissue fixation. J Biophys Biochem Cytol 3:827, 1957 Dalton AJ, Kahier H, Striebich M, Lloyd BJ:

Downloaded from jhc.sagepub.com at GEORGIAN COURT UNIV on April 18, 2015

ACROLEIN Fine

structure

cells 8.

of the

scope.

J Nat

Dawson

IMP:

of hepatic, mouse

intestinal

as revealed

Cancer

Inst

Fixation:

AS A FIXATIVE

by

11:439, What

and

electron

FOR

patholo-

tion

On the fine

structural

of glucose-6-phosphatase.

Flitney

FW:

The

time

J Histochem

course

Cyto-

of

the

fixation

of

albumin by formaldehyde, glutaraldehyde, acrolein and other higher aldehydes. J Roy Micr Soc 85:353, 1966 13. Glaumann H: Ultrastructural demonstration of phosphatases by perfusion fixation followed by perfusion incubation of rat liver. Histochemistry 44:169, 1975 14.

Histochemistry:

Princi-

ples and Practice. Chicago, University Press, 1952 15. Hopwood D: Some aspects of fixation aldehyde. J Anat 101:83, 1967

of Chicago

16.

Gomori

G:

Microscopic

with

phatase

activity

in

hepatocytes.

Res 15:242, 1966 Studies on seeds. I. Biol 48:387, 1971 5: Nucleosidediphos-

Totten

of seeds.

usefulness Sci US

C:

J Cell

Goldfischer

for 47:802,

in the

Golgi

cytological

apparatus

studies.

and

Proc

Nat

its

Acad

1961 Orrenius 5, Ericsson JLE: On the relationship of liver glucose-6-phosphatase to the proliferation of endoplasmic reticulum in phenobarbital induction. J Cell Biol 31:423, 1966 26. Palay SL, Palade GE: The fine structure of neurons. J Biophys Biochem Cytol 1:69, 1955 27. Palay SL, McGee-Russell SM, Gordon S, Grillo MA: Fixation of neural tissues for electron microscopy of perfusion with solutions of osmium tetroxide. J Cell Biol 12:385, 1962 28. Reynolds ES:The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J 29.

30.

32.

Kalt MR and Tandler B: A study of fixation of early amphibian embryos for electron microscopy. J Ultrastruct Res 36:633, 1971 18. Karnovsky MJ: A formaldehyde-glutaraldehyde fixative of high osmolality for use in electron microscopy. J Cell Biol 27:137A, 1965 19. Leskes A, Siekevits P and Palade GE: Differentiareticulum

AB,

31.

glutar-

17.

of endoplasmic

Novikoff

J Ultrastruct

HH,

Cell Biol 10:208, 1963 Sabatini DD, Bensch K, Barrnett istry and electron microscopy.

I.

Glucose-6-phosphatase distribution in situ. J Cell Biol 49:264, 1971 20. Luft JH: The use of acrolein as a fixative for light and electron microscopy. Anat Rec 133:305, 1950 21. Luft JH: Improvements in epoxy resin embedding methods. J Biophys Biochem Cytol 9:409, 1961 22. Maunsbach AB: The influence of different fixatives and fixation methods on the ultrastructure of rat kidney proximal tubule cells. I. Comparison of different perfusion fixation methods and of glutaraldehyde, formaldehyde and osmium te-

33.

34.

Ed: The

studies

with

the

electron

Saito T, Ogawa K: Lysosomal changes hepatic parenchymal cells after glucagon istration. Acta Histochem Cytochem 7:1,

Sandborn

E: Electron

microscopy

membrane

systems

and

Pharmacol

44:329,

1966

Schwab DW, fine structures

filaments.

Singal

PK,

37.

38.

Smith ments

RE: and

in rat admin1974

of the Can

neuron

J Physiol

Janney AH, Scala J: Preservation in yeast by fixation in a dimethyl

Sanders

of the first cleavage Ultrastruct Res 47:433, 36.

microscope.

J Histochem Cytochem 12:57, 1963 Saito T, Iwata K, Yamazaki Y: Ultracytochemical study of the blastocyst development. I. Acid phosphatase. J Electron Micros 22:289, 1973

sulfoxide-acrolein-glutaraldehyde Technol 45:143, 1970 35.

Cytochempreservation

of cellular ultrastructure and enzyme activity by aldehyde fixation. J Cell Biol 17:19, 1963 Sabatini DD, Miller F, Barrnett RJ: Aldehyde fixation for morphological and enzyme histochemical

Kalt MR: The relationship between cleavage and blastocoel formation in Xenopus laevis. II Electron microscopic observations. J Emb Exp Morphol 26:51, 1971

tion

24.

1269

25.

demonstra-

chem 14:361, 1966 10. Fahimi HD: Perfusion and immersion fixation of rat liver with glutaraldehyde. Lab Invest 16:736, 1967 11. Feustel Von E-M, Geyer G: Zur eignung der acrolein fixierung f#{252}r histochemische Untersuchungen. H. Lipide, Enzyme. Acta Histochem 25:219, 1966 12.

Mollenhauer

Edited by PJ Hall, 1973, p

193-197

JLE:

fixatives.

23.

Fixation the

do? Fixation in Histochemistry. Stoward, London, Chapman and

CYTOCHEMISTRY

troxide

renal micro-

1950

should

gist

9. Ericsson

ENZYME

Comparative procedures

solution.

EJ:

An

1974 evaluation to cut

Stain

ultrastructural

of Xenopus

of

study

embryos. of two

J

instru-

nonfrozen sections. J Histochem Cytochem 18:590, 1970 Wachstein M, Meisel E: On the histochemical demonstration of glucose-6-phosphatase. J Histochem Cytochem 4:592, 1956 Watson ML: Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol 4:475, 1958

Downloaded from jhc.sagepub.com at GEORGIAN COURT UNIV on April 18, 2015