[22] AUTORADIOGRAPHY 279

[22] Autoradiography
By GRETCHEN H. STEIN and ROSALIND YANISHEVSKY

Autoradiography is a technique for detecting radioactivity through the

formation of silver grains in a photographic emulsion. An emulsion con-
sists of crystals of silver halide, usually silver bromide, suspended in
gelatin. To increase their sensitivity, nuclear emulsions designed for au-
toradiography have a higher ratio of silver halide to gelatin than do emul-
sions designed for light photography. They have a very high efficiency for
low-energy 13 panicles such as those emitted by tritium (18 keVmax),
carbon-14 (155 keVmax), phosphorous-33 (250 keVmax), sulfur-35 (167
keVma~), and iodine-125 (35 keVmax). The efficiency is somewhat less for
high-energy /3 panicles, such as those emitted by phosphorus-32 (1710
keVmax).
Autoradiography is based on the same principle as photography ex-
cept that the energy for conversion of silver bromide to metallic silver is
derived from ionizing radiation rather than from photons of light. As /3
panicles pass through an emulsion layer, they set electrons free in the
silver bromide crystals. These crystals contain sensitivity specks, which
are irregularities in the crystal lattices. The free electrons migrate to
sensitivity specks and attract silver ions, which combine with the elec-
trons to form atoms of silver. A latent image of the autoradiogram is
formed when enough silver atoms accumulate at a sensitivity speck to
form a nucleus of metallic silver that catalyzes the conversion of the entire
crystal into a silver grain during development. Developers are reducing
agents that furnish electrons to reduce silver ions to silver atoms. Because
crystals that have latent images are reduced to metallic silver more
quickly than other crystals, development can be stopped when only the
latent images have developed into silver grains. Unreduced crystals are
preferentially dissolved in the fixer, leaving behind a pattern of silver
grains that denotes the presence of radioactive material.1

Materials
Nuclear Emulsions. Eastman Kodak (Rochester, N e w York) manufac-
tures four liquid emulsions. NTB, NTB-2, and NTB-3 produce large
grains suitable for light microscope autoradiography. NTB grains are the
largest (approximately 2700/~ diameter) and NTB-3 grains are the smal-
l w. D. Gude, "Autoradiographic Techniques: Localization of Radioisotopesin Biological
Material." Prentice-Hall, Englewood Cliffs, New Jersey, 1968.

Copyright © 1979 by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. LVIII All fights of reproduction in any form reserved.
ISBN 0-12-181958-2
280 SPECIALIZED TECHNIQUES [22]

lest (approximately 2300 /~ diameter) of the three, z NTE, which is de-

signed for electron microscope autoradiography, produces very small
grains (approximately 500/~ diameter). 3 NTB-3 is approximately twice as
sensitive as NTB-2, which is twice as sensitive as NTB or NTE. 4,s Be-
cause increased sensitivity means that less energy is needed to make an
activated crystal develop into a silver grain, the background increases
with the sensitivity.
Ilford Ltd. (Ilford, Essex, Great Britain) also manufactures nuclear
emulsions that cover a range of sensitivities and grain sizes. Ilford L4 is
frequently used for electron microscope autoradiography because it has a
small grain size (1200 A) and is much more sensitive than NTE. Recently,
Kodak has produced an improved emulsion for electron microscope au-
toradiography, which is also more sensitive than NTE. It is called Kodak
Special Product Type 129-01.
Dipping Vessels. We use Lab-Tek No. 4310 Cyto-Mailers as dipping
vessels (see Fig. 1). These vessels are thrown away when the emulsion is
discarded.
Slide Boxes. Small black Bakelite slide boxes (Scientific Products),
which hold 25 slides, are used for storing the slides during the exposure
period. Some investigators use this type of slide box as a light-tight box,
provided that it is sealed with tape. However, we prefer to store the taped
slide boxes inside a second light-tight box.
Desiccant. We include small packets of Drierite in the slide boxes
during the exposure period because nuclear emulsion is most sensitive
when it is dry and because moisture can cause latent image fading. The
packets of Drierite, including some blue indicator Drierite, are wrapped in
several layers of cheesecloth and secured with tape.

Autoradiography Darkroom
An autoradiography darkroom should exclude all light. This demand
exceeds the normal requirements of a photographic darkroom. (A model
darkroom for autoradiography is described by Kopriwa.6) The darkroom
should be inspected for light leaks when the eyes have adapted to dark-
ness (a minimum of 15 min). Sources of light entry may be window and
door frames, door steps, ventilation shafts, or where pipes pass through
floors. Light may also come from electrical contacts that spark on switch-

2 L. G. Caro, Methods Cell Physiol. I, 327-363 (1964).

a M. M. Salpeter, Methods Cell Physiol. 2, 229-253 (1966).
4 A. Ron and D. M. Prescott, Methods Cell Biol. 4, 231-240 (1970).
5 A. W. Rogers, "Techniques of Autoradiography." Am. Elsevier, New York, 1973.
6 B. M. Kopriwa, J. Histochem. Cytochem. 11,553 (1963).
[2 ]2] AUTORADIOGRAPHY 281

FIc. 1. A Lab-Tek Cyto-Mailer, which is used as a dipping vessel, is shown on the left.
The position of a slide rack used for drying emulsion-coated slides is shown on the right.

ing, equipment containing vacuum tubes that emit light from the rear,
clocks and watches that have luminous dials, and indicator lights such as
those on waterbaths. These objects should be removed or taped over to
shut out the light. Fluorescent lights continue to glow for an appreciable
time after being turned off and, if possible, should not be used in the
darkroom. The darkroom should have an entrance corridor with double
doors so that one may enter or leave without letting light into the dark-
room.
A safelight may be used during autoradiography according to the
emulsion manufacturer's recommendations. For example, a dark red
Wratten No. 2 filter can be used for Kodak NTB, NTB-2, and NTB-3
emulsions. The power of the light bulb should be no greater than 15 W.
The safelight housing must be light tight so that light escapes only through
the filter. Safelights and filters may be purchased from Kodak. The
safelight should be mounted about 1 m above the working space. Turn off
the safelight when it is not needed, especially when the slides are drying,
because dry emulsion is the most sensitive.

Basic Procedure for Autoradiography of Tissue Culture Cells

1. Cell Culture
Cells are grown on 22 x 22 mm glass coverslips, thickness number 1½,
in 35-mm plastic tissue culture petri dishes or on 22 × 50 mm coverslips in
four-well multiplates (Lux No. 5278, available from Flow Laboratories).
282 SPECIALIZED TECHNIQUES [22]

The coverslips are cleaned before use by: (1) 5 min in 95% ethanol to
remove grease; (2) 5 min in 1 N HCI; (3) 5 min in deionized water, fol-
lowed by four short rinses in deionized water; (4) two rinses in 95%
ethanol; and (5) two rinses in 100% ethanol. They are sterilized in a 250°
oven for 7 hr.
Cells seeded in small petri dishes and in four-well multiplates tend to
settle more heavily in the middle of these vessels. This creates different
growth conditions at the center and periphery. In addition, the cells may
be too crowded in the center for single cell analysis by autoradiography.
We have found that filling the vessels very full when the cells are seeded
reduces this problem. After the cells have attached, the volume of me-
dium may be reduced. We use 5 ml medium to seed cells in 35-mm petri
dishes and 12 ml medium per well in four-well multiplates.
Tissue sections fixed to slides, or cell suspensions smeared on slides,
may be processed for autoradiography in the same way as cells grown on
coverslips. 1The use of a cytocentrifuge to flatten cells in suspension onto
slides is discussed in the section on self-absorption.

2. Fixation
We fix the cells by adding to the growth medium an equal volume of
3 : 1 methanol : acetic acid fixative that has been freshly prepared. After 10
min, the half-strength fixative is removed by aspiration and replaced with
an equal volume of undiluted methanol: acetic acid fixative. After 10 rain,
the coverslips are removed and allowed to air dry. Methanol : acetic acid
fixative can cause some bursting of cell plasma membranes; however, it
flattens the cells well. By adding the fixative to the medium first, we have
eliminated this problem for our experiments with human cells.

3. Removal of Soluble Pools of Radioactively Labeled Precursors

For many experiments using radioactive nucleotides, fixation with 3 : 1
methanol • acetic acid adequately extracts the soluble pools of these pre-
cursors. For other experiments, such as when the cells are relatively
crowded (Fig. 2) or when they are labeled with radioactive amino acid~,
the cells are: (1) extracted with 5% trichloracetic acid (TCA) at 4° for 5
min; (2) washed 3 times with 70% ethanol, for 5 min each; (3) rinsed 2
times with 95% ethanol; (4) rinsed 2 times with 100% ethanol; and (5) air
dried, r TCA extraction is preferable to perchloracetic acid (PCA) extrac-
tion because PCA is difficult to wash out of cells and will inactivate the
emulsion if not completely removed.
r D. M. Prescott, Methods Cell Physiol. 1, 365-370 (1964).
[22] AUTORADIOGRAPHY 283

FIG. 2. T98G human glioblastoma multiforma cells were labeled with 4 /~Ci/ml
[aH]thymidine(specific activity 50 Ci/mM) for 10 min. The cells were fixed in methanol : ace-
tic acid (3 : 1) and extracted with 5% TCA. The slides were processed according to our basic
procedure, using an exposure time of 3 days. The cells were stained with Giemsa stain.
Silver grains are concentrated over the nuclei that synthesized DNA during the labeling
period.

4. Coverslip Mounting
The coverslips are mounted cell side up on clean microscope slides
using a drop or two o f P e r m o u n t (Fisher) or Euparol (Carolina Biological).
Generally we let the mounting medium dry for several hours at 60 ° , or
overnight at r o o m temperature, before the slides are dipped.

5. Preparation of Emulsion
We routinely use K o d a k NTB-2 for analysis of [aH]thymidine incorpo-
ration into tissue culture cells because it has low background, produces
grains large enough to count at x400 magnification, and needs only 3-4
days exposure to develop 25-50 grains/labeled nucleus under our experi-
mental conditions (0.01 /~Ci/ml [aH]thymidine for 24 hr).
The emulsion is a gel at room temperature and is melted in a waterbath
at 39-43 °. Although NTB-2 can be handled under a K o d a k Wratten No. 2
284 SPECIALIZED TECHNIQUES [22]

safelight filter, we suggest keeping the light off whenever possible, e.g.,
when the emulsion is melting. Gently stir the emulsion with a clean glass
rod to check its fluidity after approximately 30 min, being careful not to
create air bubbles.
It is useful to test the background of a new bottle of emulsion by
dipping a clean slide into the bottle, letting the slide dry for 2-3 hr in a
light-tight box, and then developing it. One bottle of emulsion (118 ml) is
enough to coat 1000-3000 slides. 7
Because repeated heating of the emulsion to melt it causes an increase
in the background, we dispense new emulsion into Lab-Tek Cyto-Mailers
and store them at 4°. For subsequent experiments, a single Cyto-Mailer of
emulsion is melted for 15 min and used as a dipping vessel. In general, 10
ml of NTB-2 and 10 ml of distilled water are added to each vessel and
mixed gently. For relatively flat samples, diluted emulsion forms an ade-
quate layer of emulsion. The major advantage of using diluted emulsion is
that subsequent staining is easier. For preparations that have an uneven
surface, undiluted emulsion covers better.

6. Dipping and Drying

A Cyto-Mailer of melted emulsion is held upright on the workbench in
a small beaker of 39-43 ° water or in the waterbath. We dip one slide at a
time, remove it with a slow even motion, and touch the bottom end of the
slide to a paper towel to drain the excess emulsion. The slide is allowed to
dry horizontally with the cell side up by supporting it in a test-tube rack
that has been turned on its side and tilted at a 45 ° angle (Fig. 1). In this
way, the wet slide is supported by its edges. The safelight is turned off
while the slides are drying or the test-tube rack with slides is placed in a
large light-tight box containing Drierite until the slides are dry, i.e., about
1 hr. They are placed in slide boxes containing packets of Drierite and
sealed with black electrical tape to exclude moisture and light. The sealed
slide boxes are placed inside a second light-tight box and exposed at 4°.
There are many variations of this dipping procedure. Two slides can
be placed back to back to be dipped simultaneously and then pulled apart
to dry separately. 7 Slides can be dried vertically in a test-tube rack. How-
ever, vertical drying can cause a small gradient of increasing emulsion
thickness from top to bottom, and it may cause artifacts as discussed
below under "Autoradiographic Background." The emulsion can be
wiped off the back of the slide immediately after dipping rather than after
development. A drop of emulsion can be placed on the cells and spread
over the surface using the side of a 9" Pasteur pipette tip as a roller, s This
8L. E. Allred, personal communication.
[22] AUTORADIOGRAPHY, 285

is an easy way to coat small samples, e.g., coverslip fragments. It can be

used without waiting for the coverslip mounting medium to dry. For large
coverslips, however, it is difficult to spread the emulsion evenly and with-
out causing mechanically induced background.

7. Exposure
We expose autoradiograms at 4° to minimize latent image fading. 5
However, for short-term exposures room temperature is also satisfactory.
The duration of the exposure depends on the experimental procedure
used and the grain dengity desired. The way to determine an exposure
time is to include several test slides in the experiment. These can be
developed at various times and used to estimate the correct exposure time
for the experiment.

8. Development
We use Kodak D19 developer and Kodak Fixer 197-1746, a general-
purpose hardening fixer. These solutions are stored in brown bottles and
are discarded after 2 months. If the D19 turns yellow before then, it is
discarded. To develop slides, unused developer and fixer are poured into
staining dishes and are used at 18-20 ° maximum. The slides are allowed to
warm to room temperature and are placed in staining racks. Slides coated
with either NTB, NTB-2, or NTB-3 are developed for 2 min in D19, rinsed
10-30 sec in 1% acetic acid stop bath, and fixed for 5 min in Fixer. After
fixing, the slides are rinsed gently in running tap water for 20 min, given a
final rinse in distilled water, and air dried. Ideally, the running tap water
should be at the same temperature as the other solutions used to avoid
stressing the gelatin by temperature changes. A thermostatic regulator,
which may be purchased from a photographic supply store, can be used to
maintain the appropriate temperature. However, a regulator is a conveni-
ence rather than a necessity.

9. Staining
We use Giemsa Blood Staining Stock Solution (J. T. Baker Chemical
Co.) diluted 1:25 in 0.01 M sodium phosphate at pH 7.1. Developed
autoradiographs are stained for 50 min and destained for 5 min in the same
buffer. Thereafter, the film of emulsion on the back of the slide is wiped
off with a damp Kimwipe. This procedure gives good definition of the
nucleus and cytoplasm without staining the nucleus so darkly that grains
over it are difficult to count. Much shorter staining and destaining periods
286 SPECIALIZED TECHNIQUES [22]

may also be used successfully. The choice of a staining procedure de-

pends on the nature of the experiment. Other stains are discussed below.

Other Techniques

Prestaining
Prestaining a specimen before applying nuclear emulsion generally
yields a clearer image than poststaining the developed autoradiogram.
This is so because, in prestaining, the gelatin of the emulsion layer is not
stained and the specimen stains more vividly and precisely. However,
only a few stains may be used for prestaining because most stains cause
increased background (positive chemography) even if they are removed
before the emulsion is applied. Prestains are useful for observing mor-
phological details that may be obscured by overlying silver grains after
autoradiography. For example, chromosomes can be more easily iden-
tified before autoradiography. Stains used to detect enzymic reactions are
often used as prestains because the autoradiographic process alters or
destroys the enzymic reaction of interest)
Basic fuchsin, used in the Feulgen procedure, and aceto-orcein are the
most commonly used prestains. Even these acceptable prestains can
cause a high background if they are of low purity or are old. Loss of
radioactive material during staining is also a potential problem in using
prestains. The hydrolysis step of the Feulgen procedure may remove
some radioactivity from the specimen; however, for most qualitative stud-
ies, the loss of radioactivity is insufficient to alter the interpretation of the
results. ~,~0Aceto-orcein, used as a prestain, fades during the photographic
processing. Furthermore, aceto-orcein cannot be used as a poststain be-
cause it removes developed silver grains. The Feulgen procedure also
cannot be used for poststaining because grains are removed during the
hydrolysis step.

Poststaining
An effective poststain must overcome the presence of a layer of emul-
sion over the specimen and the chemical changes brought about by the
photographic processing. If the gelatin takes up too much stain, there will
be little contrast between the specimen and the stained gelatin. If the
gelatin affects the staining reagents, or provides an environment of inap-

R. Baserga and K. Nemeroff, Stain Technol. 37, 21 (1962).

10 W. Lang and W. Maurer, Exp. Cell Res. 39, 1 (1965).
[22] AUTORADIOGRAPHY 287

propriate p H , or if the reactive groups on the specimen are altered, the

stains m a y be rendered totally ineffective.
M a n y o f the methylene blue--eosin type stains (Giemsa, Wright) are
effective poststains. The p r o c e d u r e for G i e m s a staining used in our labo-
ratory is presented a b o v e in " B a s i c P r o c e d u r e . " H e m a t o x y l i n and eosin
m a y be preferable for greater cytological detail although this p r o c e d u r e is
m o r e time consuming. T h e r e are several lists o f stains that h a v e been used
with autoradiography, 11-14 and recipes for t h e m can be found in basic
histology t e x t b o o k s . 15-1r

R e m o v a l o f Emulsion
Occasionally, it m a y be useful to r e m o v e the emulsion f r o m a com-
pleted autoradiograph, e.g., if the b a c k g r o u n d is too high or if visualiza-
tion of the underlying specimen is to be facilitated. Techniques for re-
m o v a l of emulsion h a v e been described by Bianchi et al. 18 and Rogers. 5

Stripping Film
K o d a k AR- I0 stripping film is a p r e p a r e d l a y e r o f gelled emulsion that
is 5 / z m thick, has a p p r o x i m a t e l y the same sensitivity as N T B , and pro-
duces grains suitable for light m i c r o s c o p e autoradiography. Its principal
a d v a n t a g e is that it gives a uniform layer of emulsion. Its disadvantages
are that it is m o r e difficult to apply than liquid emulsion, the stripping
process creates some static discharges that can cause background, and it
is m o r e difficult to stain the specimen through 5 /zm of emulsion. A de-
tailed description of the technique for using AR-10 has been presented. ~

Double Isotope Autoradiography

T w o isotopes that h a v e different energies can be analyzed in a single
specimen. T h e y are distinguished b y the distance that their 0-particles
H c. P. Leblond, B. M. Kopriwa, and B. Messier, in "Histochemistry and Cytochemistry"
(R. Wegmann, ed.). Pergamon, Oxford, 1963.
12j. M. Thurston and D. L. Joftes, Stain Technol. 38, 231 (1963).
13R. Baserga, Methods Cancer Res. 1, 45-116 (1967).
~4L. K. Schneider and S. L. Pierce, Stain TechnoL48, 69 (1973).
15S. N. Thompson, "Selected Histochemical and Histopathological Methods." Thomas,
Springfield, Illinois, 1966.
le A. G. Pearse, "Histochemistry: Theoretical and Applied," 3rd ed. Longmans, Green,
New York, 1969.
~r G. L. Humason, "Animal Tissue Techniques," 3rd ed. Freeman, San Francisco, Califor-
nia, 1972.
~8N. Bianchi, A. Lima-de-Faria, and H. Joworska, Hereditas 51,207 (1964).
288 SPECIALIZED TECHNIQUES [22]

penetrate the emulsion. The most frequently used combination of isotopes

is tritium and carbon-14. The 13particles from tritium range in energy from
zero to 18 keV and those from carbon-14 range from zero to 155 keV.
Consequently, the discrimination between the two isotopes cannot be
complete because approximately 15% of the/3 particles from carbon-14
have the same energy as tritium's 13 particles.
In the double-exposure method, 19 slides are dipped in emulsion, ex-
posed, and developed after a period of time appropriate for the amount of
tritium in the specimen. The ratio of tritium to carbon- 14 should be very
high, so that only a small fraction of the grains produced during this short
exposure are from carbon-14. The slides are then dipped in celloidin em-
bedding solution (no. M-4700, Randolph Products Co., Carlstadt, New
Jersey) that has been diluted 1 : 2 in alcohol-ether. Finally, the slides are
dipped a second time for a period appropriate for the amount of carbon-14
in the specimen. The 13particles from tritium are not sufficiently energetic
to penetrate to the second layer of emulsion. Therefore, grains in the
second layer are produced only by carbon-14. The focal plane of the
grains in the two layers will be different and can be distinguished. Two
emulsions that produce different-size grains may be used for each layer to
allow easier discrimination between them. Alternatively, the silver grains
in one layer can be colored by dye-coupling. 2°
A new procedure uses only a single layer of emulsion, which is ex-
panded in glycerol after the silver grains have been developed. 21 Silver
grains at different levels above the cells can be counted separately as in
the double-exposure method.

High-Speed Scintillation Autoradiography

High-speed scintillation autoradiography is a technique for amplifying
the number of silver grains produced per radioactive disintegration. Sev-
eral procedures have been published for the application of this method to
cells fixed on slides, zz-26 The basic procedure involves immersion of the
emulsion-coated specimen in a solution of scintillator, e.g., PPO (2,5-
diphenyloxazole) plus dimethyl POPOP [14-bis-2-(4-methyl-5-phenyl-

19 R. Baserga, J. Cell Biol. 12, 633 (1962).

20 E. O. Field, K. B. Dawson, and J. E. Gibbs, Stain Technol. 40, 295 (1965).
2~ S. W. Perdue, R. F. Kimball, and A. W. Hsie, Exp. Cell Res. 107, 47 (1977).
22 R. J. Przyblyski, J. Cell Biol. 43, 108a (1969).
23 G. S. Panayi and W. A. Neill, J. Immunol. Methods 2, 115 (1972).
24 B. G. M. Durie and S. E. Salmon, Science 190, 1093 (1975); B. G. M. Durie,ibid. 195, 208
(1977). Includes a correction to preceding paper.
25 A. S. Mukherjee and R. N. Chatterjee, Histochemistry 52, 73 (1977).
26 W. Sawicki, K. Ostrowski, and E. Platkowska, Histochemistry 52, 341 (1977).
[22] AUTORADIOGRAPHY 289

oxazolyl)benzene] dissolved in dioxane. The emulsion becomes impreg-

nated with scintillator molecules. Photons are released as /3 particles
pass through the scintillator. These photons activate more silver crystals
in the emulsion than would have been activated by the/3 particles alone.
Theoretically, the efficiency of the high-speed method of tritium-
labeled molecules could be 30-fold that of conventional autoradio-
graphy. 26 However, different investigators have had variable success with
this technique; in some cases only a small increase in efficiency was
produced and was accompanied by an increase in the background.

Autoradiography of Diffusible Substances

There are several techniques for preparing autoradiographs of cells
containing radioactivity in diffusible substances. Briefly, cells can be
freeze-dried and placed under a film of dried emulsion. 27 Cells can be
frozen and kept at low temperature until completion of the autoradiogram,
thus preventing solute redistribution during the process, z8 Cells can be
applied directly to dry emulsion-coated slides and air-dried although con-
trois to monitor positive or negative chemography are necessary because
the wet specimen can interact with the emulsion, z9

Potential Problems

Self-Absorption
Increasing the thickness of a radioactive specimen beyond a certain
point does not increase the number of/3 particles entering the emulsion.
This phenomenon is due to self-absorption of the/3 particles by the speci-
men. The distance that/3 particles can penetrate depends on their initial
energies and on the density of the specimen. Because most of tritium' s /3
particles penetrate less than 3 /zm through cellular material, 3° self-
absorption is an important factor in autoradiography of tritium-labeled
whole cells. Nevertheless, tritium is useful for autoradiography because it
has a short pathlength and, therefore, can be localized better than more
energetic isotopes.
Tissue culture cells are sufficiently large that they must be in a flat
configuration for tritium autoradiography in order to minimize self-

~70. L. Miller, Jr., G. E. Stone, and D. M. Prescott, Methods Cell Physiol. 1, 371-379
(1964).
zs S. B. Horowitz, Methods Cell Biol. 8, 249-275 (1974).
2a W. E. Stumpf, Methods Cell Biol. 13, 171-193 (1976).
30 W. Maurer and E. Primbsch, Exp. Cell Res. 33, 8 (1964).
290 SPECIALIZED TECHNIQUES [22]

absorption. Many types of cells flatten out on the solid substrate on which
they are grown. Other types of cells are round, e.g., cells that are
grown in suspension. In addition, flat cells become round when they are in
mitosis and when they are removed from the substrate, e.g., with trypsin.
Spreading may take from 2-12 hr, depending on the cell type, the sub-
strate, and the culture conditions. Generally, cells spread more rapidly on
tissue culture plasticware than on glassware. Coating glass with a layer of
evaporated carbon enhances spreading, al When round cells are to be used
for autoradiography, they can be flattened onto microscope slides with a
cytocentrifuge (Shandon Southern Instruments, Sewickley, Pennsyl-
vania). The chambers of a cytocentrifuge are designed so that the cen-
trifugal force drives the cells against a slide at the back of the chamber.
The cells are flattened on the slide and are then fixed as usual.
The choice of fixative affects the degree of flattening of the cells. Even
cells that are well spread in culture are several micrometers thick. For
example, Chinese hamster ovary cells in monolayer culture are about 7
/~m thick and can benefit from additional flattening. We have found that
3 : 1 methanol : acetic acid flattens cells much more effectively than neu-
tral formalin fixative. The ratio of methanol : acetic acid may have to be
modulated to obtain an intact, well-flattened specimen; increasing the
concentration of acetic acid will flatten cells but may disrupt cellular
integrity.
Uniform self-absorption in flattened cells simply reduces the efficiency
at which/3 particles are detected by the emulsion, whereas nonuniform
self-absorption can create artifacts. For example, cells increase in size
during the cell cycle, and large G2 cells may not be flattened to the same
thickness as small G1 cells. Consequently, the efficiency could be less in
the G2 cells. Nonuniform self-absorption is also inherent within individual
cells because the density of various organelles differs: the nucleus is less
dense than the cytoplasm, which is less dense than the nucleolus, z°

Autoradiographic Background
The most common causes of background are listed and discussed
briefly. More detailed discussions are given by Boyd, n2 Baserga, a3 and
Rogers. 5
1. Photographic Processing. Excessive background will occur if the
temperature of the developing solutions is high, or if the duration of
development is long. This problem can usually be recognized because
31G. A. Meek, "Practical Electron Microscopyfor Biologists." Wiley,New York, 1970.
32G. A. Boyd, "Autoradiographyin Biologyand Medicine." Academic Press, New York,
1955.
[22] AUTORADIOGRAPHY 291

background grains will be scattered randomly throughout the emulsion

and will be smaller than the grains that arose from the radioactive material
in the specimen.
2. Chemography. Chemical reactants, particularly reducing agents,
present in the biological specimen or in solutions that come in contact
with the undeveloped autoradiograph, can produce a latent image in the
emulsion. This is termed positive chemography. The opposite, negative
chemography, occurs when chemical groups cause rapid latent image
fading, i.e., loss of the nucleus of metallic silver by recombination of the
silver atoms with bromine atoms to form silver bromide. Chemography is
uncommon in cell culture experiments unless caused by prestaining with
an inappropriate stain.
Chemography due to the specimen itself may be looked for by expos-
ing unlabeled specimens to normal emulsion and to emulsion that has been
fogged by a brief exposure to light. Positive chemography will produce
grain densities higher than background in the normal emulsion. These
grains are often large and irregular and sometimes clumped. Negative
chemography will produce faded areas having grain densities lower than
the uniformly black background in the fogged emulsion.
3. Light Exposure. A high background may occur if the emulsion has
inadvertently been exposed to light, e.g., if the storage facilities for slides
are inadequate or if the darkroom has light leaks. A proper safelight is
especially important because the emulsion will be exposed under it for a
number of minutes.
4. Static Discharges. Electrostatic discharges may be emitted when
tape is stripped from its roll and used to seal a slide box or when it is
stripped from the slide box itself. Similarly, there may be electrostatic
discharges when stripping film is stripped from its support. Sometimes the
discharges may be accompanied by flashes of light. The discharges and
the light can cause background, which often appears as streaks of silver
grains running parallel to one another across the developed emulsion. This
problem can be controlled by cutting strips of tape before the emulsion is
removed from its light-tight bo.x and by untaping slide boxes slowly.
Increasing humidity and decreasing temperature in the darkroom also
help reduce these effects.
5. Pressure Effects. Emulsion is sensitive to pressure, e.g., from
scratches or fingerprints. Contraction of the emulsion as it dries also can
produce a high background if it occurs too quickly or goes too far. When
the emulsion layer is very thin, it may dry too fast. Drying slides coated
with dilute emulsion in the horizontal position rather than in the vertical
position is useful in avoiding the creation of a very thin layer of emulsion
at the top of the slide.
292 SPECIALIZED TECHNIQUES [23]

6. Environmental Radiation. Radiation from the environment, e.g.,

cosmic rays, is usually a relatively minor source of background and is
acceptable for most work. However, if desired, the background may be
reduced by shielding the emulsion and coated slides.
7. Other. Occasionally a latent image speck will form spontaneously.
This will occur more often with the more sensitive emulsions. Hence, the
choice of emulsion should be based on the least sensitive one that is
adequate for the experiment. Although the rate of formation of spontane-
ous background increases with age of the emulsion, we find a reasonable
background for 2-3 months after the expiration date suggested by the
manufacturer. However, one should always dip a test slide in the emul-
sion and check it before use in experiments in which a low background is
important.
Finally, everything from the coverslips used for cell culture to the
darkroom itself should be meticulously clean. Dust, spilled chemicals
including developer and fixer, and materials that come in contact with the
emulsion all may cause background. Glass, plastic, or high-grade
stainless-steel utensils are generally safe to use in contact with the emul-
sion but other metals, particularly copper, are to be avoided.

Acknowledgments
We wish to thank Dr. David M. Prescott, Dr. John Heumann, and Dr. DorothyPumo for
their help in preparing this manuscript. It was writtenwhile we were supported by American
Cancer Societygrants NP-156B (G.H.S.) and PF 1292(R.Y.).

[23] I n d u c t i o n a n d P r o d u c t i o n o f I n t e r f e r o n
By ROBERT M. FRIEDMAN

Many cells in culture can be stimulated to produce at least some

interferon. This is a convenient property because it permits the study of a
useful cell product with a specific biological activity, inhibition of virus
growth. Stimulation of interferon by cells in culture appears to be due to
induction of a protein that is not ordinarily made. Recent studies have
suggested that interferon production is controlled by a repressor ~ and is
determined by specific loci on chromosomes that have in some cases been
identified. 2
I R. L. Cavalieri, E. A. Havell, J. Vil~ek, and S. Pestka, Proc. Natl. Acad. Sci. U.S.A. 74,
4415 (1977).
2 p. p. Creagan, Y. H. Tan, S. Chen, and F. H. Ruddle, Fed. Proc., Fed. Am. Soc. Exp. Biol.
34, 2222 (1975).

Copyright ¢ 1979by Academic Press, Inc.

METHODS IN ENZYMOLOGY, VOL. LVIII All rights of reproduction in any form reserved.
ISBN 0-12-181958-2