Use of Ultrathin Cryo- and Plastic
Sections for Immunocytochemistry
The identification of cell surface and intracellular
molecules for light and electron microscopic observations
is an important technique for studying their location
and function in the cell. A wide range of methods
has therefore been developed to identify and localize
molecules at the subcellular level. The term "immunocytochemistry"
describes a set of methods that use
molecules with specific binding ability to other
molecules. The binding molecules, called "affinity
markers," are diverse, but have in common the ability
to specifically bind, and thus identify, other molecules.
One special class of affinity markers that are widely
used are antibodies. These are produced naturally by
mammals and can be produced to bind to a large range
of molecules, called antigens (e.g., a protein in the cell
membrane) for indirect visualization. The antibodies
are applied to specimens so that they will bind to its
target molecules. For examination in an electron
microscope, the antibody molecules are subsequently
labelled with an electron-opaque marker, usually colloidal
gold. However, for light microscopic examination,
the use of fluorescent markers is recommended.
As a prerequisite, these methods have to ensure the
accessibility of the antigen to the antibody, preferably
without compromising either the structure or the original
localization of the target molecule. For electron
microscopic examination, the specimen has to be thin
(usually ultrathin sections of 50-100nm), stained
(heavy metal salts), dehydrated, and embedded in a
supporting and stabilizing matrix.
Immunocytochemical methods can be divided
into two classes: preembedding and postembedding
methods. Preembedding methods are those in which
the cell, or isolated organelle, is labelled before embedding
and sectioning. While the outside layer of the
cell/organelle may be accessible to antibodies, the
labelling of intracellular structures must be preceded
by a solvent or detergent permeabilization step. It is
also possible to immunolabel purified small particles
(e.g., viruses, coated vesicle) or cell fragments
adsorbed onto the support film of a metal specimen
grid. Although these are very useful methods for
cytoskeletal elements (by definition, detergent/solvent
insoluble), these approachs cannot be recommended
as a general method for all antigens and are not
considered further here (for a review, see Griffiths,
The term "postembedding" refers to techniques in
which the material of interest (may it be tissues, cells,
or purified organelles) is usually either frozen rapidly
or fixed chemically, embedded in either a resin or a
concentrated solution of cryoprotectant, and hardened
by either polymerizing the resin or freezing the
cryoprotected sample in order to be sectioned. Sections
are subsequently labelled using specific antibodies and
a visualization marker. There is now a general consensus
that the labelling of sections is the best general
approach to take for any immunocytochemical study
(for more theoretical background, see Griffiths, 1993).
The two most important advantages of postembedding
over preembedding are (1) when the labelling is
carried out on thin sections, the whole surface of the
section has equal access to the reagents and (2) there
is significantly less need to improve the accesibility of
the reagents to the antigen by using a permeabilization
protocol, which may destroy fine structural details or
even remove or redistribute the antigen under study.
This article discusses the practical aspects involved in
the labelling of thin sections for electron microscopy. Essentially all the reagents mentioned are widely
available from all EM supply companies.
A. Conventional Preparation Procedures
For conventional electron microscopy preparation
procedures, the cells or tissues are fixed chemically
using aldehydes (e.g., glutaraldehyde and/or formaldehyde).
This is usually followed by postfixating
with osmium tetroxide, dehydrating in an organic
solvent (ethanol or acetone), and subsequent embedding
in a hydrophobic, epoxy resin such as Epon or
Spurr. The resin is polymerized at elevated temperatures
(60° to -70°C), and the cured, hardened blocks
are sectioned at room temperature. The ultrathin
sections are placed on metal support grids, stained
using heavy metal salt solutions (uranyl acetate/lead
citrate) and observed in the electron microscope. In
general, because of the harsh treatment applied to
the specimens, this approach is not useful for immunolabelling.
However, there are exceptions to this statement.
The most striking example of this involves
an extensive and elegant series of studies by Ottersen
and colleagues (1992) who performed immunolocalization
experiments of amino acid neurotransmitters
in brain tissue.
During the 1980s a new class of acrylic or methacrylate-
based resins were developed for immunocytochemistry.
The two key advantages of these resins over
epoxy-based resins are that they are more hydrophilic
and, in some cases, can be polymerized at low temperatures.
There are two main "families" of these
resins, namely Lowicryl resins and London resins (LR).
In conjunction with the cryosectioning technique pioneered
by Tokuyasu (1973, 1978), these resins have
become widely and successfully applied for immunolabelling
since the early 1990s. We will now point out
the key practical features involved in using these two
B. Embedding in Acrylic Resins
London resins are perhaps the simplest to use, as in
the case of LR white, the resin can be heat polymerized.
Both LR white and LR gold are now widely used,
and the manufacturers give detailed instructions on
their use. A protocol for LR gold is as follows. In this
example, infiltration and embedding are carried out at
- 0.1M phosphate-buffered saline (PBS) buffer, pH 7.4:
To make 1 liter, dissolve 2.25g of Na2HPO4·2H2O,
0.257g of NaHPO4·H2O, and 8.767g NaCl in
1000 ml distilled water
- 0.5M ammonium chloride: To make 1 liter, dissolve
26.75 g of NH4Cl in 1 liter PBS
- Fix tissue pieces (less than 1 mm3) with the fixative
of your choice, e.g., 0.5% glutaraldehyde in phosphate
buffer for 30min to 2h [note that for all
immunocytochemical techniques the fixation step
is critical [see Griffiths (1993) for discussion)].
- Immerse in 0.5M ammonium chloride in PBS (to
quench free aldehyde groups) for 30min.
- Immerse in PBS for 15-60min.
- Immerse in 50% (vol) methanol at 0°C for 10min.
- Immerse in 80% (vol) methanol at -20°C or at 4°C for 60 min.
- Immerse in 90% (vol) methanol at -20°C or at 4°C for 60 min.
- Immerse in methanol and LR gold (1:1) at -20°C or at 4°C overnight.
- Immerse in methanol and LR gold (1:2) at -20°C or at 4°C for 4h.
- Immerse in pure LR gold resin at -20°C or at 4°C for 2 h.
- Immerse in pure LR gold resin + catalyst at -20°C or at 4°C for 2h.
- Immerse in pure LR gold resin + catalyst at -20°C or at 4°C overnight.
- Immerse in pure LR gold resin + catalyst at -20°C or at 4°C and polymerize for 24h.
LR white is similar but can be polymerized by heat
(50°C), by a chemical accelerator at 4-20°C, or by UV
light (see manufacturer's instructions). Heat polymerization
in a microwave oven, where fully polymerized
blocks are available in less than an hour, is also possible
with LR white (McDonald, 1999). Polymerization
using methods other than UV light enables specimens
to be contrasted with osmium tetroxide. For impressive
examples of combining LR white with the use of
osmium tetroxide, see Tanaki and Yamashina (1994).
C. Progressive Lowering of Temperature
(PLT) in Lowicryl Resins
The specimen is fixed chemically with aldehydes
(postfixation with osmium tetroxide is omitted, as it
can interfere with the polymerization process) and
dehydrated with increasing concentrations of alcohol.
During dehydration the temperature is lowered progressively to finally reach -25 to -35°C. Infiltration of
the specimens with resin and subsequent polymerization
can be performed at low temperatures,
as Lowicryl resins have been designed to have an
extremely low viscosity at low temperatures and can
be polymerized using UV light instead of heat. The
blocks can be sectioned at room temperature using
standard equipment. The PLT method preserves antigenicity
much better than conventional preparation
methods and has been used extensively. A PLT regime
is summarized here.
- Use any of the standard aldehyde fixation
- Immerse in 30% (vol) ethanol at 0°C for 30min.
- Immerse in 50% (vol) ethanol at -20°C for 60min.
- Immerse in 70% (vol) ethanol at -35°C (-50°C) 1 for
- Immerse in 95% (vol) ethanol at -35°C (-50°C to
-70°C) 1 for 60min.
- Immerse in 100% (vol) ethanol at -35°C (-50 to
-70°C) 1 for 60min.
- Immerse in 100% (vol) ethanol at -35°C (-50 to
-70°C) 1 for 60min.
- Immerse in ethanol and resin (1:1) at -35°C (-50
to -70°C) 1 for 60min.
- Immerse in ethanol and resin (1:2) at -35°C (-50
to -70°C) 1 for 60min.
- Immerse in pure resin at -35°C (-50 to -70°C) 1 for
- Immerse in pure resin at -35°C (-50 to -70°C) 1 overnight.
- Polymerize for 2 days at low temperature (-50 and
-70°C, respectively) 1 with UV light.
The whole procedure can be performed with homemade
equipment or, more reproducibly, using commercially
available systems from Balzers or Leica
for HM23 and K11M.
D. Cryopreparation Methods (See Fig 1)
1. Rapid Freezing
|FIGURE 1 Cryoimmuno-EM of BHK-21 cells incubated
equine arteritis virus (EAV), Bucyrus strain. Cells
sectioned, and labelled for the EAV surface
glycoprotein GP5. Bar:
Chemical fixation of biological specimens is invariably
prone to lead to the formation of artifacts. These
artifacts can be avoided if biological structures are
physically fixed by very rapid freezing. Rapid freezing
is a prerequisite for obtaining a frozen specimen that
is not destroyed by ice crystals, which would inevitably
grow at too low cooling rates. Typically, biological
specimens have to be cooled at a rate of 105
K/s in order to obtain vitrification, i.e., a state of ice in the
cell that is amorphous, i.e., lacks ice crystals. Different
freezing techniques have been developed to achieve
this goal and the reader is referred to Robards and
Sleyter (1985) and to Roos and Morgan (1989) for
detailed descriptions of the available methods.
However, to summarize, it is possible to freeze biological
specimens rapidly with most of the available
freezing techniques, provided that they are thin
enough (virus suspensions, etc.). The only freezing
method that allows bulk specimens to be vitrified is
the high-pressure freezing technique (see Studer et al.
1989; Dahl and Staehlin, 1989).
2. Freeze Substitution
Bulk-vitrified specimens can be subjected to a dehydration
regime called freeze substitution. In this procedure,
the vitrified ice is slowly substituted by an
organic solvent, preferably at low temperatures. The
cold organic solvent is then subsequently replaced by
a suitable resin that is kept at temperatures between
-40 and -85°C, followed by polymerization with UV
light at the same temperature. The resulting resin
blocks can be sectioned and the sections labelled at
room temperature. However, high-pressure freezing
devices are not standard equipment for the average
electron microscopy facility and one may have to resort to a method that allows vitrification of the specimen
at lower cooling rates by employing cryoprotectants,
such as sucrose. Cryoprotectants used at
reasonably high concentrations will prevent the formation
of ice crystals in the specimen even at very low
cooling, such as those achieved by immersion in liquid
nitrogen. However, in order to allow the cryoprotectant
to infiltrate the entire specimen, it has to be fixed
chemically prior to infiltration. A freeze-substitution
schedule for Lowicryl HM20 (van Genderen et al.
1991) is as follows.
2.3M sucrose solution in PBS
: To make 500ml, dissolve
393.64 g of sucrose in PBS and adjust volume to
3. The Cryosectioning Technique
- Fix cell pellets and grow cells on filters or tissue
(e.g., with 4% formaldehyde ± 0.1% glutaraldehyde
in buffer) for 1 h. A recommended alternative
is to add the fixative for 5-10min to the culture
medium at the culture temperature before switching
to having the fix in a buffer.
- Infiltrate the specimen with a cryoprotectant
(e.g., 2.3M sucrose) for 30-60 min depending on its
- Cut specimen into small pieces (no more than
- Mount specimen on specimen stubs and freeze in
- Transfer to methanol at -90°C supplemented with
0.5% uranyl acetate for 36 h.
- Raise temperature to -45°C (at about 5°C/h).
- Rinse several times with fresh methanol at -45°C.
- Infiltrate with Lowicryl HM20 in the following
series of Lowicryl/methanol mixtures: 1 : 1 for 2 h,
2:1 for 2h, pure Lowicryl for 2h, and than
- Polymerize for 2 days at -45°C with UV light.
- Section block at room temperature using a glass or
preferably a diamond knife in an ultramicrotome.
- Label with antibodies and gold (see later); all subsequent
steps are done by floating the grids on
drops as small as 5 µl.
- Stain with 4% aqueous uranyl acetate for 10min.
- Rinse with distilled water.
- Stain with lead citrate for 1-5 min.
- Rinse in water, dry, and examine.
The thawed frozen section technique offers a
number of advantages over most other methods for
high-resolution immunolabelling. For more details,
see Griffiths (1993).
- It is potentially the most sensitive technique for
immunolabelling, as initial aldehyde fixation is the
only denaturation step for the antigen (freezing and
thawing of the specimen do not seem to affect the
- Because the sections are not embedded in resin,
they offer the highest access of the antigen to the antibody
relative to other techniques. Consequently, this
approach is the most sensitive one for those antigens
that are retained in the section; this is an important
point, as many small soluble antigens may be very difficult
to maintain in the sections.
- The possibilities for staining/contrasting are
greater than for any other method.
- The entire procedure, including photographic
documentation, can be performed in one working day.
As mentioned earlier, chemical fixation of the specimen
prior to infiltration is a prerequisite. Usually a
solution containing 4-8% paraformaldehyde in phosphate,
HEPES, or PIPES buffer is used (pH between 6
and 8, determined by the buffer and the specimen to
be fixed). The formaldehyde is often supplemented
with 0.1-0.5% glutaraldehyde, which is a stronger
cross-linker. Cryoprotected, vitrified biological specimens
can be sectioned easily at low temperatures,
provided the choice of cryoprotectant does not
compromise the sectioning properties of the block. The
most widely used cryoprotectant is sucrose. Employed
at concentrations between 2.1 and 2.3M
, the blocks are
usually easy to section. High concentrations of sucrose
will give softer tissue blocks and will have to be
sectioned at lower temperatures. For more difficult
specimens (such as plant or insect tissues), an alternative
cryoprotectant mixture developed by Tokuyasu
consisting of 1.8M
sucrose and 20% (w/v)
polyvinylpyrrolidone (PVP, MW 10,000) is recommended.
Detailed descriptions of the cryosection
method are available (Webster, 1999).
Sucrose/PVP infusion mixture
: To make 100ml,
prepare a paste consisting of 20g of PVP, 4ml of 1.1M
in a buffer such as 0.1M
(total volume 20ml), prepare 80ml of 2.3M
the same buffer, mix the paste and the sucrose solution
thoroughly, cover the mixture and leave at room temperature
overnight so that minute air bubbles can
escape, and adjust pH to neutrality using 1M
(use pH indicator paper).
Infusion of small tissue blocks with sucrose
solutions usually takes 15-60min, whereas with sucrose/PVP, at least 2h are required. Overnight
infiltration may offer more uniform infiltration of the
cryoprotectant. Infiltrated specimens are cut to size
and mounted on specimen stubs made of aluminium,
silver, or copper and frozen by simply plunging them
into liquid nitrogen. The stub is inserted into its position
in the specimen arm of the cryomicrotome and the
block is sectioned at temperatures in the range of -60
to -80°C for semithin sections or -80 to -120°C for
Sections are picked up using a wire loop (any
pliable metal with a diameter of 1-2mm) containing a
drop of 2.3M
sucrose in PBS. Ideally, the sucrose is still
fluid at the moment it makes contact with the
section(s). The surface tension of the fluid will help
stretch the compressed and wrinkly section(s). (Note
Stretching will not occur if picking up is done with an
already frozen droplet, even if it is subsequently
warmed up to room temperature.) Sections picked up
in this way have one face exposed to the sucrose and
the other face to the air. The latter will stick avidly to
the surface of a formvar/carbon-coated EM specimen
grid or a glass slide. One significant problem with pure
sucrose pick up is that some parts of cells, notably the
Golgi complex and endosomes, tend to overstretch
during the picking up stage. In order to reduce the
magnitude of this problem, Liu et al.
the technique of picking up sections with a loop containing
a 1:1 mixture of methyl cellulose (exactly the
same solution used for embedding, see later) and
sucrose. This is now the method of choice for
picking up cryosections. One added advantage of
retrieving sections with the sucrose/
mixture is that they can be stored, attached to specimen
grids, and covered with dried sucrose/
cellulose for up to 6 months (Griffith and Postuma,
The sections are usually stained after labelling,
embedded, and dried. Staining and embedding are
achieved by exposing the sections to an inert organic
polymer mixed with the stain, usually uranyl acetate.
The most used polymer for embedding is methyl cellulose
(polyvinyl alcohol and even resins have also
been used for this purpose) containing 0.2-0.3% uranyl
0.2% methyl cellulose solution
: Mix low-viscosity
(25centipoise) methyl cellulose powder with cold
triple-distilled water to make a 2% solution, leave
refrigerated (methyl cellulose is more soluble at low
temperatures) for 2-3 days, centrifuge the mixture at
at 40°C for 1 h, store the solution in the fridge,
where it will be stable for up to 6 weeks, and mix nine
parts of the solution with one part of a 3% solution of
uranyl acetate in water for contrasting/embedding
Use the following a labelling procedure for electron
microscopy, in all cases, float the grids on drops (as
little as 5µl) on a strip of parafilm. The upper (nonsection)
side of the grids must be kept clean and dry,
while the lower surface, with sections attached, should
always be kept hydrated.
- Collect grids by floating them on 1-5% fetal calf
serum(FCS)/PBS on ice; wash in PBS once before
going further. Note that many other reagents can be
used to block nonspecific binding of antibodies such
as 1% fish skin gelatin or 2% gelatin.
- If the cells have been glutaraldehyde fixed, free
aldehyde groups can be quenched in 0.02M glycine in
5-10% FCS/PBS for 10min; rinse twice in PBS for a
total of 5 min.
- Centrifuge antibody solution (lmin at 13,000g),
dilute in 1-5% FCS/PBS, and incubate sections for
15-60min. Use the highest concentration of antibody
that does not give background labelling over structures
that do not contain the antigen.
- Wash six times in PBS for a total of 15 min.
- Incubate grids in protein A-gold for 20-30min.
Dilute protein A-gold in 1-5% FCS/PBS. The concentration
is critical. Too high a concentration gives nonspecific
- Wash six times in PBS for a total of 25 min.
- Wash four times in distilled water for a total of
- Incubate three times with 2% methyl cellulose
solution (25cp) containing 0.1-0.4% uranyl acetate for
10-20min (on ice!).
- Pick the grid up with a 3-mm loop and remove
excess fluid with filter paper.
- Air dry the grid suspended in the loop.
- The thickness of the methyl cellulose film determines
the contrast and the extent of drying artefacts.
- The grids can now be examined.
For double labelling, after step 6, float the grids on 1%
glutaraldehyde in PBS for 5min followed by many
rinses in PBS (Slot et al.
, 1991). Repeat steps 2 to 6 using
a different size of gold, followed by steps 7 to 12.
III. COLLOIDAL GOLD
Colloidal gold coupled to antibodies or other
affinity markers is now the marker of choice for EM
immunolabelling, as it is very electron opaque and easily prepared reproducibly in a range of sizes. The
gold particles can be conjugated to antibodies directly
or to protein A. We prefer the latter, as we find it more
stable and reproducible than IgG gold. However, a disadvantage
of protein A is that it binds strongly only to
certain species of IgG (rabbit, human, pig, and guinea
pig always work; for a more detailed list, see Griffiths,
1993). When using a species of antibody that binds
poorly (such as those from rat and mouse), an intermediate
antibody step, such as a rabbit antimouse antibody,
extends the usefulness of protein A gold. These
reagents are now widely available from many commercial
sources. For more details on the preparation of
colloidal gold markers, see Griffiths (1993).
One common reason why EM methods are not
applied more routinely is the amount of time and effort
required to obtain a result. In recent years there have
been many experiments using microwave ovens to
aid in chemical fixation and embedding protocols.
Although the effect of microwaves on biological
specimens is still not fully understood, it is clear that
routine embedding into epoxy resin or LR white is possible
in less than 4h (Giberson and Demaree, 1999).
V. FINAL COMMENT
- There is no labelling. The first thing to consider
is to try immunolabelling with a light microscopical
approach (e.g., cryostat sections). If thick sections of
lightly fixed specimens do not work (provided the
secondary, visualizing antibodies are good), there is
usually no point in continuing to the EM level. One
exception to this statement is for small antigens
that may be lost during the preparation for light
microscopy. For these it is better to use an embedding
approach, perhaps after freeze substitution. Provided
one has a strong positive, specific signal at the LM
level, a negative result at the EM level can be due
to a number of reasons. In the case of plastic (e.g.,
Lowicryl) sections, a common reason is that the antibody
has no access to the fixed antigen on the surface
of the section. Usually, this is less of a problem with
thawed cryosections. In the latter approach, fixation
conditions can be reduced drastically (e.g., 5 min in 2%
formaldehyde). While this will deleteriously affect the
structure, it will help the investigator to decide if the
problem is due to the fixation preventing access to
the antigen. A second reason for a negative signal at
the EM level, already alluded to, is that the antigen
may have been washed away during the rinsing steps.
This is a problem for small molecules, especially with
the Tokuyasu approach. In this case one should crosslink
the cells/tissues more severely (1-2% glutaraldehyde).
For such antigens the resin approach is
preferred. The third reason for lack of labelling is the
quantitative aspect: the concentration of the antigens
may be too low to detect. Note that the surface of a thin
section provides a very small amount of antigen for the
antibody when compared to, say, a whole cell at the
immunofluorescent level. For more details, see
- There is too much labelling/"nonspecific"
binding. Both the antibody and the gold may not have
been diluted sufficiently. It is best to use a standardized
gold reagent at its optimal concentration with a
characterized primary antibody. If both are unknown,
measure the OD520 of the diluted gold reagent (e.g., a
1 : 100 to 1:500 dilution). The commonly used range of
final concentration gives an adsorbance of 0.1 at a 520-
nm wavelength (generally 1:10 to 1:100 final concentration).
The optimal concentration is the highest that
does not give background labelling in the absence of a
primary antibody. When the gold concentration has
been standardized, combine this with a dilution series
of the antibody and use the highest concentration that
does not give background labelling. The definition of
background is, of course, at the discretion of the investigator
and assumes some knowledge of the antigen:
a membrane protein, for example, would not be
expected in the nucleoplasm. Background labelling
can often be due to impurities in the antiserum. In this
instance, the only solution is to purify the antibody,
either by preparing an IgG fraction (e.g., ammonium
sulfate precipitation) or by affinity purification. It
should be noted that the latter approach often results
in a significant, or even total, loss of the highest titre
antibody molecules (they remain on the column
during the elution procedure).
A positive signal in immuno-EM is only significant
when independent proof is provided that the gold
labelling one sees is really due to the antigen of interest.
Such proof of specificity should be obtained by two
different approaches (for more detail, see Griffiths
- Immunochemical characterization to show
that the antibody recognizes the antigen using an
independent method such as immunoblotting or
- Biological proof of specificity. The best control
here is to be able to correlate the labelling pattern with structures known to contain or not contain the antigen.
Any treatment that blocks or removes the antigen
should eliminate the labelling.
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