THE

JOURNAL

HI5T0cHEMI5TRY

OF

Copyright

© 1975 by The

LIGHT

Vol.

CYTOCHEMI5TRY

AND

Histochemical

Society,

MICROSCOPIC

LOCALIZATION

EPOXY-EMBEDDED

JOSE Pathology

Received

Tissues

from

localization

of

for

mice tetroxide,

sites

of reaction

were

using

a phase

in

and

curacy

in

greater

resolution

the The

in

with

use

the

localization

animal

tissues

the

introduction of larly glutaraldehyde

this

recommended

light

alkaline

has

into

fixatives, the field

level, a better understanding of these enzymes was gained

normal

tissues

tions

but

(2, 8, 9, 13,

also

is that

block an

must

initial

be

necessary zation

in order of the

select an area the electron

many

cryostat

within

of interest microscope. sections

sections

of

light

sections

way, 1

the This

with general

the

general

the

tissue

investigators

work

was

ammonium

supported

of by

Institute Contract NO1CP33347 grant from the United Foundation

and

(6).

Nat,onai

like

ac-

obtain

samples for

of direct and

to

of the electron

the

correlations

electron

present

of visualizing

a

micros-

methodology

reaction

sites

material With may be

due

to

the

section.

in i-p

using

reduction

The

the

this method, observed with

use

in

the

of toluidine

allows the investigator to in relation to the cytologic

tissue.

In

addition,

correlation

observations

ultrastructural

to

and

this

method

between electron

light

mi-

microscopic

studies. MATERIALS

AND

METHODS

Normal mammary gland, mammary tumor, kidney and intestine were removed from several female BALB/c mice. Additional mammary tissue was obtained from female BALB/C (nude/nude) mice. Intestine and testis from a male C3H/An mouse were also studied. Fixation: Immediately after removal, the tissue was placed in 1.5% glutaraldehyde in 0.1 M cacodylate,

the

the lead reaction by treating the

absence

a direct

with of the with

sulfide

of

to

similar processed

microscopy

of the

croscopic

localiand

and

for were

resolution

permits

previously

incubations

localization

details

is

in of

microscope.

blue as a counterstain observe the reaction

Thus,

of the

degree

of plastic-embedded

thickness

sites with

phosphatases

phase contrast microscope. the enzyme localization

tissue

microscope

appropriate substrates. Afterwards, phosphate produced by the enzymatic was visualized for light microscopy the

the

these

light

the

of the distrinot only in

for examination In the case for

light would

examination.

the

to observe

enzyme

phosphatases, used

for

with

and

greater

portion

selected

sections between

consists

the mi-

the

studied

The two major problems with this the lack of resolution in the cryostat

which

condi-

blue,

a high

be estimated tissue that

copy. We

pathologic

The

toluidine

microscopy the

the the techultrastruc-

of

main problems with using the electron

a small

screening

particuof histo-

of

electron

post-

microscopy. between

sites

achieve

with

ultrastructural

with

study

microscopy. method are

23).

However, one of the study of these enzymes croscope

in

18-20,

for

could same

The

the

electron

the

to

enzymes

widely

(22).

the

1975

triphosphatase,

correlation

order

sample

phos-

been

chemistry facilitated the observation localization of these enzymes using nique of electron microscopy. At an tural bution

tissue

for

for

and

for in

of these

microscope

dialdehyde (27),

technique

of adenosine

and

a close

Foundation,

26,

counterstained

microscope

is

the

in plastic

show

August

treated

sections

data

of of

form

Cancer

adenosine

embedded

contrast

localization

Michigan

in revised

f-dependent

plastic

The

a portion in

(ATPases)

studied

1-p

phase

tissues

selecting

histochemical

phatases

in

and

IN

MICROSCOPY’

WELLS

glutaraldehyde, Mg

and

microscope. with

pathologic

triphosphatases

1.5% or

visualized

PETER

AND

2, 1975,

dehydrated

microscope.

normal

The

were

contrast

observed

electron

May

phosphatase

in osmium

12, pp. 921-931, 1975 Printed in U.S.A.

REACTIONS

ELECTRON

Department of Biology, Detroit, Michigan 48201

publication

fixed

CYTOCHEMICAL

FOR

RUSSO

Laboratory,

alkaline

fixed

reaction

OF

MATERIAL

Experimental

23, No.

Inc.

In this reaction Cancer

pH

7.2,

containing

4#{176}C. The

an institutional of Detroit.

was diluted

1%

glutaraldehyde

with

sucrose, used

0.1 M cacodylate

921

Downloaded from jhc.sagepub.com at WAYNE STATE UNIVERSITY on March 21, 2015

and

fixed

to prepare from

for the 25%

2 hr fixative glutanal-

at

4.

#{149}

-,-,--

‘..

4.’

#{149}“-.

FK;.

1. Cross-section A i-p

phosphatase. x720.

of section

a

mammary stained with

gland 0.5%

duct toluidine

from

a blue,

BALB/c mouse. Incubated 3 hr for alkaline photographed using bright-field illumination.

FIG. 2. Phase contrast micrograph of the same field as in Figure 1. Arrows indicate sites of positive reaction for alkaline phosphatase in the plasma membrane of the myoepithelial cells and in the epithelial border of the lumen. x720. FIG. 3. Phase contrast micrograph of a mammary gland ductule from a BALB/c mouse. Incubated for 1 hr in control medium without Na-fl-glycerophosphate. A i-p section stained with 0.5% toluidine blue. x480. FIG. 4. Electron micrograph of a portion of a mammary gland duct from a BALB/c mouse. Incubated 1 hr for alkaline phosphatase. Arrows indicate localizations in the epithelial border toward the lumen (L), in the close junctions between the epithelial cells (E) and in the plasma membrane of the myoepithelial cells (M). Stained with uranyl acetate and lead citrate. x 10,000.

922

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CYTOCHEMICAL

REACTIONS

IN

EPOXY-EMBEDDED

MATERIAL.

923

5 and 6. Mammary gland ducts from a BALB/c mouse. Incubated 20 mm fr Mg + -dependent ATPase. 5. Light micrograph of a i-p section showing a mammary duct in cross-section; stained with 0.5% toluidine blue, photographed using bright-field illumination. x 1080. FIG. 6. Phase contrast micrograph of the same field as in Figure 5; arrows indicate sites of positive reaction for Mg -dependent ATPase in the plasma membrane of the myoepithelial cells, the epithelial border of the lumen and between the epithelial cells. 500. FIG. 7. Cross-section of a mammary gland duct from a BALB/c mouse. Incubated 20 mm in control medium that did not contain ATP. A i-p section stained with 0.5% toluidine blue and photographed with phase contrast. x1080. FIG. 8. Higher magnification of a portion of a mammary duct incubated 20 mm for Mg -dependent ATPase. Arrows indicate the sites of positive reaction in the epithelial border of the lumen (L). in the close junctions of the epithelial cells (E) and in the plasma membrane of the myoepithelial cells (M). Uranyl acetate and lead citrate staining. 8400. FIGS. FIG.

Downloaded from jhc.sagepub.com at WAYNE STATE UNIVERSITY on March 21, 2015

924

RUSSO

AND

dehyde in glass ampules (Electron Microscopy Science Inc., Fort Washington, Pa.). After fixation, the tissue was washed with three changes of 0.1 M cacodylate, pH 7.2, containing 6% sucrose. The tissue was usually held at 4#{176}C in the last buffer wash until the histochemical incubations were to be run. Histoehemistry: The tissue was removed from buffer at 4#{176}C and either minced by hand with razor blades on sectioned in a Smith-Farquhar microtome, using 7% agar

as

a supporting

medium.

These

slices

Light microscopy: Thick (l-p) sections were cut with glass knives mounted on an LKB Pyramitome from the Epon-Araldite blocks used for the electron microscopy study. These sections were mounted on glass slides, stained with 0.5% toluidine blue, decolonized with methanol and cover-slipped with Harleco tiple

incubated

for

1 hr at 37#{176}C with

of tissue

sites

were

priate

incubated

substrate

was

in media replaced

in which

with

agitation.

the

an equal

appro-

volume

of

distilled water. After the incubations, the reaction mixtures were drawn off and the tissue was washed three times at 4#{176}C with 0.1 M cacodylate containing 6% sucrose. The tissue was postfixed at 4#{176}C using 1% osmium tetnoxide in 0.1 M cacodylate for 2 hr. The tissue was dehydrated using a graded series of ethanol solutions, infiltrated with Epon-Araldite (iS) and acetone in 1:1 ratio, transferred to pure Epon-Araldite, embedded and polymenized at 60#{176}C for 48 hr.

and

FIGs.9

dependent FIG.

10.

Plastic

sections

ATPase. 9. Light micrograph

Proximal

tubules

appear

of

of a i-p denser

kidney section darker

and

condenser.

in

xylene.

The

slides

Photomicroscope

When

the

were

fitted

sections

were

ob-

with

a mul-

observed

with

of reaction

could

be

detected.

Whenever

speci-

RESULTS

Alkaline phosphatase gland is localized in the

in mouse mammary plasma membranes

the myoepithelial and epithelial cells. that, in i-p sections of plastic-embedded

We

rial

substrate

previously

mixture reaction

removed stained than

a Zeiss

mens were photographed in either bright-field or phase contrast, a green interference filter 546 nm ± 20 nm was inserted just below the condenser in order to increase the contrast for black and white film. Electron microscopy: Thin sections were cut using diamond knives with an LKB Ultrotome, mounted on copper mesh grids, and examined at 60 kV in a Siemens Elmiskop 1A. Most of the sections that were observed and photographed were stained with 2% uranyl acetate and Reynold’s lead citrate (24). However, sections stained with 2% unanyl acetate alone were examined in order to eliminate the possibility of lead artifact from the staining procedure causing a misinterpretation of histochemical localization.

The Mg k-dependent ATPase reaction was accomplished using the conventional method of Wachstein and Meisel (29). The material was incubated for 20 mm at 37#{176}C with agitation. Control sections for each enzyme

resin

with

bright-field illumination, the histochemical reaction appeared negative and reaction sites were not observed. However, when the phase contrast condenser and Neofluor phase objectives were used, specific

The alkaline phosphatase localization was done using the incubation mixture of Mayahara and Ogawa (14). An initial assay was made, during which the tissue sample was incubated for 3 hr at 37#{176}C. All other were

synthetic

served

were then washed two more times at 4#{176}C using 0.1 M cacodylate containing 6% sucrose. To start the histochemical reactions, these slices were transferred from buffer at 4#{176}C to one of the incubation mixtures at room temperature. All of the incubation mixtures were used immediately after final mixing and discarded after one use.

samples

WELLS

from

incubated

with

for 3 hr, it was possible in the myoepithelial

a BALB/c

with 0.5% toluidine the distal tubule in

mouse.

to visualize the cells with bright-

Incubated

blue, the

using center

the

20

mm

bright-field of

of

found mate-

the

for

Mg-

illumination.

micrograph.

x600.

Phase contrast micrograph of the same field as in Figure 9. Arrows indicate localizations for Mg + -dependent ATPase in lumen of the proximal tubules and in the region of the basal lamina surrounding the proximal tubules. .K600. FIG. ii. Phase contrast micrograph of a portion of mouse kidney which was incubated 20 mm in control media without ATP. A i-p section stained with toluidine blue. x600. FIG. 12. Electron micrograph of a proximal tubule showing localizations for Mg4 -dependent ATPase in the FIG.

brush

tubule.

10.

border

area

to

Figure

responds FIG.

between Uranyl FIG.

indicates

of the

The

lumen

within

and

the

14. Uranyl

in the

larger

box

acetate

region

of the

corresponds and

lead

basal

citrate

Higher magnification electron micrograph the processes at the base of the epithelium and acetate and lead citrate staining. 21,000. 13.

14.

Electron that the

micrograph reaction occurs

of

a cross-section between the

lamina.

to Figure staining.

PT denotes 13, while the x5600.

the epithelium area within the

showing localizations in the basal lamina (BL).

through microvilli.

the Uranyl

brush acetate

border and

of the smaller

of Mg + -dependent M. mitochondria;

proximal box cor-

ATPase N, nucleus.

of the proximal tubule. Arrow lead citrate staining. 36,00O.

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CYTOCHEMICAL

REACTIONS

IN

EPOXY-EMBEDDED

MATERIAL.

-.

D1?

‘

#{149}:

,FU -

.

. ,#,

‘‘#{176}:

Ih

V.t

:

‘I

.,q

-f -

-,

,

1

,,

-.

,

.

,

-

Downloaded from jhc.sagepub.com at WAYNE STATE UNIVERSITY on March 21, 2015

925

926

RUSSO

field

light

time

was

microscopy

reaction tron

to

product

with

1 hr

which

microscopy,

ble

(Fig.

reduced

the

was

cells,

where

the

est, but also between the border of the cells When

slides

areas

blue.

A

were

which

that

did

When

this

trast,

the

phase duced We

for

not

in

was

not

contain

phase

strong-

appeared distinguished with

in

in

viewed

(Fig.

study of black areas

phenomena

that

gland.

The

to

negative

illumination optics were

i-p

we plastic when

had

seen sections

viewed

(Fig. i9). When used to observe the

with phase same

(Fig.

i-p

testis, to be the same

mammary appear

bright-field

cells

(Fig.

phosphatase

exhibit

any

phase

contrast

(Fig. phase the

reaction (Fig.

mouse.

Wachstein

mammary

myoepithelial

Sites

of positive

was

also

the

material

in

was used, the the plasma

ductule in

epithelial were

was cells

the and

-dependent

and

in the

contained

ATPase

positive of origin,

field

were

negative.

could

be

visualized (Fig.

plasma host

The

20 mm

the reac-

and

in

in

the

A similar

female ATP-

membranes cells

in

reaction

using

of

border

induced Mg-dependent

while

localized

lumen.

tumor was incubated in control out ATP, and the cells of origin

bright-field

was

luminal

in the

23).

made with between

membranes cells. Some

in the

carcinomas (26), the

in

contrast

of a mammary +

plasma epithelial

appeared

not

incubated phase

micrographs for Mg

not

microscopy

and

interdigitations

In scirrhous BA.LB/c mice

Incubated

ATP-

8) confirmed the observations contrast. The reaction appeared in

Meisel

reaction

viewed

Electron

cellular

the the

was

light

of the

incubated

tion

villi using

and gland

bright-field

media 7).

we detected ATPase, using

of Mg-dependent

a mammary

specifically myoepithelial

ase the

gland

when phase contrast was demonstrated

6).

microscope

contrast field, the

15-18. Small intestine from a BALB/c FIG. 15. Light micrograph of several intestinal 0.5% toluidine blue. The upper circle encloses FIGs.

but

when

ductule

in

not

by

mouse with

(Fig.

The

phospha-

of testis

the

(Fig. 5), localization

con3).

laminar

alkaline

with

localization

control

mouse

in the

in

visible

this material observed with

In

ase

cells

medium

in phase

negative

material.

did

described

The

membranes

normal

where the alkaline phosphatase is known localized in the laminar cells that surround seminiferous tubules (25), we observed the

be

as

toluidine

which

(29).

visualized

contrast,

tissues in order to see if the alkaline tase reaction sites could be observed of different

testis

the

observed

technique

the

contrast correspond to lead deposits proby alkaline phosphatase activity (Fig. 4). decided to expand this method to other

sections

in when

in the

for

21).

intro-

fi-glycerophosphate.

was

reaction sites

visualized

control

was

incubated

was

was The

In the mammary activity of Mg4-dependent

stained made

tissue

microscopic that the

elec-

20).

allowed us to in the myoepi-

optics only

reaction easily be

were

material

a

detecta-

The

reaction

was

reaction

electron established

dense

was

observed

control

mammary

too

WELLS

reaction

the

avoid

the epithelial cells and in toward the lumen (Fig. 2).

the sites of enzymatic black areas and could from

when to

illumination.

of phase contrast the reaction not

thelial

But,

in order

reaction

bright-field

duction visualize

i).

AND

the

in these phase

of same

cells

contrast

portion

of the

medium withwere negative,

for Mg -dependent ATPase. illumination. A i-p section stained

with brush border that appears positive for Mg -dependent ATPase in the phase contrast micrograph in Figure 16 and that corresponds to the electron micrograph in Figure 18. The lower circle indicates a second area of the intestinal brush border that appears negative in the phase contrast micrograph in Figure 16 and that corresponds to the electron micrograph in Figure 17. x390. FIG. 16. Phase contrast micrograph of the same field as in Figure 15. Note that the positive reaction for Mg 4-dependent ATPase appears in the brush border in the apical portion of the villi, while the lower portion of the villi appears to be negative. x390. FIG. 17. Electron micrograph of a portion of a villus similar to the area enclosed by the lower circle in Figure an

area

of the

intestinal

16. The reaction for Mg-dependent ATPase is absent in the microvilli Uranyl acetate and lead citrate staining. x 14,000. FIG. 18. Electron micrograph of a portion of a single villus corresponding

and

in the

intestinal

epithelium.

to the area indicated by the upper circle in Figure 16. The Mg4 f-dependent ATPase is localized in the microvilli of the intestinal epithelium. Note that where a single microvillus is cut in a perfect longitudinal section the reaction appears in the plasma membrane and in the space between the microvilli and not inside the microvilli themselves. Uranyl acetate and

lead

citrate

staining.

14,000.

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CYTOCHEMICAL

REACTIONS

IN EPOXY-EMBEDDED

MATERIAL

W.34

-II 1.

r (

-.ci

I’

-..

-.

-

-

w

#{231}

‘F..

-‘.

.,..

.r-_T .

-.J

...-..

:‘$ .,o-

.-‘..

r...

-

Downloaded from jhc.sagepub.com at WAYNE STATE UNIVERSITY on March 21, 2015

927

928

RUSSO

when

this

material

(Fig. 24). The negative when nation

was

used

In mouse ase ma

with

10), the

the

tected.

two

when

When

i-p

ii).

Electron that

the

any reaction However, when amined zyme

in phase

was

the

electron

localized

used

without proximal

method

did

phosphatase

for human malignant

testis,

the

brane

of

localization the

confirmed i-p

with

plastic

electron

the

en-

places: the basal with the

(6, 25). lead

floating

the

fide for However,

varying we

with with used

we have localized plastic-embedded

of the villi (Fig. 16), while the basal of the villi near the crypts are negative. the same specimen is observed with

bright-field

illumination

(Fig.

15),

is not detectable in any portions electron microscope confirms shown

in

the

microvilli presented those

apical

phase

the

reaction

of the villi. The the localization

contrast

micrograph.

The

of the apical portion of a a positive reaction (Fig. 18), of the

basal

portion

were

villus while

negative

(Fig.

DISCUSSION

The

methodology

reported

of

phosphastase

degree

of

accuracy

embedded

been

The

used

by

visualization from sections.

in

material

microscope.

et

sections

the

visuali-

of

phase

al.

(1,

reactions preparations

4)

of the epithelial may be considered plasma mary

to the

the

scope.

precipitates

This

in

cytochemical

frozen

obtained

and

are

results

other

we i-p

tried

of by

sections

lead

sulfide,

ammonium time to

sul-

up to visualize

4 hr. the

illumination sulfide. microscope

our

in using

for

adenosine in frozen

methodology, i-p sections phase contrast

same portion microscope.

of

of the In the

mice, Mg-dependent in the plasma membranes myoepithelial an integral

of these and

cells and part of the

cells. humans,

Mg-dependent

In the the

mamsame

ATPase

in agreement

with

of our method the localization

obtained

technique

which ammo-

has

16). Many reports exist about localization of Mg-dependand alkaline phosphatase in the and in the kidney (5, 7, 9, i2, 29).

Our

has

rats

of

(ii,

The advantage accurately relate

for

of

as

for

results

to

With

and

membrane glands of

localization

in

produced by sarcolemma

ATPases material

glands localized

was

sections with

polarizing

(2i).

use contrast

laboratory,

1%

and then have studied the block with the electron mammary ATPase

The phase

bright-field ammonium

the

material

epoxycontrast

our

reaction of muscle

a high

microscope

phosphate

phosphatase

the with

the

using

Barter

However,

i-p

interference

of calcium

alkaline

allows

reactions

fixed

been reported the histochemical ent ATPase small intestine

17).

zation

and

the

the was

of the alkaline greater resolution

phosphate material on

mem-

localization

the

lengths of were unable

reaction sites after treatment Padykula

the

In

was (11)

rat

surround

microscopy.

sections

in

portions portions When

plasma

sulfide

reaction

localized

the

nium

have

correlating triphosphatase

be

16,

this

and

in

with benign 17). In the

11,

that

allows a localization reaction with

ATPbrush border contrast, the

to

and

sections

microscope phosphatase

in

cells

tubules,

localized myoepithelial

glands 10,

was

laminar

seminiferous

the

close

observaglands

same localization gland of the

mammary lesions (3,

the intestine, the Mg-dependent ase appears in the microvilli of the of the intestinal villi. With phase appears

of

and epithelial cells. The reported for the mammary and and

and

was

membranes

to convert the epoxy-embedded

same

In

alkaline

plasma

material

tubules the

permanent

not

microscope,

in exactly

not

that possible with frozen incubated and then treated

ATPase. were ex-

the brush border (Figs. 12 and 14) and lamina (Figs. 12 and 13), as localized phase contrast microscope.

the

were

with electron microscopic not possible. In the mammary

than were

(Fig.

of this

tubules

for Mg-dependent the proximal

with

was

contrast

study

distal

de-

completely negadistal tubules. A

was incubated reaction in the

microscopic

confirmed

same be-

easily

illumination

observed

obThe

in the differences are

reaction appears the proximal and

when

are

microscope.

of tubules

section of kidney which ATP does not show any tubules

sections

negative and the

bright-field

(Fig. 9), the tive in both

ATP-

in the basal lamof the proximal

contrast

types

this

correlations tions were of mice,

Mg-dependent

phase

WELLS

with

contrast

22). the

distal tubules appear preparation (Fig. 10) tween

in phase

to be completely bright-field illumi-

appears positive the brush border

(Fig.

served

viewed

(Fig.

kidney,

reaction and in

tubules

was

tumor appears conventional

AND

was

with also

localization heterogeneous

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these

reports.

is the ability to in i-p sections the

used

electron for studying

of ATPase tissues.

microthe

in tumors In

a line

of

CYTOCHEMICAL

FIG.

19.

A i-p section seminiferous FIG.

reaction vessel.

20.

Seminiferous was tubule;

Phase

tubules

stained BV, contrast

for alkaline xil6O. FIG. 21. Seminiferous Na-fl-glycerophosphate.

REACTIONS

from

with 0.5% blood vessel. micrograph

phosphatase

in the

the

testis

IN EPOXY-EMBEDDED

of a C3H/An

toluidine x 1160. of the

blue same

laminar

tubules (ST) of A i-p section stained

mouse with

and field

cells

that

mouse.

Incubated

photographed as

in

surround

929

MATERIAl.

1 hr for alkaline

using

Figure the

testis, incubated 0.5% toluidine blue

19.

The

bright-field arrows

seminiferous for and

phosphatase.

illumination. indicate

tubules

1 hr in control photographed

the (ST).

with

ST,

medium phase

positive BV,

blood

without contrast.

xilOO. FIG. 22. A carcinoma induced by the inoculation of D2 epithelial cells into a female BALB/c mouse. Incubated 20 mm for Mg-dependent ATPase. A i-p section stained with 0.5% toluidine blue and photographed using bright-field illumination. -615. FIG. 23. Phase contrast micrograph of the same field as in Figure 22. Arrows indicate positive sites of reaction in the plasma membrane of the D2 cells in the carcinoma. Note that the majority of the cells which belong to the host are negative for the Mg -dependent ATPase. >6i5. FIG. 24. A portion of the same tumor which was incubated for 20 mm in control medium without ATP. A i-p section was stained with toluidine blue and photographed using phase contrast. Note that the reaction does not appear in the plasma membrane of the D2 cells. 615.

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930

RUSSO

cells

obtained

cinoma

from

brane

of the

ent

a spontaneous

in BALB/c cells

ATPase.2

culture mouse, use

for

these

injected induce

marker cells

components

contrast

is

background

are

taken

host

female BALB/c carcinoma (26).

of cytological

structures

within

physical

the

interpretation

it

provides

lead phosphate of phase contrast

reaction

sites

higher

refractive

index

embedded

tissue

transmission

of

will

be

that

with prepa-

visualiza-

deposits follows the microscopy. If the

deposits)

have

compared

to the

surrounds

the

and lead

The use of phase simple methodology tion of the phosphatase

that

ultrast

ruct

ural

localization

microscope

method

for other tissues that react with

systems

electron-dense

with

the

te

and for other heavy metals

and

Mr.

of the his

William

wish

Isenberg

manuscript

skillful ‘Unpublished

thank

and

photographic results.

Dr.

for editorial Mr.

Robert

assistance.

applied 1954

on the

to

histo-

hydrolyzing Cytochem

of

identification Biophys Biochem

PrinciChicago

of alkaCytol

on the

magnesium

in arteries.

ultrastructural

dependent

Histochemie

neutral

18:210,

1969

T: Stromal estimated

Kon

K,

Fujii

activity. 1971 T,

in

Doi

response

by

the

Acta

aid

Pathol

Y, Tominaga

localization dysfunctional

and

in breast of the

alka-

Microbiol

5, Nakajima

T:

of alkaline

phospha-

neoplastic

glandular

epithelium of the mammary ductulus of female rats, Histochemistry and Cytochemistry. Edited by T Takeuchi, K Ogawa, S Fujita. Japan Society

of Histochemistry

Japan, 12. Makita

and

1972, p 413-414 T, Sandborn EB:

Cytochemistry,

Aldehyde

Kyoto,

fixation

fine structural localization of alkaline tase activity in intestinal epithelial cells, ings of the Twenty Seventh Annual Electron Society of America. Edited

the

Arceneaux. La., 1969,

Russo criticism

Kaspark

A quantitative

Studies

studies a

H, Schidt

tases

enzyme to form

Irma

of

Ultrastructural

13.

to

Further

line phosphatase Scand 79:321,

14.

authors

Los

1961

HF:

carcinomatosis,

ACK NOWLEDGMENTS

The

la

en

vitro” por la fitohe1968 acid and alkaline histologic diagnosis.

N:

AJ: Historadiographic phosphatase. J

10. Jensen

of this

markers.

de

tnifosfatasa

cytochemistry Nature 173:1234,

Kaplan

DG,

alkaline [Biol]

Lond

J, Borgers M: Ultrastructural localization of alkaline phosphatase activity in the absorbing cells of the duodenum of mouse. J Histochem Cytochem 14:629, 1966

11.

observed

encourages

A quantitative

chemical differentiation of enzymes adenosine tniphosphate. J Histochem 8:159, 1960 Gomoni G: Microscopic Histochemistry, ples and Practice. The University Press, Chicago, 1964, p 167-219

ATPase 9. Hugon

contrast optics provides a that permits the visualizareactions with a high

electron

Freiman

11:488,

is trans-

degree of accuracy in i-p sections of plasticembedded material. The close correlation between the histochemical localization shown with the phase contrast microscope and the

5’

“in 13:419, Histochemical an aid to the

for enzyme phosphatase.

7. Hale line

lead particles phase differ-

the light particles.

HG:

Proc Inst Med Chic 28:75, 1970 Davies HG, Barter R, Danielli JF:

localization

plastic-

use of phase contrast optics allows the of this small difference between the is transmitted through the plastic-

embedded material mitted through the

6.

adenosina

GW:

method alkaline 5.

Davies

Sangre

Changus phosphatases:

8. Hoff

a slightly them,

light through the causing a slight

retarded

ence. The detection light that

lead

a

4.

that can be tissue. Thus, we

for the

tion of principles

(i.e.,

3.

JF,

transformados

maglutinina.

the are

reaction.

since

de

linfocitos

as a histo-

are able to localize the enzymatic reaction more accuracy as compared to unstained rations. The

2.

cytochemical method for estimating phosphatase activity. Proc R Soc 144:412, 1956 Boven FG: Localizaci#{243}n ultraestructural

actividad

blue as a routine stain for be observed with phase

advantageous,

for orientation

Barter

from

R, Danielli

CITED

1.

-depend-

ATPase

of toluidine that will

LITERATURE

mem-

allows us to differentiate from the other cells that

of the

The use i-p sections

plasma Mg

cells

into a a scirrhous

WELLS

adenocar-

the

of Mg-dependent

chemical neoplastic

used

(28),

is positive

When

and they

The

mice

AND

for

Publishers,

Baton

Rouge,

Makita T, Sandborn EB: The ultrastructural localization of adenosine tniphosphatase and alkaline phosphatase activity in eosinophil leukocytes. Histochemie 24:99, 1970 Mayahara H, Ogawa K: The effect of thickness of specimen on the ultrastructural localization of alkaline phosphatase activity in the rat proximal convoluted tubule. J Histochem Cytochem

16:721, 15.

Claitor’s p 280-281

and

phosphaProceedMeeting, by CJ

1968

Mollenhauer use in electron 1964

DM: Plastic microscopy.

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embedding Stain

mixtures for Technol 39:111,

CYTOCHEMICAL

REACTIONS

IN

16. Murad TM: A proposed histochemical and electron microscopic classification of human breast cancer according to cell of origin. Cancer 27:288, 1971 17. Murata I, Kawashima H, Mon M: Histochemical

18.

19.

20.

study on alkaline phosphatase activity in human neoplasms. Gann 57:513, 1966 Nada 0, Ulano Y: Adenosine tniphosphatase activity in the carotid body of the cat. Z Zellforsch Mikrosk Anat 130:455, 1972 Nakamuna 5, Miyagami M, Moniyasu N: Electron microscopy study of ATPase activity in human brain tumors. J Neurosurg 38:420, 1973 Novikoff 5, Biempica L: Cytochemical and electron microscopic examination of Morris 5123 and

Reuver H 35 hepatomas after several transplantation. Gann Monograph 1:65, 21. Padykula HA, Gauthier GF: Cytochemical ies

22.

23.

of adenosine

tniphosphatases

years of 1966 stud-

in skeletal

and

role

in

cancerogenic

931

MATERIAL

Folia Histochem Cytochem (Krakow) 8:117, 1970 24. Reynolds ES: The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J Cell Biol 17:208, 1963 25. Russo J: Acid and alkaline phosphatase of the mouse

26.

testis

27.

A model

stages

of

Am

post-natal

17:302,

1967

carcinoma in the mammary carcinoma,

for human

of ultrastructure

aldehyde Soule

cell

29.

different

XI International Cancer Congress, Florence, Italy, 1974. Excerpta Medica, Amsterdam, 1975 p 468 Sabatini DD, Bensh KG, Barnett RI: Preservation

28.

at

development. Acta Physiol Lat Russo J, McGrath C: Scirrhous mouse:

mus-

cle fibers. J Cell Biol 18:87, 1963 Pearse AGE: Histochemistry: Theoretical and Applied. Williams and Wilkins Co., Baltimore, i972, p 475-575 Raikhlin NT: Plasma membranes: their ATPase activity

EPOXY-EMBEDDED

fixation. HD,

line

Maloney

from

hyperplastic 14:90, 1973 Wachstein enzymatic

a primary nodule.

M, Meisel staining

with necrobiosis rotoxic agents.

and enzymatic activity J Cell Biol 17:19, 1963 T, Vasquez J, Long Proc

Am

Assoc

by ischaemia

Histochem

1957

mechanism.

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A: A in a D2

arising

E: A comparative reactions in the

induced J

tumor

Cytochem

by

Cancer

rat

Res

study of kidney

and

neph5:204,