1280

LETTERS

TO

describe acid phosphatase activity against PC and DEP as “PAP-bike” would be anomalous. Since it is apparent that PC and DEP are not totally specific substrates for either prostate gland or non-bysosomal acid phosphatase, but may be specific for secreted acid phosphatase, it might be more appropriate to use the term “secreted acid phosphatase” which would describe both lysosomab and non-lysosomab secreted acid phosphatase activity against these substrates. However, “secreted acid phosphatase” is also inadequate since secretion of acid phosphatase by every cell type known to hydrobize PC and DEP has not been conclusively demonstrated. We agree that naming an enzyme in reference to the organ or organs in which it is found, or that describing enzyme activity in reference to substrate hydrolysis when more than one substrate is hydrolyzed by the same enzyme would bead to confusion. Serrano et al. appear to assume that PC and DEP are in fact hydrolyzed by the same enzyme. However, our comparative study of enzyme activity against these substrates has revealed distinct differences in histochemicab staining of rat tissues. Sebaceous glands are more active against PC than DEP, while ventral prostate epithelia are more active against DEP than PC, and osteoclasts appear equably active against both substrates. These results suggest that more than one enzyme or isoenzyme are involved in the hydrolysis of PC and DEP. Therefore, until further characterization of the A

SENSITIVE

HISTOCHEMICAL

MICROSCOPIC

Horseradish to

peroxidase Graham

and

macromolecular

(HRP)

histochemistry (4)

This

method

of choice functional to obtain neunonab as much histochemical

(5,

7)

has

mw.

rapidly

40 become

ac-

has

been

of

000

(S,

the

6,

9).

method

for many investigators interested in the anatomy of the nervous system. In order most precise information about various connections the fixative used should retain enzymatic activity as possible and the procedure

bightmicroscopical Until now, most

should

visualization investigators

result

LITERATURE

in

of neuronal have with

optimal

HRP. minor

‘Supported by Grants from the Swedish Medical Research Council Project B7812X0302009A and by Grant 1F32NS0S3471, United States NINCDS, NIH. Technical assistance mainly provided by Madeleine Janild.

CITED

1. Helms SR, Brattain MG, Pretbow II TG, Kreisberg JI: “Prostatic acid phosphatase?” a comparison of acid phosphatase activities in epithebiab cells, granubocytes, monocytes, lymphocytes, and platelets purified by velocity sedimentation in isokinetic gradients of Ficoll in tissue culture medium. Am J Pathol 88:S29, 1977 2. Schofield BH, Mulhern HL, McDonald DF: Acid phosphorylcholine phosphatase of sebaceous glands 25:309,

and 1977

osteoclasts.

J Histochem

Cytochem

3. Sebigman AJ, Sternberger NJ, Paul BD, Friedman AE, Shannon WA Jr., Wasserkrug HL, Plapinger RE, Lynm D: Design of spindle poisons activated specifically by prostatic acid phosphatase (PAP) and new methods for PAP cytochemistry. Cancer Chemother Rep 59:233, 197S BRIAN

H.

SCHOFIELD

F. MCDONALD

DANIEL

Orthopaedic Johns

for publication

Karnovsky

tracer

acid phosphatase activities against PC and DEP has been carried out, it would be best to describe this possibly “secreted”, possibly “PAP-like” enzyme activity as APCP (acid phosphorylcholine phosphatase) and ADEP (acid r-ephedrine-o-phosphatase).

FOR

OF

great value in many fields of research such as vascular permeability, spread of vasogenic edema and localization of cellular antigens. We have been particularly interested in basic mechanisms concerning uptake and retrograde axonab transport of this

EDITOR

METHOD

DEMONSTRATION Received

cording

THE

School

of Medicine Maryland

LIGHT-AND

ELECTRON-

21, 1977

tronmicroscopicab

escape

the method proposed by Gra(4) which gives excellent ebec-

pictures

action product but neurons containing In an retrograde

21205

PEROXIDASE’

modifications followed ham and Karnovsky

well

Laboratory

University

Baltimore,

HORSERADISH July

Research

Hopkins

of the

electron

due to limitations low amounts

dense

re-

in sensitivity of enzyme may

detection.

attempt HRP

quantitative

to increase the sensitivity of the method we have made a systematic,

study

on the

effect

of fixation

and

have

also examined the effect of variations in other steps of the histochemical procedure to arrive at a method which gives optimal detection of retrogradeby transported HRP in hypogbossal neurons (9). In our lightmicroscopicab study (9) the following experiments were done. Twenty-four hours after injection of 1.S-3 mg HRP type II (Sigma) into the tongue, adult mice were fixed by vascular perfusion through the heart. We used abdehyde fixatives made up in 0.1 M phosphate buffer, pH 7.4. Concentration, type

of abdehyde,

of buffer results

rinse were

duration before

of fixation

incubation

evaluated

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 12, 2015

after

were counting

and

duration

varied. the

The no

of

LETTER

TO

labelled hypogbossal neurons. Our results showed that paraformaldehyde in the fixative reduces the number oflabelled neurons as compared to 1.5-2.5% glutaraldehyde (GA). Fixation in 1.5% GA for 4 hr is recommended

followed

by

a wash

in

5%

sucrose

phosphate buffer over night. Variables in the histochemicab procedure were systematically studied in order to determine optimal pH, buffer type, buffer concentration and substrate concentration of material fixed in 1.5% GA (9). Equivalent groups of sections from each animal were treated in different ways and the results were obtained by comparing the no of labelled hypogbossal

neurons

with

that

found

in the

group

incubated

according to Graham and Karnovsky (4). The best results were obtained by a modification that involves incubation of sections in the dark for 30

mm

in

10 ml

cacodylate

buffer

(2)

(pH

5. 1; 0.1

M), 20 mg diaminobenzidine tetrahydrochboride (DAB) and 0.1 ml 1% hydrogen peroxide. This procedure resulted in a considerable increase in the no of detectable HRP-containing neurons. Of particular importance is that incubation occurs at the pH optimum of the enzyme which has also recently been stressed by Streit and Reubi (11). The choice of buffer is also essential. As compared to other buffers at the same pH and ionic strength (tris; acetate; citrate) cacodylate buffer in the medium considerably increased the no of detectable cells. Other details of the lightmicroscopical study will be presented elsewhere (9). The method described by Graham and Karnovsky (4) involves incubation in a Tris-buffer containing medium at pH 7.6. In neuroanatomy the retrograde HRP method is usually based on lightmicroscopical enzyme histochemistry alone. For certain purposes ebectronmicroscopicab visualization of the enzyme might be needed and the question therefore arose, could our modification with incubation at a low pH in cacodylate buffer also be a feasabbe alternative for ultrastructurab studies when high sensitivity is required. Six adult mice received 1.S mg HRP type VI (Sigma) by injection into the tongue. Twenty-four hours later they were perfused and fixed by immersion in 1.5% GA for 4 hr as in the lightmicroscopical study. After wash in the sucrose buffer over night multiple sections from the hypoglossal nucleus of each mouse were cut on a tissue sectioner and divided into three groups which varied only in the type of histochemicab incubation used. Group “GK” was incubated according to Graham and Kannovsky (4) (5 mg DAB, 0.1 ml 1% H202, 10 ml Tris-HC1 buffer pH 7.6). Group “MO” according to our lightmicroscopical (9) procedure (20 mg DAB, 0.1 ml 1% H202, 10 ml cacodylate buffer, pH 5.1). Group “MOIm” according to our modification at pH 7.0 with addition of 0.1 M imidazole to the medium (E. Merck, Darmstadt). The sections were all preincu-

THE

1281

EDITOR

bated for 30 mm in DAB-buffer and the final incubation occurred in the dark for 60 mm. After wash in cacodylate buffer, pH 7.2, they were osmificated for 1.5 hr (1 ml 4% OsO, 1 ml aq. dest, 2 ml cacodylate buffer, pH 7.2). They were then dehydrated in ethanol, passed through propyleneoxide and embedded in Epon. Thick sections were stained with toluidine blue. Areas containing labelled neurons were selected for thin sections. They were collected on single hole grids, stained with lead citrate and uranyl acetate and viewed in a JEOL 100 C electron microscope. In the thick sections HRP-containing neurons were easiest to identify in the MO group due to the presence of numerous black cytoplasmic granules. The localization of the electron dense reaction product in the hypogbossal neurons in the GK group was the same as has been described in other ubtnastructural papers on retnogradely transported HRP i.e., mainly in various lysosomal structures (6, 8). We could not find any obvious difference with regard to the lysosomal localization of HRP between the three groups but many mitochondria had reaction product along their internal christae in the MO, and MOIm treated sectioiis. Such mitochondnia were present both in labelled and unlabelled cells. Similar deposits of reaction product have been observed previously,

for

instance

in renal

tubular

cells

incu-

bated for demonstration of exogenous myogbobin (1) and might be related to the use of a high concentration of DAB. The ultrastructural appearance of the labelled neurons did not differ very much between the three groups of sections incubated in different ways (Fig. 1). Incubation in cacodylate buffer at pH 5.1 did not change

the

morphology

of cellular

onganelbes

but

in

small areas they were clumped together leaving clear spaces in the cytoplasm. Generally there was a good ultrastructural preservation of the tissue. Our results show that the modified and sensitive lightmicroscopical procedure for demonstrating netrogradely transported HRP can also be used for electnonmicroscopicab visualization of the enzyme in hypoglossal neurons. The technique with incubation at pH 5.1 can be tried as an alternative to the Graham and Karnovsky procedure (4) in cases where higher sensitivity is required. Although acid phosphatases routinely are incubated at a low pH (5.1-5.2) for electronmicroscopical demonstration of the enzymes (3) it is conceivable that problems may arise in certain tissues incubated for exogenous HRP at pH Si. Incubation can then be tried at pH 7.0 with addition of 0.1 M imidazole to the medium. This substance is known to increase sensitivity for histochemical detection of hemepeptide peroxidases (10) and we have found (9) that at pH 7.0 addition of imidazole increases the no of light microscopically detectable hypoglossal neurons. However, the no never reached as high values

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 12, 2015

1282

LETTER

,.

TO

THE

EDITOR

:.‘“

,



f’’j’t 41

:‘

:-,

.

.f

\.4’

.d-

f.-

#{149}*‘‘

., ‘i

#{149}41 ‘S

5,

-

-

- -

I

.

-.

-

.

.

*

.,-.-

-:

...

.: -.

-

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S’S

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.

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-

.

.

-

/;.

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-



.

.

9

5’S

,

________ FIG.

MO.

1. Thin

4

from

hypogbossal

occurred

at

section

nucleus

incubated

for peroxidase

activity

at pH Si,

alternative

xS980.

as

when

incubation

imidazole

in the

The

use

indicated

of the by

improve

contain

only

retrograde

the

concentrations

axonal

transport.

retrograde

HRP

technique

for

situations

when

rized

the

facilitate

study of of

Mount

We and

there

neurons

would

that

therefore

studies

it could

also

is a need

application

modifications

we

using

be tried

of increas-

in “Proposed

have

Wash

buffer

over

3.

Rapidly

microscopical tioner)

for

repeatedly

In

Dehydrate

beneoxide with lead

summa-

5%

been

sucrose

7.0

phosphate

electron

sections, 40-p.

20-p. thick

thick,

section

for

light-

(tissue

1%

dry

in

H202.

Wash

sec-

microscopy.

air,

2 mm

in cacodylate in

ethanol

buffer, and

then

let

sections

in

xylene.

46 filter

to

cells.

the ml mg

0.1

pH

7.0

chem Diem

3.

Essnen

treat

to

Thereafter

5.1.

which

20:672, 1972 K: Scientific

Pharmacenticals. E: Phosphatases;

with

Contrast

Preincubate

of pH

as

propy-

thin

sections

above

but

Incubate in the 0.1 M imidazole

proceed

LITERATURE

2.

4. Lightmicroscopical procedure: Incubate in dank for 30 mm at room temperature in 10 sodium cacodylate-HC1 buffer (pH 0.1 M), 20 diaminobenzidine tetrahydnochlonide (DAB) and ml

at

series,

in Epon.

MO-IM:

instead

added.

Geigy

studies,

in ethanol

1. Anderson WA: The as an ultrastructurab

night. frozen

no.

labelled procedure:

and embed and uranyl.

Alternative

pH

Method.”

METHOD

in

Wnatten

of HRP

MO: Preincubate in the same medium as for bightmicroscopy except without H2O2 for 30 mm. Incubate in full medium at pH 5.1 for 60 mm (dark, room temperature). Wash twice in cacodylate buffer pH 7.2 for S mm. Osmificate for 1.S hr (1 ml 4% OsO, 1 ml aq. dest, 2 ml cacodylate buffer, pH 7.2).

1. Fixation in 1.5% glutanaldehyde in 0.1 M phosphate buffer pH 7.4 first by vascular perfusion at 37#{176}C and then by immersion for 4 hr at room temperature. 2.

Kodak

Electronmicroscopical

medium PROPOSED

Use

identification

Alternative

following

instance HRP immunohistopermeability studies etc.

its

in Eukit.

5.

should

HRP

neuroanatomical

ing sensitivity, for chemistry, vascular to

without

modifications

identification

low it

order

technical

lightmicroscopical

recommend in other

5.1

facilitate

various

our

greatly

pH

medium.

as for

at

same has

MO.

CITED

use of exogenous myoglobin tracer. J Histochem Cytotables.

Documents

New Electron

York,

Geigy.

1962 Microscopy

Enzymes. Edited by MA Hayat. Van Nostraud Reinhold Co., New York, 1973, 1:204 4. Graham RC, Kannovsky MJ: The early stages of absorbtion of injected horseradish peroxidase in the proximal of mouse kidney. J Histochem Cytochem 14:291, 1966 S. Knistensson K, Olsson Y: Retrograde axonal transport of protein. Brain Res 29:363, 1971 6. Kristensson K, Obsson Y: Retrograde transport

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 12, 2015

of

LETTER of horseradish 3. Entry into

localization 1976 7. La Vail, transport 176:1416,

penoxidase injured

in axons

of penikaryon. JH, La Vail, in the central 1972

TO

THE

transected axons. and subsequent

Brain

Res

115:201,

MM: Retrograde nervous system.

axonal Science

onal transport. Brain Res. In press 10. Simonescu, N, Simonescu, M, Palade, G: Permeability of muscle capillaries to small heme-peptide. J. Cell Biol. 64:586, 1976 11. Streit, P, Reubi, J C: A new and sensitive staining

study.

J.

Comp.

Neunol.

method

radish system.

8. La Vail, JH, La Vail, MM: The retrograde intnaaxonal transport of horseradish peroxidase in the chick visual system: a light and electron microscopic 1974.

1283

EDITOR

LESLIE

AND

Our

results

(5) concerning liven

support the

plasma

the

effect

findings

membrane-bound

bile

salts

et al. upon

enzymes.

We have investigated the histochemical location of y-GT (-y-gbutamybtranspeptidase, E.C.:2.3.2.2.) in over 100 human liver biopsy specimens in normal and pathologic conditions. We could classify the biopsies as normal liven, with a faint sinusoidal membranous staining and as abnormal (chronic aggressive hepatitis, cirrhosis and alcoholic liver diseases)

with

a very

strong

activity

present

SALTS June

Laboratory

Sweden

IN CHOLESTATIC

LIVER

30, 1977

with low tissue activity of y-GT can be explained by the detergent effect of bile salts accumulating in this condition in the hapatocytes. We are studying the concentrations of the different bile salts in human liver to compare these findings with the histochemical activity of y-GT in the same tissue to investigate whether there exists a strict inverse correlation between bile salt concentration and -y-GT activity in liver tissue. LITERATURE

in bile

ducts and canalicubi and a weaken activity in the sinusoidal membranes. Furthermore, in cholestatic conditions we found three different histochemical patterns. In the first group, with a slight degree of cholestasis (early and resolving phase), the y-GT activity was quite similar to the normal condition. In the second group, with established cholestasis, a very strong activity was found only in portal ducts, peniportal canaliculi and a weaker activity on the sinusoidal surfaces; the activity in the latter two locations became weaker towards the central zone, where microscopical cholestasis prevails. In the third group, with severe cholestasis, very few ducts and peniportab canaliculi were still strongly positive, while the rest of the parenchyma looked as “washed-out.” Severe cholestasis is characterized by intrahepatic accumulation of bile salts (3). Taking into account the detergent effect of bile salts (1, 2, 4) and the difference in concentration of bile salts in normal and cholestatic liven (3), we treated sections from a liver biopsy, showing strongly positive y-GT activity (cirrhosis with hepatoma), with increasing concentrations of bile salts (0-30 mM). The y-GT activity disappeared from the sections by this treatment and could be detected in the bile salt solutions. For deoxycholate the detergent effect started at 1 mM concentration, for chenodeoxycholate at 1.5 mM, while for cholate only at 6 mM. Thus the apparent paradox of high serum concentrations of y-GT in patients with severe cholestasis associated

visual

of Uppsala

Uppsala,

BILE

horse-

pigeon

of Pathology

University

for publication

of Vyvoda

of different

transported

MALMGREN

Institute

9. Mabmgren, L, Olsson, Y: A sensitive method for histochemical demonstration of horseradish penoxidase in neurons following retrograde ax-

Received

axonalby

YNGvE OlasoN Neuropathological

1S7:303,

y-GLUTAMYLTRANSPEPTIDASE

for

penoxidase (HRP) in the Brain Res. 126:530, 1977

1.

CITED

Coleman R. Holdsworth G: Effects of detergents on erythrocyte membranes: different patterns of solubilization of the membrane proteins by dihydroxy Soc

and

trihydroxy

bile

salts.

Biochem

Trans

3:747, 1975 2. Evans WH, Kremmer T, Culvenon JG: Robe membranes in bile formation. Comparison

of of

the composition of bile and a liver bile-canabicubar plasma membrane subfraction. Biochem J 154:589, 1976 3. Greim H, Tr#{252}bzsch D, Czygan P, Rudick J, Hutterer F, Schaffner F, Popper H: Mechanism of choletasis. 6. Bile acids in human livers with or without biliary obstruction. Gastroenterology

63:846,

1972

Righetti ABB, Kaplan MM: Disparate responses of serum and hepatic alkaline phosphatase and 5’ nucleotidase to bile duct obstruction in the rat. Gastroenterobogy 62:1034, 1972. 5. Vyvoda OS, Coleman R, Holdsworth G: Effects of different bile salts upon the composition and morphology of a liver plasma membrane preparation. Deoxycholate is more membrane damaging than chobate and its conjugates. Biochim Biophys Acta 465:68, 1977 4.

C. A. BusAcHI J. MEBI5

V. J. DESMET Academisch Ziekenhuis Laboratorium

voor

Sint Histochemie

Cytochemie Minderbroedersstraat

B-3000

Downloaded from jhc.sagepub.com at UCSF LIBRARY & CKM on March 12, 2015

Leuven,

12

Belgium

Rafael en

A sensitive histochemical method for light-and electron-microscopic demonstration of horseradish peroxidase.

1280 LETTERS TO describe acid phosphatase activity against PC and DEP as “PAP-bike” would be anomalous. Since it is apparent that PC and DEP are no...
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