Download Comparison of cryofixation and aldehyde fixation for plant actin

The Histochemical Journal 32: 457–466, 2000.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
Comparison of cryofixation and aldehyde fixation for
plant actin immunocytochemistry: Aldehydes do not destroy F-actin
Stanislav Vitha1,2,∗ , František Baluška3,5 , Markus Braun3 , Jozef Šamaj4 , Dieter Volkmann3 & Peter W. Barlow6
Institute of Plant Molecular Biology, Academy of Sciences of Czech Republic, České Budějovice, Czech Republic
2
Department of Biochemistry, University of Nevada, Reno, NV 89557, USA
3
Botanisches Institut der Universität Bonn, Bonn, Germany
4
Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Nitra, Slovakia
5
Institute of Botany, Slovak Academy of Sciences, Bratislava, Slovakia
6
IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of Bristol, Long Ashton, Bristol, UK
1
∗
Address for correspondence: Department of Botany and Plant Pathology, 166 Plant Biology Laboratory, Michigan State
University, East Lansing, Michigan 48824, USA
Received 11 November 1999 and in revised form 7 February 2000
Summary
For walled plant cells, the immunolocalization of actin microfilaments, also known as F-actin, has proved to be much trickier
than that of microtubules. These difficulties are commonly attributed to the high sensitivity of F-actin to aldehyde fixatives.
Therefore, most plant studies have been accomplished using fluorescent phallotoxins in fresh tissues. Nevertheless, concerns
regarding the questionable ability of phallotoxins to bind the whole complement of F-actin necessitate further optimization of
actin immunofluorescence methods. We have compared two procedures: (1) formaldehyde fixation and (2) rapid freezing and
freeze substitution (cryofixation), both followed by embedding in low-melting polyester wax. Actin immunofluorescence in
sections of garden cress (Lepidium sativum L.) root gave similar results with both methods. The compatibility of aldehydes
with actin immunodetection was further confirmed by the freeze-shattering technique that does not require embedding after
aldehyde fixation. It appears that rather than aldehyde fixation, some further steps in the procedures used for actin visualization
are critical for preserving F-actin. Wax embedding, combined with formaldehyde fixation, has proved to be also suitable for
the detection of a wide range of other antigens.
Introduction
Immunocytochemistry is an invaluable tool for studying the
in situ localization of many proteins. A plethora of additional
techniques are also being used to detect distributions of relevant molecules. For instance, the natural dynamism of proteins can be visualized in vivo by using fluorescent analogues
introduced via microinjection (Hepler & Hush 1996) and by
fusion with green fluorescent protein (GFP) (e.g., Kost et al.
1998). These newer techniques are an important complement
to previously established methods of immunocytochemistry
or affinity cytochemistry in tissues that have been subjected
to fixation and/or embedding and sectioning.
Organisation of actin microfilaments is most often studied using two methods: (1) fluorescent-labelled phalloidin (rhodamine–phalloidin), and (2) immunofluorescence
with anti-actin antibodies. Each method is prone to artefacts. Therefore, critical evaluation and comparison of the
methods are essential for the correct interpretation of the
results obtained. The rhodamine–phalloidin technique is not
without problems. For example, maize root cells treated
with cytochalasin D show nuclear accumulation of maize
actin-depolymerizing factor (ADF), together with G-actin,
which form short actin–ADF rods. Rhodamine–phalloidin
does not bind to these actin–ADF rods (Jiang et al. 1997).
Moreover, at least in some cell types, phalloidin does not
bind to the whole complement of F-actin due to the masking
of phalloidin binding sites with other actin-binding proteins
(e.g. Nishida et al. 1987, Ao & Lehrer 1995, Jiang et al. 1997).
As noted by Staiger & Schliwa (1987), it is disturbing that the
staining patterns obtained with fluorescent phalloidin are of
rather a diffuse nature. Furthermore, there are often serious
problems with high background fluorescence as well as with
rapid fading of the signal with fluorescently tagged phallotoxins (La Claire 1989, McCurdy & Gunning 1990). The use
of actin antibodies is, therefore, preferable.
Classical immunochemical procedures using fixed and
embedded tissues provide only a static picture of protein
localization. The ultimate aim of chemical fixation is to
immobilize proteins in the same state and location as they
were in vivo. However, the fixation step is considered to be
the most critical and there is a justified concern about the
slowness of chemical fixation which thus leaves time for
abnormal re-arrangements of the cytoplasm and cytoskeleton
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(Mersey & McCully 1978, He & Wetzstein 1995, Doris &
Steer 1996). The slowness of fixation is especially critical
in the case of higher plant cells, which are encased within
robust cellulosic cell walls. Therefore, alternative methods
of plant tissue preparation have been sought, one of them
being rapid freezing followed by freeze-substitution (further
referred to as cryofixation; e.g. Baskin et al. 1995, Roy et al.
1997). However, proteins are not crosslinked by this procedure during the first critical fixation step. As emphasized by
Melan & Sluder (1992), unless the fixative completely immobilizes soluble proteins, further sample preparations and differential extraction could lead to artefactual redistribution of
the antigens. Furthermore, Wasteneys et al. (1996) noted that
one undesirable by-product of cryofixation was a punctatelike appearance of microtubules.
In chemical fixation, there is the serious danger that aldehydes may damage the antigenicity of some epitopes and hence
impair their binding of antibodies. Actin is traditionally considered as one of the most sensitive structures to aldehydes
(Lehrer 1981) and the early difficulties of visualizing F-actin
in plant cells were explained as being due to detrimental
effects of aldehydes on F-actin (e.g. Parthasarathy et al. 1985,
Seagull et al. 1987, Traas et al. 1987, Sonobe & Shibaoka
1989, Doris & Steer 1996). Again, cryofixation, which avoids
the use of chemical fixatives, was reported to preserve tissue
antigenicity better (Baskin et al. 1995). However, there are
often difficulties in visualizing, at the ultrastructural level,
single actin microfilaments using cryofixation (e.g. Roy et al.
1997); and attempts to immunolocalize the full extent of
F-actin in cryofixed plant and fungal samples embedded
in methacrylate have failed so far (Baskin et al. 1995,
Czymmek et al. 1996). With respect to the reliability of cryofixation for the visualization of F-actin, the use of organic
solvents, such as propane and acetone, appears to be critical. These solvents have been reported to render F-actin
unreactive towards rhodamine–phalloidin (Tang et al. 1989,
Raudaskoski et al. 1991).
Using a low-melting-point polyester wax known as
Steedman’s wax (Steedman 1957), a breakthrough was
achieved in the visualization of all classes of F-actin in
aldehyde-fixed maize root cells (Baluška et al. 1997b,
Vitha et al. 1997), using a modification of the microtubule immunostaining procedure of Brown et al. (1989).
Similar success in the immunofluorescence localization of
actin was reported previously in aldehyde-fixed plant cells
not exposed to embedding procedures (e.g. Mole-Bajer &
Bajer 1988, McCurdy et al. 1988, McCurdy & Gunning
1990, Sawitzky et al. 1996, Wasteneys et al. 1996, 1997,
Blancaflor & Hasenstein 1997, Braun & Wasteneys 1998a,b).
In some plant cell types, aldehyde fixation allowed consistent
visualization of F-actin, also using fluorescent phalloidins
(e.g. Schmit & Lambert 1987). All this indicates that the difficulties of F-actin visualization might be related not so much
to fixation as to the further processing of plant tissues. To
clarify this issue, we have compared the immunolocalization
of actin in roots of garden cress (Lepidium sativum L.) after
S. Vitha et al.
using three plant tissue preparation protocols: (1) cryofixation
according to Baskin et al. (1995), but with embedment
in Steedman’s wax; (2) formaldehyde fixation followed by
embedment in Steedman’s wax (Vitha et al. 1997); and
(3) aldehyde fixation combined with the freeze-shattering
technique which avoids embedding and sectioning of plant
tissues (Wasteneys et al. 1997). We demonstrate that both
aldehyde-based fixation and cryofixation give almost identical results in the actin immunofluorescence in all tissues of cress roots. Moreover, we have applied a wide
range of specific antibodies on Steedman’s wax sections of
formaldehyde-fixed maize root apices and show that this
technique allows excellent preservation of antigenicity of all
epitopes tested. These results also served as an internal control for possible localization artefacts and allowed us to assess
whether proteins of various molecular weights were sufficiently immobilized by the formaldehyde fixation used.
Materials and methods
Plant material
Garden cress (Lepidium sativum L.) seeds were germinated
for 24 h in darkness on vertically oriented wet filter paper.
Root tips, 5 mm long, were excised with a razor blade prior
to further processing. Maize (Zea mays L., cv. Alarik) was
germinated in the same manner and root tips were harvested
three days after germination. Arabidopsis thaliana seeds were
germinated on vertically oriented agar medium (1% w/v
agar in water) and whole seedlings were harvested when
the roots were about 5 mm long. Tradescantia virginiana
peels were obtained from the lower epidermis of young
leaves.
Formaldehyde fixation – Steedman’s wax embedding
Root apices were fixed, under vacuum-infiltration, with
1.5% formaldehyde (Sigma, Deisenhofen, Germany; F-1635)
in stabilizing buffer (50 mM PIPES, 5 mM MgSO4 , 5 mM
EGTA, pH adjusted to 6.9 with KOH; all chemicals from
Sigma) for 1 h at room temperature, except for some
maize roots which were fixed for 24 h. Then, they were
briefly washed in stabilizing buffer, dehydrated in an
ethanol series, and embedded in low-melting polyester wax
(Steedman’s wax; Steedman 1957) as described in Vitha et al.
(1997).
Cryofixation – Steedman’s wax embedding
Root apices were cryofixed in the manner described by Baskin
et al. (1995). Apices were placed on a Formvar film stretched
on a loop made of copper wire, plunged into liquid propane
and freeze-substituted in dry acetone for 48 h at −80 ◦ C, then
allowed to warm up to room temperature over a period of 18 h.
Embedding was done as for the formaldehyde-fixed roots,
except that a graded acetone/wax mixture was used instead
of ethanol/wax.
Aldehyde fixatives do not destroy plant F-actin
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Freeze-shattering
The freeze-shattering procedure was modified after Braun &
Wasteneys (1998a). Roots were split into halves prior to fixation, then fixed for 30 min with 1% w/v formaldehyde, 1%
v/v glutaraldehyde (Sigma; Grade I) in stabilizing buffer.
After three rinses in thin buffer, they were washed in a mixture of equal volumes of stabilizing buffer and phosphatebuffered saline (PBS; 140 mM NaCl, 2.7 mM KCl, 6.5 mM
Na2 HPO4 , 1.5 mM KH2 PO4 , pH 7.3) for 5 min and then in
PBS alone for 10 min. Tissues were then incubated three
times with freshly prepared 1 mg/ml NaBH4 , washed again
in PBS, placed in cold (−10 ◦ C) methanol for 5 min, rinsed
with PBS containing 50 mM glycine and transferred to PBS
containing 0.05% pectolyase w/v (ICN Biochemicals, Irvine,
CA; Cat. no. 151804) and 4 mM mannitol for 60 min. After
permeabilization for 1 h with PBS/glycine containing 1%
Triton X-100, the roots were gently squashed between two
polyethyleneimine-coated microscope slides and frozen in
liquid nitrogen for 1 min. The frozen slides were ripped apart,
then put back together again, and pressure was then applied to
fracture the frozen tissue. After thawing, the root fragments
were incubated with the primary antibody.
Sectioning of wax-embedded tissues
Seven micrometre-thick longitudinal wax sections were cut
on a rotary microtome, attached to slides coated with
glycerol–albumen and dewaxed as described in Baluška et al.
(1997b) except that after rehydration sections were left in
stabilizing buffer for 90 min instead of 30 min. This ensured
better preservation of F-actin (unpublished results).
Immunocytochemistry
Sections from wax-embedded tissues were processed for
actin immunocytochemistry as described in Baluška et al.
(1997b). Briefly, sections (7 µm-thick) were incubated for
60 min at room temperature with the primary antibody
(see Table 1), washed 2 × 10 min in stabilizing buffer and
incubated for 60 min at room temperature with secondary
antibody which was either fluorescein isothiocyanate (FITC)
conjugated goat anti-mouse IgG (F-9005; Sigma), anti-rat
IgG (Sigma), or anti-rabbit FITC conjugate (F-6005; Sigma),
depending on the primary antibody source, at 1 : 100 dilution in PBS for anti-mouse and anti-rabbit antibodies and
at 1 : 20 dilution for anti-rat antibodies. Sections were then
washed in PBS for 10 min, stained for 10 min in 0.01%
w/v toluidine blue in PBS, washed in PBS for 10 min and
mounted in an anti-fade mountant containing 0.1% (w/v)
p-phenylenediamine (Johnson & Nogueira Araujo 1981). For
immunofluorescence of endoplasmic microtubules, the cold
methanol step after dewaxing was omitted and a Triton X-100
treatment was added (Baluška et al. 1992). Controls were
treated in the same manner as other sections, except that the
pre-immune rat or rabbit sera or normal mouse IgG were used
instead of the primary polyclonal or monoclonal antibodies,
respectively.
Freeze-shattered specimens were incubated with mouse
anti-tyrosine tubulin (Sigma) diluted 1 : 100 with PBS/glycine
or mouse anti-actin antibody (Table 1) at 1 : 200 dilution
for 2 h at 37 ◦ C, or overnight at room temperature. After
three rinses in the PBS/glycine buffer, cells were incubated
with FITC-conjugated secondary antibody (Sigma, F-9006,
1 : 100) for 2 h at room temperature. Stained cells were rinsed
three times with PBS/glycine and mounted in an anti-fade
mountant.
Fluorescence microscopy
Sections were viewed with a Zeiss Axiovert 405M microscope equipped with epifluorescence and standard FITC-filter
set (BP450-490, LP 520). Photomicrographs were taken on
Kodak T-Max 400 film. Freeze-shattered specimens were
viewed with a Leica confocal microscope TCS4D (Leica,
Heidelberg, Germany) using 488 nm laser for excitation,
dichroic mirror 505 nm and emission filter 515–545 nm. Confocal images are a projection of 10 optical sections taken
at 0.5 µm steps. Individual projections were then cropped
and assembled into Figure 2 and the brightness and contrast
adjusted, using Adobe Photoshop graphics package (Adobe
Systems Inc., Mountain View, CA, USA).
Table 1. Primary antibodies used on formaldehyde-fixed, wax-embedded tissues.
Antigen
Antibody
Dilution
Source
Obtained from
Actin
α-tubulin
PM-ATPasea
Profilin, ZmPRO3
PIP2b
AGPc
AGP
AGP
Calreticulin
HDEL-proteins
C4
N356
1 : 200
1 : 100
1 : 200
1 : 100
1 : 200
1 : 20
1 : 20
1 : 20
1 : 20
1 : 10
Mouse
Mouse
Mouse
Rabbit
Mouse
Rat
Rat
Rat
Rabbit
Mouse
ICN Biochemicals, Meckenheim, Germany
Amersham Life Science, Arlington Heights, IL, USA
W. Michalke
C.J. Staiger
Perseptive Biosystems, Framingham, MA, USA
P. Knox
P. Knox
P. Knox
R. Napier
R. Napier
a
8-1502
LM2
MAC207
JIM8
Plasma-membrane-ATPase; b phosphatidylinositol-4-,5-bisphosphate; c arabinogalactan-protein.
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S. Vitha et al.
Figure 1. Actin immunofluorescence in longitudinal sections from Lepidium roots. Samples were fixed in formaldehyde for 1 h (A–D, G, H) or cryofixed
(E, F, I, J), and embedded in Steedman’s wax. Formaldehyde-fixed samples show identical actin staining patterns to those obtained in cryofixed samples.
Shown are cells of cortex (A–F), epidermis (G, I), and stele (H, J). Photographs were taken using an epifluorescence microscope. Scale bar in J = 10 µm.
(A) Cell in prophase cut in the median displays a perinuclear (asterisk) actin network. (B) Cell in prophase. Actin microfilaments disintegrate in the
middle of the cell (asterisk) and actin accumulates at the end walls, forming characteristic ‘brackets’ (arrowheads). (C) Young phragmoplast (arrow).
At this stage, accumulation of actin along the end walls is still prominent. The cell at a somewhat later stage of division has a phragmoplast (arrowhead)
extended more towards the side walls. (D) Mature phragmoplast stains for actin preferentially at its periphery (arrowheads) while its central part,
where presumably the cell plate has already formed, is actin-depleted. (E) Cell in prophase with a central actin-depleted zone (asterisk) and with
accumulation of F-actin at the end walls (arrows). (F) Young phragmoplast (arrow; similar to in C) and mature phragmoplast actin-stained at the cell
periphery (arrowheads) but not the centre. The two daughter nuclei can be seen as dark areas on the opposite sides of the newly formed cell plate. (G, I)
F-actin in the root epidermis. Nucleus indicated by asterisk. (H, J) In the stele, nuclei (asterisks) are encased within a perinuclear basket of F-actin
(arrowheads). Shown are the cells in the root elongation zone, the marked cells belong to the outermost layer of stele, pericycle.
Results
Aldehyde fixation versus cryofixation using
Steedman’s wax embedding
Both fixation methods, aldehydes and cryofixation, show
essentially identical immunolocalization patterns of F-actin.
Actin microfilaments could be detected in all tissues of the
root tip of Lepidium: in cells of cortex (Figure 1A–F), in epidermis (Figure 1G, I) and in stele (Figure 1H, J), but not
in the quiescent centre or root cap. Both methods showed
identical localization of actin in mitotic cells: the accumulation of actin along the end walls and the appearance of an
actin-free zone in prophase (Figure 1B, E), labelling of young
Aldehyde fixatives do not destroy plant F-actin
phragmoplasts (Figure 1A, C, F), and the lack of fluorescence
signal in the middle of mature phragmoplasts where the cell
plate has already formed (Figure 1D, F). Both aldehyde- and
cryo-fixation enabled visualization of perinuclear F-actin baskets in the pericycle (Figure 1H, J). There was one minor
difference between the result of the two methods: whereas
the chemically fixed tissues showed diffuse fluorescence of
the nuclei, there was no fluorescence in nuclei of cryofixed
cells (Figure 1G, I). Control sections incubated with normal
mouse IgG instead of the anti-actin antibody showed only
negligible, diffuse fluorescence (not shown).
F-actin in aldehyde-fixed, freeze-shattered cells
Tissues prepared by freeze-shattering after aldehyde fixation
allowed visualization of dense F-actin networks in diverse
cell types of the Lepidium root (Figure 2A–C, F). The same
technique was also compatible with the immunolocalization of microtubules (Figure 2D). In addition, F-actin was
detected in root hairs of several species (Figure 2E–G) and in
Tradescantia epidermal peels (Figure 2H). It should be noted
that actin filaments were observed in the very tip of the root
hair (Figure 2E–G). Epidermis and root hairs were chosen
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because they are in immediate contact with fixatives and thus
are expected to be the most affected by the aldehydes.
Localization of various antigens in
formaldehyde-fixed and wax-embedded maize roots
The procedure, optimized originally for F-actin, proved
suitable for other antigens without any further modification, except for endoplasmic microtubules (see Methods).
Immunofluorescence with antibodies directed against different epitopes gave characteristic and mutually different localization patterns. Figure 3 shows that, besides
actin (Figure 3C), it was possible to localize microtubules
(Figure 3A, B), plasma membrane ATPase (Figure 3D, E),
profilins (Figure 3F), PIP2 (Figure 3G) and different
arabinogalactan-protein (AGP) epitopes (Figure 3H–J). Furthermore, antibodies against different AGP epitopes gave
rather different localization patterns (compare Figure 3H–J),
some of them being highly tissue-specific, as shown here
for sieve element specific AGPs recognized with JIM8 antibody (Figure 3J, K). Calreticulin was associated with distinct
punctate domains at cellular peripheries (which correspond
to plasmodesmata as revealed with immunogold electron
Figure 2. Actin and tubulin immunofluorescence in aldehyde-fixed, freeze-shattered tissues, viewed with a confocal laser scanning microscope. Each
image is a projection of 10 optical sections taken at 0.5 µm steps. Scale bars = 10 µm. (A) Optical section through the cortical cytoplasm shows a fine
network of F-actin in the cortex of Lepidium roots. (B) Median optical section through the same cells as shown in A. Nuclei (asterisks) are enmeshed
by F-actin. Also notable in these rapidly elongating cells is the enrichment of the end walls by actin (arrowheads). (C) Lepidium root epidermis with
abundant, mostly longitudinal, bundles of F-actin. (D) The stele of the elongation zone of Lepidium root immunostained for α-tubulin shows labelling
in all cell types. Shown are the wide cells of the young metaxylem (centre) surrounded by the phloem. (E–G) F-actin in root hairs of Arabidopsis (E),
Lepidium (F), and Zea (G). (H) Stomatal cells of Tradescantia. The non-fluorescing structure in the centre are the guard cells. The adjacent three
contact cells show dense network of F-actin. Note the localization of F-actin around the nucleus of the contact cell (arrow). The surrounding pavement
cells of the epidermis showed actin network that was less dense and stained with much lower intensity than the contact cells. The lack of staining in
guard cells is probably due to insufficient penetration of the antibody, while the difference in staining intensity between the contact and pavement cells
presumably reflects differences in actin abundance, even though differential antibody penetration may have contributed as well.
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S. Vitha et al.
Figure 3. Immunolocalization of various antigens in Steedman’s wax-embedded maize root sections viewed with an epifluorescence microscope.
Roots were fixed in formaldehyde for 1 h, except for C where the fixation was 24 h. Stars indicate positions of nuclei. Scale bar = 8µm (A–C,
F–N) and 15µm (D, E). (A) Cortical microtubules (indicated by arrow) in cells of cortex in the root elongation zone. (B) Endoplasmic microtubules in cells of root cortex in the elongation zone. (C) F-actin in stelar cells of the elongation zone, in a root that has been fixed in formaldehyde for 24 h.
Aldehyde fixatives do not destroy plant F-actin
microscopy, data not shown) and to recently formed cell walls
(Figure 3L). Proteins with the HDEL-retention sequence were
localized to nuclear envelopes, perinuclear endoplasmic reticulum networks, and assembling cell plates during cytokinesis (Figure 3M, N). In all cases, control sections showed
only very faint, diffuse fluorescence (not shown). Interestingly, F-actin survived even 24 h formaldehyde fixation
(Figure 3C).
Discussion
There are several strategies for the preparation of multicellular plant material for immunofluorescence. These include
whole mounts (Ericson & Carter 1996, Harper et al. 1996),
sectioning without embedment (Blancaflor & Hasenstein
1993, 1997), and embedding and microtome sectioning
(Baskin et al. 1992, 1995, Baluška et al. 1992, 1996,
1997a,b). In each of these strategies, there is usually a tradeoff between preservation of antigenicity, the degree of protein
immobilization and spatial resolution, and structural preservation of the tissue.
Contrary to many previous reports and also to common
general opinion, we were previously able to immunolocalize the whole complement of F-actin networks in
all the diverse cells and tissues of root apices after
formaldehyde fixation (Baluška et al. 1997a,b, Vitha et
al. 1997). Moreover, pre-treatment with the protein crosslinking agent m-maleimidobenzoyl-hydroxylsuccinimide
ester (MBS) (Sonobe & Shibaoka 1989) proved to be unnecessary, as the same immunostaining patterns were seen with
or without MBS. In cytokinetic cells, prolonged MBS treatment actually prevented visualization of the phragmoplast
actin (Vitha et al. 1997). Moreover, our current results show
that F-actin can be localized even after long (24 h) formaldehyde fixation (Figure 3C) in maize roots. However, we did
not test prolonged fixation on other tissues and species.
We report here that both cryofixation and aldehyde fixation, combined with the Steedman’s wax embedment reveal
the same actin microfilament networks. This achievement
stands in contrast to cryofixation combined with methacrylate
embedment (Baskin et al. 1995). We were able to visualize the
full range of F-actin arrays of dividing plant cells. The validity
of presented F-actin localization is supported by the fact that
the individual F-actin arrays observed in our specimens were
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also reported by other authors who used different methods:
e.g. perinuclear basket of interphase cells (e.g. Seagull et al.
1987, Traas et al. 1987), cortical transverse microfilaments
(McCurdy et al. 1988, Baluška et al. 1997b), the dismantling of F-actin in pre-mitotic cells (e.g. McCurdy & Gunning
1990), actin-depleted zones of mitotic cells (Mineyuki &
Palevitz 1990, Cleary et al. 1992, Liu & Palevitz 1992,
Braun & Wasteneys 1998b), accumulation of F-actin near
cell walls facing the spindle poles during mitosis and cytokinesis (e.g. Cho & Wick 1990, 1991, Kennard & Cleary 1997),
accumulation of F-actin within phragmoplasts (Palevitz 1987,
Braun & Wasteneys 1998b), breakdown of F-actin in the
central part of more advanced phragmoplasts (Valster &
Hepler 1997). However, we could not detect microfilament
arrays linking expanding edges of phragmoplasts with actin
patches at division sites defined by pre-prophase bands. Since
these authors (Valster & Hepler 1997) used unusually high
phalloidin concentrations, some of the fluorescence can be
probably attributed to phalloidin-induced polymerization of
G-actin. Alternatively, this so-called ‘snap-shot’ technique
may simply ‘freeze and amplify’ the overall pattern of F-actin
distribution revealing some aspect which would otherwise be
difficult to visualize. In support of this, all F-actin distribution
patterns were obvious immediately after the microinjection
of high levels of fluorescein-conjugated phalloidin (Valster &
Hepler 1997).
The actin-distribution patterns provided by the freezeshattering procedure further confirm that the use of aldehyde
fixatives per se does not destroy F-actin. The cells observed
were root hairs and epidermis cells, which were in immediate contact with the fixative. If aldehydes were destroying F-actin or its immunoreactivity, these cells would have
been affected most severely. However, fine networks of actin
microfilaments were immunolocalized in tissues subjected
to 30-min fixation in a mixture of formaldehyde and glutaraldehyde, without any pretreatment with MBS (Figure 2).
It is remarkable that actin filaments were seen not only in
the basal part of the root hair, but some of them reached to
the very tip (Figure 2E–G). This actin staining pattern differs from the findings of Miller et al. (1999), who showed an
actin-free zone in the tip of elongating root hairs of Vicia and
concluded that bundles of actin filaments would inhibit exocytosis in the root hair tip. However, Small et al. (1999) noted
that during fixation and namely permeabilization, the fine
meshwork of F-actin disappears first, while the actin bundles
(D) Plasma membrane H+ -ATPase at the root tip (asterisk indicates the root cap junction, arrows deliminate the quiescent centre). (E) Plasma membrane
H+ -ATPase within the transition zone at the cortex/stele interface (small asterisk indicates position of the endodermis, large asterisk indicates the position
of narrow metaxylem). (F) Profilins identified with maize specific anti-profilin ZmPRO3 antibody in epidermal cells. (G) Phosphatidylinositol-4-,5bisphosphate in the pericycle cell (arrow highlights the plasma membrane-associated labelling). (H) Arabinogalactan-protein epitope recognized by
LM2 antibody. (I) Arabinoglactan-protein epitope recogized by MAC207 antibody. (J) Arabinogalactan-protein epitope recognized by JIM8 antibody.
Only the sieve elements are labelled (asterisk). (K) Differential interference contrast image of J. (L) Calreticulin labelled with maize-specific anticalreticulin antibody (arrow points to the cytokinetic cell plate that is enriched with calreticulin). The distinct punctate domains at cellular peripheries
correspond to plasmodesmata, as revealed with immunogold electron microscopy (data not shown). (M) HDEL proteins labelled in dividing cells of
the cortex. The larger black asterisk denotes the position of a cell undergoing mitosis. The smaller asterisk indicates young, HDEL-positive cytokinetic
plate. Note the increased HDEL fluorescence at cell periphery domains facing spindle poles during mitosis. This feature persists also during cytokinesis.
(N) HDEL proteins in a cell of the pericycle in the post-mitotic transition zone, showing the perinuclear ER networks.
464
are relatively resistant to those treatments. Since Miller et al.
(1999) applied MBS to living, unfixed cells and used rather
strong permeabilizing agents, it is possible that the fine actin
network of the root hair tip was not preserved with their
procedure.
Based on our results where we obtained good actin
preservation with either cryofixation or chemical fixation and Steedman’s wax embedding, we suggest that the
poor preservation of the actin cytoskeleton after cryo- or
aldehyde fixation combined with methacrylate embedment
(Baskin et al. 1992, 1995) is caused either by the methacrylate
embedding medium or by some other step in this procedure,
but not by the use of aldehyde fixative as such. However,
Chaffey et al. (1997a,b) obtained good actin preservation with
aldehyde fixation and methacrylate embedding of secondary
tissues in hardwood tree species. Although we cannot explain
this contradiction, it is possible that primary tissue (used here
and by Baskin) somehow differs from the secondary tissue in
its reaction. This may be because the secondary tissue is more
robust and withstands methacrylate de-embedmnent and/or
acetone–resin removal better.
The use of a range of different antibodies on sections from
formaldehyde-fixed and Steedman’s wax-embedded roots
provided us with an internal control for the immunofluorescence procedure, as antibodies to different antigens gave
different localization patterns (see Figure 3). For instance,
accumulation of small soluble proteins in phragmoplasts
was interpreted as a typical sign of a localization artefact
(Vos & Hepler 1998). Since some of the cytoplasmic proteins that we detected are rather small (e.g. profilins are
about 15 kDa in size), their rapid redistribution to other subcellular compartments is likely if the antigen immobilization is insufficient. Surprisingly, antibodies raised against
different isoforms of maize profilins produce clearly different labelling patterns with the Steedman’s wax technique
(von Witsch et al. 1998). The same was shown for the
whole complement of maize cyclins (Mews et al. 1997). This
suggests that even small and soluble cytoplasmic proteins
are sufficiently immobilized by the formaldehyde fixation.
The membrane-bound PIP2, which is only about 1 kDa in
size, was localized preferentially at the plasma membrane
(Figure 3G) and did not accumulate in the phragmoplast, as
might have been expected if PIP2, or the protein to which PIP2
binds, was also redistributed by the procedure. Localization
of calreticulin in a punctate pattern close to cell walls, and
along freshly formed cell wall, and lack thereof in an endoplasmic reticulum throughout the cytoplasm (Figure 3L),
is consistent with the finding that calreticulin, at least in
maize roots, is preferentially located in cortical endoplasmic reticulum associated with plasmodesmata (Baluška et al.
1999). Although calreticulin has an HDEL motif, it constitutes only a fraction of the total HDEL proteins in the cell
and that is probably why the anti-HDEL antibodies do not
reveal calreticulin staining pattern (Figure 3M, N). The results
with various antibodies presented in Figure 3 also show that
the antigens of interest are not destroyed by formaldehyde
fixation.
S. Vitha et al.
While the cryofixation procedure has the advantage of
being fast acting, avoiding thus the danger of cytoplasmic rearrangements during fixation, it may not be suited
for the localization of small, soluble proteins. Since proteins are not chemically crosslinked, they can redistribute
or be extracted during further sample handling, thus giving an artefactual image of the protein’s localization and/or
abundance. However, we did not evaluate antigen redistribution in the cryofixed samples as we did in the aldehyde
fixed maize roots. Lastly, cryofixation of large objects can be
problematic because of insufficient freezing rates and, in turn,
damage of tissues by ice crystals. In fact, we noticed some
structural damage to cells and tissue disintegration in several
cress roots that were cryofixed. Formaldehyde fixation followed by Steedman’s wax embedding seems to be well suited
for actin immunolocalization in whole, robust organs which
cannot be conveniently cryofixed, e.g. maize root apices.
Whole-mount methods (Ericson & Carter 1996, Harper et al.
1996) often require very long incubation times and use of
permeabilization and extracting agents. The suitability of our
technique also extends to the immunocytochemistry of plant
secondary tissues (Chaffey et al. 1996), though some of the
woody cell types create difficulties for the cutting of good
sections.
It should be noted that Steedman’s wax method permits application of many histochemical staining techniques
besides immunocytochemistry, and is comparable to the
paraffin embedding method in this regard. For example, sections taken from formaldehyde-fixed and Steedman’s waxembedded plant tissues have been successfully stained for
AGPs with β-glucosyl Yariv phenylglycoside (Šamaj et al.
1999), and for callose using decolourized aniline-blue
(unpublished data) without any significant background staining. These results are in general agreement with those
reported by Baird (1967) for other standard histochemical
staining techniques.
Last but not least, because Steedman’s wax is soluble in
ethanol, the use of hazardous organic solvents, often used
with other embedding media, can be completely avoided, thus
minimizing health risks and waste disposal costs.
Acknowledgements
We thank Chris J. Staiger, Richard Napier, and Paul Knox for
providing us with antibodies raised against profilins, ER proteins, and arabinogalactan-proteins. The research was supported by a fellowship from the Alexander von Humboldt
Foundation (Bonn, Germany) to J.S. and, partially, to F.B.
who also receives support from the Slovak Academy of
Sciences, Grant Agency Vega, Bratislava. Financial support
to AGRAVIS (Bonn, Germany) by the Deutsche Agentur
für Raumfahrtangelegenheiten (DARA, Bonn, Germany) and
the Ministerium für Wissenschaft und Forschung (MWF,
Düsseldorf, Germany) is gratefully acknowledged. IACR
receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.
Aldehyde fixatives do not destroy plant F-actin
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