Cryostat technique

Cryostat technique
WOLF D. KUHLMANN, M.D.
Division of Radiooncology, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany
Since the early times of enzyme cytochemistry, cryostat sections have been used for all types
of histological staining including histochemical and immunological staining. Indeed, the
cryostat technique is an alternative to paraffin histology when with the latter immuno-staining
or other molecular specific stains are difficult to achieve. All experimental beginning has to
consider the possibly deleterious effects of tissue fixation, dehydration and embedment for the
desired stainings.
With cryostat sections, antigen preservation is usually much better than with paraffin sections,
however, morphological detail is most often inferior. A prerequisite for reliable cryosectioning
is the use of a good microtome in a cryo-chamber cooled by refrigeration, capable
of reaching at least -30°C. An important feature of the cryomicrotome is the anti-roll plate in
order to allow flat sections to be cut. Several cryostat systems can be purchased from the
market. In our hands, the Linde Kryostat (System Dittes-Duspiva; Walter Dittes, Heidelberg)
proves to be excellent, but others may work as well.
There exist almost as many cryostat techniques as laboratories, so the individual laboratory
has to determine what sequential steps (prefixation or not, freeze protection and sectioning
followed by fixation and drying etc.) will give the best results with the molecules to be
detected. Even “minimal” fixation will greatly improve the structural preservation of cells.
Also, many antigens will survive some fixation. Some common experimental steps for the
preparation of cryostat sections are given in Table 1.
Table 1: Preparation of cryostat sections for light and electron microscopy
Procedure Description Remarks
Fixation Buffered formaldehyde (e.g. 1-4%)
prepared from paraformaldehyde
Formaldehyde-glutaraldehyde mixture
Other fixatives
Alternatively: no fixation
Light microscopy: fixation prior to
cryo-sectioning not necessary, only
optional step
Electron microscopy: important step for
tissue stabilization in preembedding
immuno-staining technique and in
cryo-ultramicrotomy
Freeze protection Dimethylsulfoxide (DMSO), f.e. 10%
DMSO in cacodylate buffer
Sucrose, f.e. 30% in cacodylate buffer
Glycerol, f.e. 30% in cycodylate buffer
Other freeze protection
Light microscopy: not necessary, only
optional step
Electron microscopy: important step for
tissue stabilization in preembedding
immuno-staining technique and in
cryo-ultramicrotomy

Snap-freezing
Liquid nitrogen cooled isopentane:
immersion of tissue block mounted on
a tissue holder with a drop of Tissue
Tek® as binding matrix 1)
Direct snap-freezing in liquid nitrogen
Frozen tissue blocks are transferred into
the cryochamber or stored in liquid
nitrogen
Cryo-sectioning
Light microscopy: sections are cut at
4-8 µm thickness
Electron microscopy: sections are cut at
10-40 µm thickness for preembedding
immuno-staining
Electron microscopy: cryo-ultramicrotomy
for obtaining ultrathin frozen
sections
Light microscopy: cryochamber at -30°C
Electron microscopy: cryochamber at
-30°C for preparing thick frozen
sections (preembedding immunostaining)
Electron microscopy: cryochamber at
-90°C to -120°C for ultrathin cryosectioning
Collection of
sections
Light microscopy: frozen sections are
mounted in the cryochamber on clean,
BSA or silane conditioned glass slides
and transferred to room temperature;
slides can be stored for several days
at -20 to -80°C before staining
Electron microscopy: frozen cut sections
are dropped straight as free-floating
sections into vials with 10% DMSO
Electron microscopy: ultrathin frozen
sections are collected dry on nickel
grids or spread on 50% DMSO and
transferred with a wire loop onto a
drop of water at room temperature;
optionally: sections are picked up with
a loop containing a drop of 2.3 mol/L
sucrose or 1% methyl cellulose; the
loop is then touched onto the surface of
a grid to deposit the sections
Light microscopy: cryosections may be
either air-dried (variable times), then
fixed (in acetone or ethanol) at 4°C
or fixed at -20°C for 5, 10 or 15 min
Electron microscopy: sections for
preembedding immuno-staining
are kept free-floating in the vials
during all subsequent steps until final
resin embedment
Electron microscopy: ultrathin sections
are cut at a thickness of about 50-70
nm; sections are picked up and
transferred for direct imaging or
immuno-staining by a variety of
methods
Fixation
Light microscopy: air-dried sections are
fixed in acetone at 4°C or at -20°C for
5 or 10 or 15 min, then allowed to dry
Alternatively: other type of fixation
Light microscopy: different methods
may be tried depending on the
molecules to be studied
Electron microscopy: usually no further
fixation needed; postfixation (OsO 4 )
of immuno-stained thick frozen
sections is useful for further resin
embedment
Immuno-staining
Light microscopy: see Immuno-staining
with paraffin embedded tissue sections
and Immunofluorescence staining of
cryostat sections
Electron microscopy: see Preembedding
immuno-staining for electron
microscopy
Electron microscopy: see Miscellaneous
immuno-staining techniques
Light microscopy: some differences in
immuno-staining of paraffin and
cryostat sections, e.g. antigen retrieval
steps in paraffin preparations, enzyme
inhibition schedules
Electron microscopy: staining of
ultrathin
frozen sections is quite different from
procedures of preembedding immunostaining
of thick frozen sections

1)
Tissu Tek is a formulation of water-soluble compounds (polyvinyl alcohol, polyethelene glycol and other
non-reactive ingredients (Sakura Inc.)
Frozen sections for light microscopy
The concept of first cutting cryo-sections which are then fixed is different from the classical
paraffin embedment procedure (where fixation is first and followed by embedment). This can
involve problems inasmuch as soluble antigens may leak away from the cryo-sections before
sufficient tissue stabilization is achieved.
Usually, snap-frozen tissue blocks are cut on a standard cryostat with a clean blade, and
sections are mounted on acetone cleaned or specially coated glass slides. Coating is done for
better adhesion of sections, f.e. with poly-L-lysine, bovine serum albumin, silane or other
molecules. Unfixed sections may be immediately stored at -80ºC. Frozen sections are thawed
at room temperature and immersed immediately into a fixative. For fixation purposes, widely
employed solutions are (a) cold acetone for 5 min; (b) cold methanol for 5 min; (c) 70%
ethanol for 15-30 sec; (d) 4% formaldehyde freshly prepared from paraformaldehyde for 5
min; or (d) mixtures from ethanol and formaldehyde either followed by acetone or not. One
one has to reconcile that acetone is not a real fixative like aldehydes. Acetone will solve fatty
structures, and associated antigens may solve, too.
After fixation, slides are rinsed briefly in phosphate buffered saline at pH 7.4 and subjected to
immuno-staining. Cryo-sections being fixed with acetone alone are quite vulnerable with
respect to surfactants (Tween etc.) or substances for blocking endogenous enzyme activities
(methanol etc.). Endogenous peroxidase activity can be inhibited (with the exception of
neutrophilic granulocytes) by use of 0.1% sodium azide plus 0.3% hydrogen peroxide in
phosphate buffered saline (PBS). Optionally, inhibition of peroxidase activity can be tried by
a glucose oxidase blocking procedure, i.e. incubation of cryo-sections in PBS containing D-
glucose, the enzyme GOD and sodium azide.
The use of distilled water as final step after all immunohistological procedures including
chromogen development should be avoided because distilled water can generally be harmful
to acetone fixed cryo-sections; use tap water instead.
Cryo-techniques for electron microscopy
Cryo-techniques are a group of related procedures for the stabilisation of biological
specimens for microscopic observations. Tissue samples (lightly fixed or not and
cryoprotected) are frozen to maintain morphological structure and composition as it exists
under physiological conditions. Thus, cryopreserved samples exhibit the best antigenicity,
essential for successful immuno-electron microscopy.
The method of ultrathin frozen sectioning has been primarily proposed as an alternative to
conventional resin embedded tissue sections for ultrastructural studies. The concept is to
avoid morphological changes and denaturation effects of both dehydration and resin
embedment procedures for enzyme cytochemical and immunocytochemical research. The
advantage of cryo-ultramicrotomy for such approaches over resin ultramicrotomy is selfunderstanding.
From the very beginning of cryo-ultramicrotomy (FERNÁNDEZ-MORÁN H,

1952; BERNHARD W and NANCY MT, 1964; DOLLHOPF FL and SITTE H, 1969; HODSON S and
MARSHALL J, 1970; KOLEHMAINEN-SEVÉUS L, 1970; TOKUYASU KT, 1973; TOKUYASU KT
and SINGER SJ, 1976; TOKUYASU KT, 1986) to the point where this technique has become an
appropriate method for ultrastructural studies, it has taken about 20 years of development.
Examples of cryo-techniques for immunocytochemical studies include
• Cryo-ultramicrotomy: ultrathin frozen sections (50-70 nm thick) are cut on an
ultramicrotome in a cryochamber. Sections are thawed on grids for transmission
electron microscopy and subsequently immuno-stained, for example with ferritin or
gold conjugated antibodies.
• Cryo-substitution: this is a cryo-variant of conventional resin embedment. The method
avoids denaturing effects of room temperature dehydration and resin embedment.
Samples are frozen, then dehydration and resin embedment are performed in a cryosubstitution
unit, for example at -90°c in acetone. At this temperature the water ice is
replaced by acetone in a sublimation type process which may minimize adverse effects
on antigenicity. The sample is then infiltrated with low viscosity resins (such as the
Lowicryl series) and cured at low temperatures with UV light. Ultrathin sections are
cut from the embedded tissue blocks with conventional ultramicrotomes for
subsequent
immuno-staining.
Apart from the above cryo-techniques we have to mention cryo-electron microscopy which is
used to observe molecules, viruses and tissues in their native state without fixation or
staining. A typical application is called frozen hydrated transmission electron microscopy. A
thin layer of unfixed aqueous sample is frozen to give a sheet of vitreous ice with the sample
suspended in it. The preparation is observed at low temperature in the electron microscope
equipped with a special cold stage. Samples prepared in this way are very sensitive to the
electron beam. All immuno-stainings may be done prior to freezing. In a similar way, samples
are prepared for cryo-SEM (scanning electron microscopy) where specimens are rapidly
frozen, sputter coated and transferred to the SEM for imaging.
Selected publications for further readings
Fernández-Morán H (1952)
Bernhard W and Nancy MT (1964)
Dollhopf FL and Sitte H (1969)
Hodson S and Marshall J (1970)
Kolehmainen-Sevéus L (1970)
Kuhlmann WD and Viron A (1972)
Tokuyasu KT (1973)
Tokuyasu KT and Singer SJ (1976)
Tokuyasu KT (1986)
Full version of citations in chapter References.
© Prof. Dr. Wolf D. Kuhlmann, Heidelberg 20.09.2008