THE Jouas or HISTOCHEMISTRY im Copyright © 1977 by The Histochemical

Vol.

CYTOCHEMISTRY

Society,

Brief

POLYETHYLENEIMINE

AS

25, No.

5, pp. 384-387, 1977 Printed in U.S.A.

Inc.

Report

TRACER

PARTICLE

FOR

(IMMUNO)

ELECTRON

MICROSCOPY’ J. Centre

for

Medical

W.

SCHURER,

Electron

Received

PH.

Microscopy

for

J.

and

publication

HOEDEMAEKER

Department of Pathology, The Netherlands

February

9, 1977,

I. MOLENAAR

AND

and

of Groningen,

University

in revised

form

March

Groningen,

2, 1977

Polyethyleneimine (PE!) is proposed as a tracer for use in electron microscopical investigations. Relative small molecules are available (molecular weight 600-60,000). PEI is soluble in water; it is not visible in the electron microscope without further treatment, but can easily be detected as a particle by contrasting it with phosphotungstic acid or 0s04. Using PEI of a molecular weight of 40,000, particles of 10 nm diameter can be produced. The strong cationic character of PEI results in electrostatical binding to anionic sites. Hence perfusion and immersion of tissues with PEI of various molecular weights offers possibilities to either study the location of anionic sites or pathways of transport. Anionic sites could be demonstrated in the normal and pathologic glomerular basement membrane. Work on the use of PE! as a marker particle in immunoelectronmicroscopy is in progress.

The

possibility

tron

to

microscopic

rived

from

are

their

detectable

structural

certain

use

tracers

in

molecules

biologic

characteristic against

details

property

the

of the

tissues.

These

studies they can

muno-electron

to detect

as

microscopy (14).

tracer

enzyme quently. Ferritin 600,000),

Many

molecules

the

de-

locate been

tissue proposed

conjugation

and the most fre-

ference with cific binding

polypeptide

a core

hydroxyphosphate).

the

However, molecule cule

of about With

(mole. 2000

the

iron

electron

wt.

=

specificity

of

the

atoms

antigen-binding

since the total diameter is about 12 nm, the transport

across

cellular

membranes

and

the of

investigation Organization

has

been supported for the Advancement

by

larger.

An

of ferritin

is possible

has

the

enzyme

molecules

uct does

not present

an

of density

area

diameter.

is its

without

inter-

capacity. Nonspeconstitutes no

no electron

density

of its

This

is

capable

of substrate,

of the

converting

reaction

prod-

itself

as a particle,

but rather

measuring

at

40 to 50 nm

means

that

least

accurate

as

localization

in will

be difficult and quantitative studies almost impossible. Another disadvantage of the use of the horseradish peroxidase as a tracer molecule is that many biological tissues contain endogenous peroxidase ac-

barriers

‘This Netherlands

antibodies

molecule

of

numerous

sites.

imof

use

the antigen-binding to tissue components

The

molecule

of the ferritin of this moleother

is even

of the

own, but its enzymatic activity may be used to create an electron dense product by adding hydrogen peroxide in the presence of 3.3. diaminobenzidine tetra-hydrochloride (11). This compound becomes even more dense after fixation in 0804. Since one

micro-

will be difficult. This problem also arises in muno-electron microscopy, in which the diameter

Pure

to

problem.

is a globular

conjugate

disadvantage

binding to several tissue components, which makes numerous washing procedures necessary. The molecule of horseradish peroxidase is relatively small (mole. wt. = 40,000) as compared to ferritin, which to some extent facilitates its transport across cellular membranes. Also in this case

they ultra-

scope this core may be recognized as a characteristic particle with a diameter of 5.5 nm, which makes detection and quantitative studies easy. For the use in immuno-electron microscopy ferritin can be conjugated to antibodies (1, 12) without interference with

globulin

ferritin

additional

tracers can be (2, 4) or, when be used in im-

(7, 13, 15), but nowadays ferritin horseradish peroxidase are used

containing

(ferric

of the

and

have

elecis

that

background

used as such in penetration conjugated to antibodies, antigens

as

studies

tivity,

making

blocking

control

studies

necessary. be able

In

Research.

tracer

order

to

molecules

it could

384

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procedures to

and

numerous

get

optimal

results

be

of interest

to

with combine

BRIEF

the

above

cules

in

mentioned use

and

advantages at

the

of the

same

time

tracer leave

Small

the

Water-soluble

nonspecific

binding

for conjugation to avoid

tibody molecule in conjugation the use in in vivo experiments. 6.

p in

(r)

which

radius the

-p--- (

=

P

is

to tissue

compo-

to antibodies.

denaturation

procedures

of the

an-

and

for

the

[-C-C-N-C--C-N-I,, C

H

C

of

R can

3C212

-

2>

segments

and

be calculated

R

is

by means

of

relation



=

1/6

Pb2

relation b = ab,, is the corrected length of a polymer segment. Assuming R is the most important measure of the polymer coil and assuming that we work with statistical folded polymer segments (0 conditions, Flory a = 1), then P should be about 940 in which

=

A combination of properties of such a wide range demands a molecule like a polymer, in which various chemical groups are iterated. The available biopolymers would certainly meet the requirements mentioned to some extent, but synthetic polymers offer more possibilities to add specific groups in a large amount. Although synthetic polymers do not have a defined molecular weight and structure, these vary rather within a limited range. A polymer that would qualify as a tracer particle should contain: 1. NH2-groups for conjugation to antibodies. 2. Complexing groups for contrast purposes. 3. Polar groups for solubility. 4. A cationic character to avoid an excessive binding to tissue components. Considering a choice from the available synthetic polymers, good results are to be expected with polyvinylamine and polyethyleneimine, because both molecules contain the above mentioned properties. Since the latter polymer is commercially available in various molecular weights, this molecule was selected. Polyethyleneimine is a branched polymerisation product of ethyleneimine with the global structure formula:

312e

number

of inertia.

in the

Fixation-proof.

\

3

rr3/22)

in size.

3. Minimal nents. 4. Possibilities 5.

385

moleout

disadvantages. To achieve this it would be necessary to search for a tracer having the following specifications: 1. Detectable in the electron microscope as a particle. 2.

REPORT

desired

case

of a sphere

of 5 mm

diameter

(R

2, 5 nm).

As to the visibility of a particle microscope, it is known that the mined by the relation: NZ43/S

> 4700

with the electron contrast is deter-

nm2

in which N = number of atoms, Z = atom-number and S = area in nm2. It can be calculated that for adequate contrast with iron, silver, mercury or uranium, in the cross-section of a sphere of 5 nm diameter, respectively 1280, 540, 263 and 222 atoms are necessary. This means that in case of a chemical bond between this number of metal atoms and a polymer of which P = 940, a loss of specific groups will

be the

the

polymer.

result,

leading

to decrease

to guarantee in electron microscopy nearly massive metal In

order

of solubility

of

the visibility of it should form a and will it stay

the polymer colloid with soluble. In our experiment PEI with a molecular weight of 30,000-40,000 is used (P - 1500-2000 gives R (0) = 3.15-3.65 silver

nm), may

lead

which to

in colloids

combination of 3 nm

with diameter.

gold

or An

alternative is to confer the contrast to the molecule in a later stage of the procedure; in tissues containing pure PEI, the polymer could be contrasted with Os04 or phosphotungstic acid, and poststained with uranyl acetate and lead citrate, giving rise to particles with a diameter of about 10 nm. These two staining procedures were first tested in an in vitro system.

NH2

It has many primary, secondary and tertiary amino groups (10). The free electron orbitals of nitrogen are available for complex binding with heavy metals for contrasting purposes, while NH2 groups can be useful for conjugation with globulins. The size of such a polymer can be calculated from a given molecular weight and from the number of polymer segments of the molecule. From the model system of Kuhn (8, 9) and the theory of Flory (6) the mass distribution in a polymer coil is given by the following relation:

Colloid system: In this staining procedure heavy metal ions are reduced to conglomerates of pure metal atoms in the presence of the water-soluble polymer. In this way the metal particles remain colloidally in solution by means of a polymer layer. Best results were obtained with 1% PEI solution and 0.1% AgNO, or AuCl, solution reduced by NaBH4 or glutaraldehyde. The glutaraldehyde has two functions: it reduces the ions and crosslinks the polymer around the metal particle, thus stabilizing the colbid in order that the earlier-mentioned requirements of a tracer particle can be met. Using this method dense particles may be produced having a

Downloaded from jhc.sagepub.com at University of Otago Library on April 22, 2015

BRIEF

386

REPORT

diameter of about 3 nm and which are visible on formvar film (and also in tissues). However, in purification procedures the colloid appears unstable. Disperse system: In this staining procedure heavy metal is bound to the polymer by means of ionogenic

bonds. As explained earlier, this may result in a decreased solubility of the complex. Therefore, the polymer is to be stained at the moment when it has reached the “desired” spot in the tissue (described under in vivo), where a reaction between the poly-

S.

-‘

-

s---

. 1’

1-4. Electron micrographs of the gbomerular basement membrane of rats injected with 0.2 ml of a solution in diluted HC1 (pH 7.3). FIG. 1. After 1 hr electron dense particles are observed in the lamina rara interna and externa with regular intervals. Phosphotungstic acid/uranyb acetate/lead citrate. x 30,000 FIG. 2. After 2 hr the number of electron dense particles in the lamina rara interna is somewhat decreased, while only occasionally a particle is observed in the lamina rara externa. 0s04/uranyl acetate/ lead citrate. x30,000. FIG. 3. After 4 hr electron dense particles are observed lining up in the filtration-slits. Os04/uranyl acetate/lead citrate. x 130,000. FIG. 4. One hour after PEI injection in rats with autobogous immune complex gbomerubonephritis, electron dense particles are present in the lamina rara interna. The lamina rara externa shows immune aggregates (asterisk) but only a few electron dense particles are present. 0s04/uranyl acetate/lead citrate. x 70,000 FIGs.

0.5%

PEI

Downloaded from jhc.sagepub.com at University of Otago Library on April 22, 2015

BRIEF

mer

and

heavy

complex. and

It

0504

are

solutions As tion

metal turned suitable

the

appear

in

an

insoluble

phosphotungstic in this

to be

for in vivo

acid

way

with

PEI

unstable

experiments:

30,000-40,000)

was

Wistar

rats,

Free injected

the

LITERATURE

disperse

weighing

cationic

PEI

intravenously approximately

(mole

1. wt

into

nor-

200

g. 0.2

in diluted HC1 (pH 7.3) was injected, which was well tolerated by the animals. Using this amount, the PEI particles could be detected only in reasonable amounts in the glomerubar basement membranes of the kidney. Until 1 hr after the injection, PEI particles are found in the lamina rara interna and the lamina rara externa of the glomerular basement membrane with a fairly regular interspacing (Fig. 1). Occasionally a particle may be seen lying in the filtration slit or in Bowman’s capsule, sometimes adhering to the cell membrane of the epithelial cells. In the following hour the number of PEI particles, present in the lamina rara externa, decreases, while the number of PEI particles in the lamina rara interna does not seem to change very much (Fig. 2). During this period only a few particles could be demonstrated in the slit pores. Four hours after the injection the number of PEI particles, present in the lamina rara interna, also decreases and particles may be seen lining up in the filtration slits (Fig. 3). At none of the time intervals PEI particles are detected in the lamina densa of the GBM. Preliminary experiments with PEI injections in animals, which were made nephrotic, either by deposition of immune complexes or by injections of aminonucleoside of puromycin (5), showed that in these cases the localization of PEI particles was different. One hour after injection the particles could be found in a considerable amount in the lamina rara interna of the glomerular basement membrane (GBM), whereas the lamina rara externa only occasionally showed the presence of a particle (Fig. 4). The typical localization of PEI particles in the glomerular basement membrane of normal animals suggests that the injected PEI molecules are bound to regular interspaced sites of negative charge, present in the lamina rara externa and the lamina rara interna of the GBM. This was also demonstrated after injection of lysozyme (3). The results of these preliminary experiments with nephrotic rats sugml

gests

of a 0.5%

that

PEI

in

case

solution

of

proteinuria

cate the PEI is suitable to trace anionic sites and certain pathways in tissues. Further studies on its use as a tracer and as a marker-particle in immunoelectron microscopy are in progress.

in purifica-

experiments

chosen.

In vivo mal

result

concentrations.

colboids was

to that

to react

in different

procedures,

system

has out

387

REPORT

the

areas

with

negative charge of the lamina rara externa of the GBM are decreased. These results and the localization of PEI particles, as found for example in brain and liver, mdi-

Avrameas

with the

5:

Coupling

of enzymes

glutaraldehyde. detection

JP,

and

to

proteins

conjugates

for

antibodies.

MG: The

capillaries to graded

rats

142:61, Cauldfield anionic

of the

6:43, 1969 Farquhar

of gbomerular

phrotic

Use

of antigens

nochemistry 2. Cauldfield

3.

CITED

Immu-

permeability

of aminonucleoside-nedextrans. J Exp

Med

1975 JP, sites

in

Farquhar gbomerular

MG:

Distribution basement

of mem-

branes. Their possible robe in filtration and attachment. Proc Soc Nat Acad Sci 73: 1646, 1976 4. Clementi F, Palade GE: Intestinal capillaries. I. Permeability to peroxidase and ferritin. J Cell Biob 41:33, 1969 5.

Fleuren

ment

GJ:

Studies

ubonephritis.

6.

7.

8. 9.

on

of experimental

pathogenesis

immune

Doctoral

and

complex

Thesis,

treat-

glomer-

University

of

Groningen, 1976 Fbory PJ: Principles of Polymer Chemistry. Cornell University Press, Ithaca, New York. 1953, p 399-431 K#{252}hlman W, Avrameas S: Glucose-oxidase as an antigen marker for bight and electron microscopic studies. J Histochem Cytochem 19:361, 1971 Kuhn W: Uber die Gestalt fadenformiger Molek#{252}lein Losungen. Kolboid Zschr 68:2, 1934 Kuhn W: Beziehung zwischen Molekulgrosze, statischer

MolekUlgestalt

und

elastische

Eigen-

schaften hochpolymere Stoffe. Kolboid Zschr 76:258, 1936 10. Meltzer YL: Water-Soluble Polymers, Technology and Applications. Noyes Data Corporation, Noyes Building, Park Ridge, New Jersey. 1972, p 162-193 11. Seligman AM, Karnovsky MG, Wasserkrug HL, Hanker stration

JS: Nondroplet of cytochrome

lymerizing dine (DAB). 12.

Siess for

E, the

ultrastructural oxidase activity

demonwith po-

osmophilic reagent, diaminobenziJ Cell Biol 38:1, 1968

Wieland

0,

preparation

Miller of pure

F: A simple and

active

method y-gbobu-

lin-ferritin conjugates using glutaraldehyde. Immunology 20:659, 1971 13. Singer WJ: Preparation of an electron dense antibody conjugate. Nature 183:1523, 1959 14. Sternberger LA: Immunocytochemistry. Prentice-Hall, Inc. Englewood Cliffs, New Jersey. 1974

15.

Venkatachalam MA, Karnovsky MJ, Fahimi HD, Cotran RS: An ultrastructural study of gbmerular permeability using catalase and peroxidase as tracer proteins. J Exp Med 132:1153, 1970

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