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1985 33: 345J Histochem CytochemD H Simmonds, R W Seagull and G Setterfield

Evaluation of techniques for immunofluorescent staining of microtubules in cultured plant cells.  

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‘Supported by grants from the Natural Sciences and Engineering

Research Council of Canada.2Present address: Chemistry and Biology Research Institute, Ag-

riculture Canada, Ottawa, Ontario, Canada K 1 A 0C6.

345

0022-1554/85/83.30The Journal of Histochemistry and CytochemistryCopyright © 1985 by The Histochemical Society, Inc.

Vol. 33, No. 4, pp. 345-352, 1985Printed in U.S.A.

Original Articles

Evaluation of Techniques for Immunofluorescent

Staining of Microtubules in Cultured Plant Cells’

DAINA H. SIMMONDS,2 ROBERT W. SEAGULL, and GEORGE SETFERFIELD

Department ofBiology, Carleton University, Ottawa, Ontario, Canada KIS 5B6

Received for publication July 19, 1984 and in revised form November 8, 1984; accepted November 23, 1984 (4A01 71)

Various modifications to the immunofluorescent labelingprocedures for microtubules in plant cells have been corn-

pared using cell cultures of Vicia bajastana Grossh. Usingserial section electron microscopic reconstructions as a ref-erence, we have chosen as our standard procedure a methodthat maximizes both the preservation of the cytoskeletonand the proportion of cells staining, while minimizing thedegree of nonspecific staining. The critical steps of theprocedure include stabilization of the cytoskeleton, cell

wall permeabilization, and cell extraction. To maintainstructural integrity during the procedure, it is necessary

Introduction

To date the majority of information on organization of the

plant cytoskeleton has been obtained using transmission dcc-

tron microscopy (EM), with and without serial section recon-

struction (Hardham and Gunning, 1978; Seagull and Heath,

1980; Seagull, 1983). While this method yields details on the

precise organization of the cytoskeletal elements, it allows

examination of relatively few cells and usually provides infor-

mation on only a portion of an entire cell.

The production of antibodies specific to cytoskeletal struc-

tures, particularly microtubules (MT), coupled with immu-

nofluorescence microscopy has made it possible to examine

cytoskeletal organization in entire animal cells, both in culture

and organized tissues (Osborn and Weber, 1982). The appli-

cation of these techniques to plant systems has, however, been

limited, primarily because the cell wall forms a barrier to an-

tibody penetration (Knox et al., 1980) and vacuolated plant

protoplasm is difficult to preserve during staining. Recently,

modifications of animal cell procedures have been used to

produce immunofluorescent images of plant MT (Lloyd et al.,

1979; Van der Valk et al., 1980; Wick et al., 1981; Simmonds

et al., 1983; Fowke et al., 1984). While all these reports show

to stabilize the cytoskeleton with paraformaldehyde. Tofacilitate antibody penetration into the cell, it was nec-essary that the walls be made permeable via partial en-zymatic digestion. Detergent extraction of cells increased

the proportion of cells staining and decreased the level ofnonspecific binding of the antibodies. The procedures de-

tailed in this article provide a good starting point for theapplication of immunofluorescent labeling techniques to

other plant systems.KEY WORDS: Microtubules; Immunofluorescence; Electron mi-

croscopy; Plant tissue culture.

MT patterns, there is considerable variation in the detailed

MT organization described; cf. Figure id ofLloyd et al. (1979),

Figure 3 of Wick et al. (1981), and Figures 5 and 8 of Sim-

monds et al. (1983). Do such differences in detailed organi-

zation of MT reflect natural variations in different cells or

species, or do they result from differences in immunocyto-

chemical techniques?

We have examined the effects of a number of different

immunofluorescence procedures on organization ofMT in cul-

tured vetch cells. The method chosen as optimal, termed our

“standard” procedure, revealed MT in the majority of cells

with a minimum of nonspecific staining and showed MT pat-

terns comparable to those observed using reconstruction of

serial EM sections. We also show by immunofluorescence and

EM that variations in staining procedures can cause significant

disturbance of MT distributions.

Materials and Methods

Plant material. The main studies were carried out on liquid-suspension cultures ofvetch cells (Vicia hajastana Grossh.) maintained

as previously described (Simmonds et al., 1983). For most experi-

ments, cells were transferred from stock medium containing 0.5 mg/liter

2,4 dichlorophenoxyacetic acid (2,4-D) and grown in 0.025 mg/liter

gibberellic acid (GA) for 1 to 2 days. This treatment causes cessation

of mitosis with continued cell elongation and leads to consistent pat-

terns of MT oriented transversely to the axis ofelongation (Simmonds

et al., 1983). Some studies were also performed on cycling cells cul-

tured with 2,4-D, where MT arrangements are more variable.

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346 SIMMONDS, SEAGULL, SETTERFIELD

In addition to the experiments with vetch cells, the immunoflu-

orescence techniques were also applied to cell suspensions of Dat is ra

innoxia (Furner et al., 1978), tomato, tobacco, Nicotiana rustica (Douglas

et al., 1981), and eggplant (Gleddie et al., 1983) cultured with 2,4-

D, and root tips of onion, radish, and clover germinated from seed

on wet filter paper.

Standard immunofluorescence procedure. The main proce-

dures used were adapted from Bershadsky et al. ( 1978), Osborn et

al. (1978), and Rogers et al. (1981). The final result of numerous

modifications designed to optimize the proportion ofcells stained and

preservation of MT is termed the “standard” procedure and is detailed

below:

1. Wash cultured cells (centrifuge at 50 Xg for 3 mm) one time

with microtubule stabilizing buffer MSB- 1 (0. 1 M (piperazine-

N,N’-bis(2-ethanesulfonic acid)) (Pipes) buffer containing 3

mM ethyleneglycoltetraacetic acid (EGTA), pH 6.9).2. Sediment cells and resuspend for 10-15 mm in wall-digesting

enzymes [0.5% (wlv) Onozuka R-l0 celulase (Yakult Honsha

Co. Ltd., Nishinomiya, Japan), 1.0% (wlv) Rhozyme HP-iSO(Corning Biosystems, Corning, NY), 0.25% (w/v) pectinase

(ICN Nutritional Biochemicals, Cleveland, OH), 0.025% (wlv)

gelatin (Matheson, Coleman and Bell Co., Cincinnati, OH) in

MSB-1, pH 6.1].3. Wash cells two times in MSB-1 to remove enzymes.4. Resuspend cells in a minimal volume of MSB- I and layer onto

coverslips precoated with poly-L-lysine (mol wt 300 kD) (Sigma

Chemical Co., St. Louis, MO) for 5-8 mm. All subsequent

steps involve gently transferring cover glasses with adhering

cells.

5. Prefix with fresh 3% paraformaldehyde (Polysciences, Inc.

Warrington, PA) in MSB-l for 60 mm.

6. Wash in MSB-1 three times, 1 mm each.

7. Extract with 1.0% (wlv) Triton X-iOO in MSB-2 (50 mM

imidazole, 50 mM KCI, 0.5 mM MgCl2, 1 mM EGTA, 0.1

mM ethylenediaminetetraacetic acid (EDTA), 1 mM 2, mer-captoethanol, 4 M glycerol, pH 6.7) for 60 mm.

8. Wash in MSB-2 three times, I mm each.

9. Fix in 1.0% glutaraldehyde in MSB-2, 10 mm.

10. Reduce free aldehyde groups with 0. 1% NaBH4 in phosphate

buffered saline (PBS) ( 1 5 mM phosphate buffer, 0.85% NaC1,

pH 7.0), two times S mm each.1 1. Wash in PBS three times, 1 mm each.

12. Layer coverslips with 75 jal of monoclonal rat anti-yeast tub-

ulin (MAS 078, clone YOL 1/34, Cedarlane Laboratories Ltd.,

55 16-8th Line, R.R. #2, Hornby, Ont., Canada) diluted I :300

with PBS, and incubate for 45 mm in high humidity.

13. Wash in PBS three times, 4 mm each to remove unbound

antibody.

14. Layer coverslips with 75 jal of fluorescein-conjugated goat

anti-rat immunoglobulin a (IgG) (Miles Labs, Ltd., Rexdale,

Ont., Canada) diluted 1:60 with PBS and incubate as in stepI2.

15. Wash as in step 13.

16. Mount coverslips in 50% glycerol in PBS containing 0.1%

diphenylamine, pH 9.0.

Root tips (5 mm) were passed intact through the standard pro-

cedure to step I I . Following washing in PBS they were squashed on

poly-L-lysine-coated coverslips. Large debris was removed with forceps

and the coverslips with adhering cells were passed through the re-

maiming steps of the procedure.

Modifications to the standard procedure. A number of modi-fications to the standard procedure were attempted. These are listed

below, numbered according to the step in the standard procedure that

was varied:

2. Standard enzymes were replaced with 0. 1% Worthington

ultrapure cellulase (Cooper Biochemicals Ltd. , Mississauga,

Ont., Canada) in MSB-l, pH 6.1, for 5 mm.

Sa. Prefixation in paraformaldehyde was omitted.

Sb. Instead of paraformaldehyde, cells were prefixed in 1.0%

glutaraldehyde in MSB-1 for 20 mm. In this case step 9 was

omitted.

Sc. Cells were prefixed in 3% paraformaldehyde after step 1,

before treating with enzymes. Paraformaldehyde was re-

moved by washing cells five times with MSB-l and steps S

and 6 were omitted.

7a. Steps 7 and 8 were omitted, cells were fixed in 1.0% glu-

taraldehyde in MSB-1, 10 mm.

7b. In place ofTriton, cells were extracted in cold absolute meth-

anol for 6-8 mm; glutaraldehyde postfixation was omitted,

similar to the method of Wick et al. (1981).

7c. Paraformaldehyde prefixation, step 5, was omitted. Cells were

extracted in Pipes-EGTA buffer and 1.0% Triton X-100,pH 6.9, according to Lloyd et al. (1979). After extraction,

cells were fixed with 1.0% glutaraldehyde in 0. 1 M Pipes

buffer, pH 6.9.

12. Alternative antibody preparations were used:

12a. Polyclonal antibody to chick brain tubulin raised in rabbit

(Miles Laboratories, Rexdale, Ont., Canada).

i2b. Polyclonal antibody to bovine brain tubulin raised in rabbit

by K. Rogers and V. Kalnins, University of Toronto.1 2c. Monoclonal antibody to Polytomella flagellar rootlets raised

in mouse by W. Achison and DL. Brown, University of

Ottawa.

Electron microscopy. Cells in growth medium were centrifuged

(SO Xg for 3 mm), resuspended in 1.0% glutaraldehyde in 0.1 M

phosphate buffer, pH 6.8, at room temperature for 1 hr. and then

washed three times in phosphate buffer, 20 mm each. Cells were

postfixed in phosphate-buffered 1.0% osmium tetroxide (JBEM 5cr-

vices, Dorval, Que.) for 1 hr and washed as previously. Cells were

dehydrated in 2,2-dimethoxypropane (Thorpe and Harvey, 1979) and

flat embedded in Spurr’s medium between silicone-treated microscope

slides. Well-oriented cells were selected under a light microscope and

serial sections parallel to the long cell axis were cut using a diamond

knife on an LKB ultramicrotome. Sections were stained with uranyl

acetate and lead citrate and observed using a Siemens 101 electron

microscope. Three-dimensional reconstructions were prepared as pre-

viously (Seagull and Heath, 1980).

Cells processed by the standard immunofluorescence procedurewere also examined by EM. These cells were not attached to coverslips;all changes of solution were achieved by centrifugation. Cells were

transferred to the electron microscopy procedure described above

after step 9 of the standard immunofluorescence procedure. Fixation

in 1% glutaraldehyde in MSB-2 (step 9) was extended to 1 hr.

Results and Discussion

Cultured vetch cells expanding in GA are highly vacuolated.

By electron microscopy, such cells, fixed directly in glutaral-

dehyde-osmium, showed numerous cortical MT immediately

beneath the plasmalemma in the thin cytoplasmic layer (Figure

1 ). These MT were generally oriented transverse to the lon-

gitudinal axis of the cell. In serial section reconstructions (Fig-

ure 2), MT were distributed more or less evenly throughout

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IMMUNOFLUORESCENT STAINING OF PLANT MICROTUBULES 347

the length of the cells and occurred singly, in pairs, or in small

clusters (Figures 1,2). Individual MT often veered in direction

and ran between clusters. In general, the MT arrangement

revealed by electron microscopy in the elongating vetch cells

was similar to that seen in elongating cells oforganized tissues

(Hardham and Gunning, 1978; Seagull, 1983).

Interphase cells in 2,4-D cultures, viewed in the EM, showed

greater variation in MT arrangements than the cells in GA.

While some cells had transversely ordered MT similar to those

in Figure 2, others had highly disordered MT, as shown in the

serial section reconstruction of Figure 3. Cortical MT have

been reported relatively infrequently in EM sections of inter-

phases of cycling cells in culture and no obvious order was

apparent (Roberts and Northcote, 1970; Fowke et al., 1974).

Such disordered MT were not found in EM sections of GA

cells. Disordered cortical MT have also been seen in root tip

cells by both EM (Seagull, 1983) and immunofluorescence,

and in 2,4-D-cultured vetch cells by icnmunofluorescence

(Simmonds et al., 1983).

Results from the standard immunofluorescence staining

procedure and a number of variations of this procedure are

summarized semiquantitatively in Table 1. Four main attri-

butes of the preparations were assessed: proportion of cells

with MT staining; degree of aggregation of MT into thick

fluorescent strands; similarity of MT pattern to that in EM

Figure 1. Electron micrograph of a grazing longitudinal section through

the cortical cytoplasm of a vetch cell grown in GA and prepared using

routine glutaraldehyde-osmium fixation. Cortical microtubules are

organized singly (-*), in pairs (�), or in small cluster (‘._.‘). Longitudinal

axis of cell (*-*). Bar = 1 jam.

serial reconstructions; degree of nonspecific staining, both dif-

fuse and of recognizable cellular organdIes.

The standard procedure was judged to give the best results

overall with vetch cells (Treatment a, Table 1 ). The majority

of cells showed strong staining of MT networks, the MT were

generally in fine strands, indicative of little aggregation, and

the overall MT distribution was consistant with that seen by

EM. With the rat monoclonal anti-tubulin, little or no staining

of non-MT elements occurred. Typical views of cells stained

by this procedure from GA and 2,4-D cultures are presented

in Figures 4 and 5, respectively. Note the presence of cells

with both ordered and disordered MT in the cycling 2,4-D

culture. This is in agreement with previous immunofluores-

cence results (Simmonds et al., 1983) and the EM reconstruc-

tion in Figure 3.

Permeabilization of the cell wall is a major step in immu-

nofluorescence staining of plant cells. Most cells will not stain

without application of some type of wall-modifying treatment

(digestive enzymes or chelating agents). It is not clear whether

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348 SIMMONDS, SEAGULL, SETTERFIELD

Figure 2. Serial reconstruction diagram of microtubules in a vetch

cell grown in GA obtained from IS serial EM sections that glance

through the cortical cytoplasm. Microtubules form parallel arrays on-

ented perpendicular to the long axis of the cell � Microtubules

that terminate in the series are indicated with dots. Bar = I jam.

such agents simply open holes in the wall or have other actions,

such as removal of charged groups in the wall, that impede

diffusion of antibodies. Unfortunately most wall-digesting en-

zymes are impure and prolonged treatment causes loss of MT

staining, presumably because ofproteolytic activity. This harmful

effect is reduced by adding protease inhibitors or gelatin to

the enzyme solution (Simmonds et al., 1983; Wick and Duniec,

1983). Application of enzymes prior to fixation of the cells,

as in the standard procedure, also minimizes damage to the

protoplast (cf. Treatments a and e, Table I).

The response ofdifferent cells to enzyme treatment is van-

Figure 3. Serial reconstruction diagram ofmirotubules in a cell grown

in 2,4-D obtained from 20 serial EM sections through cytoplasm.

Microtubules show little overall order, although some local clustering

is evident. Microtubules that terminate in the series are indicated with

dots. Bar = 1 jam.

able. Vetch cells responded best to the three-enzyme mixture

of the standard procedure. The cellulase and hemicellulase

were essential in this mixture, while pectinase gave minor

improvements in staining. When Worthington cellulase was

substituted for the three-enzyme mixture, a reduced number

of vetch cells stained (Treatment b, Table I ). Suspension cul-

tunes of a number of other species (Datura innoxia, tomato,

tobacco, Nicotiana rustica, and eggplant) also gave good MT

staining with the standard enzyme mixture. On the other hand,

primary cultures of leaf mesophyll cells of Zinnia stained well

after Worthington cellulase treatment, but not after the stan-

dard enzyme mixture (Falconer and Seagull, 1984). Both do-

ver and radish root-tip cells exhibited fine MT arrays when

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IMMUNOFLUORESCENT STAINING OF PLANT MICROTUBULES 349

Table 1. immunofluorescent staining ofmicrotubules (MT) in vetch cells

Frequency of Lateral MT Nonspec ific fluorescence

Treatment

cells withstained MT

(1)

aggregationof MT

(2)

pattern similarto EM images

(3)

(4)

diffuse organdies

a.Standard ++++ + ++++ - -

b. Worthington cellulase + + + + + + + - -

c. No paraformaldehyde prefixation + + + + + + + + + - + +

d. Glutaraldehyde prefixation + + + + + + + + + + + + + +

e. Wall digestion after paraformaldehyde + + + + + + +

f. No Triton-glycerol extraction + + + + + + +

g. Cold methanol extraction, polyclonal + b + + + + +

anti-bovine tubulin

h. Triton-Pipes extraction, no + + + + + + + + + +

prefixation, polyclonal anti-bovine

tubulin

i. Polyclonal anti-chick tubulin + + + + + + + + + + +

j. Polyclonal anti-bovine tubulin + + + + + + + + + +

k. Monoclonal anti-Polytomella tubulin none NA’ NA + + +

�Staining in the four categories was scored semi-quantitatively between the following extremes by examining at least 100 cells per treatment: ( 1 ) ( + ) 10% cellsstained; ( + + + + ) 90% cells stained. (2) ( + ) MT as numerous line, straight fluorescent strands (e.g., Figures 4, 6); ( + + + ) MT as thickened branched strands(e.g., Figure 7). (3) Microtubule pattern similar to that of EM reconstructions in: ( + ) 10% of stained cells; ( + + + + ) 90% of stained cells. (4) ( - ) all stain in MTstrands (Figure 4); + + + strong staining of other than MT strands (Figures 6-9).

bLess than 2% of cells stained.‘NA, not applicable.

processed by the standard procedure. The proportion of cells

stained when Worthington cellulase was substituted for the

standard enzymes was, however, greater in clover and less in

radish. Recently Wick and Duniec (1983) have replaced en-

zyme treatment with EGTA for staining ofonion roots. EGTA

treatment alone is not sufficient to allow staining of cultured

vetch cells, but we found that this treatment combined with

the standard enzymes, improved cell separation in radish roots.

EGTA improved staining of Zinnia mesophyll cells cultured

for longer periods (Falconer and Seagull, 1984). Clearly the

method of wall permeabilization must be carefully considered

for each new species, and perhaps, each cell type.

Apart from permeabilization of the wall, for effective MT

staining with minimum nonspecific binding of antibodies to

other components it is necessary to permeabilize and partially

extract the protoplast. With vetch cells the best results were

obtained using 1% Triton X-100 in an MT stabilizing buffer

containing 4 M glycerol (step 7, standard procedure), as onig-

inally described by Bershadsky et al. (1978). Omission of this

extraction greatly reduced the number of cells with stained

MT. while diffuse background staining was increased (Treat-

ment f, Table 1 ). Replacement of the Triton-glycerol buffer

with cold methanol gave similar poor results (Treatment g,

Table 1 ). Methanol extraction lead to good immunostaining

of MT in small dividing cells of onion root tips (Wick et al.,

1981), but gave less satisfactory staining with radish roots.

Using EGTA, Wick and Duniec (1983) found additional pen-

meabilization ofroot tips to be unnecessary. Lloyd et al. (1980)

used Triton X-100 in Pipes buffer for extraction of cultured

cannot cells prior to MT staining. With vetch cells this method

gave fewer cells with stained MT. increased aggregation of

MT. and elevated nonspecific staining.

To minimize damage by the Triton-glycerol extraction,

cells were prefixed in paraformaldehyde (step 5, standard pro-

cedure). This gave a fine distribution of MT (Treatment a,

Table 1 ; Figures 4-6), resembling that seen in EM sections of

both GA and 2,4-D grown cells (Figures 1-3). When glutar-

aldehyde was used for prefixation good MT patterns were also

seen, but nonspecific staining increased (Treatment d, Table

1 ), possibly because Triton extraction was less effective. Stain-

ing was possible without prefixation but MT showed much

greaten lateral aggregation into thick fluorescent strands

(Treatment c, Table 1 ; Figure 7 ) or anastomosing networks

(Figure 8). Even greater distortion of MT arrangements, as

compared to EM views, was seen in GA grown cells extracted

without prefixation in Triton-Pipes (Treatment h, Table 1;

Figure 9). In addition to vetch cells, good MT staining and

preservation was obtained with the other species of cultured

cells and radish and onion roots using the prefixa-

tion-extraction combination of the standard procedure.

Electron microscopy of cells processed by immunofluones-

cence procedures prior to glutaraldehyde-osmium fixation

confirmed the effect of prefixation on preservation of MT

arrangements. Cells prefixed in paraformaldehyde prior to Tn-

ton-glycerol extraction showed MT distributed in the cyto-

plasm individually on in small clusters, as seen in cells fixed

directly from culture (cf. Figures 1 0 and 1 ). On the other hand,

cells prepared for immunofluorescence without prefixation cx-

hibited poor preservation (Figure 1 1 ). Large areas appeared

empty, while MT occurred in distinct clusters surrounded by

aggregated cytoplasmic material. These MT aggregates still

showed transverse orientation to the cell axis (Figure 1 la) and

correlated in distribution with the thick strands seen in non-

prefixed immunofluonescence preparations (Figures 7,8).

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..

� k:�

�:/e:;#’�� �U-’ �

!‘�

L+.

350

,fr. ‘r. . �..

�� ‘�-‘-�

� � � I

Figures 4-9. M icrographs showing indirect immunofluorescent stain-

ing of microtubules in cultured vetch cells. Cells in Figure 5 were

grown with 2,4-D and were cycling; all other cells were grown in GA

and ceased mitosis, but continued to elongate. Bars = 20 jam.

Figure l. Cell stained by the standard procedure. MT appear as fine,

transversely oriented strands distributed more or less evenly alongthe cell length. The cell is not squashed and blurred areas (p’.) are

outside the plane of focus.

Figure 5. Cycling interphase cells stained by the standard procedure.

Note sOme cells with ordered MT (�) and others with disordered

MT (-s.

Figure 6. Cell prepared by the standard procedure but with polyclonal

anti-bovine tubulin antibody replacing the standard monoclonal anti-

yeast tubulin antibody. The microtubules are finely dispersed and

ordered but are somewhat obscured by nonspecific diffuse fluores-

cence. The nucleus (n) shows strong nonspecific staining.

Figure 7. Cells prepared by the standard procedure without para-

formaldehyde fixation. Due to lateral aggregation, microtubules ap-

pear as thick fluorescent strands. Some disorganization of transversely

ordered microtubules has also occurred. Some nonspecific staining,

e.g. , nucleolus (0), is also present.

Figure 8. Cell prepared without paraformaldehyde fixation and stainedwith polyclonal anti-bovine tubulin antibody. Moderate lateral aggre-

gation of MT has produced a “web-like” array. Microtubules also

appear beaded, probably due to nonspecific staining of adhering col-

lapsed cytoplasm. n, nucleus.

Figure 9. Cell prepared without paraformaldehyde prefixation, cx-

tracted with Triton X-l00 in Pipes buffer and stained with polyclonal

anti-bovine tubulin antibody. Microtubules are extensively aggregated

and considerable distortion of transverse order has occurred (�). n,

nucleus.

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IMMUNOFLUORESCENT STAINING OF PLANT MICROTUBULES 351

.-�5.j’ r�.� .-�.

‘/‘

�,. .-‘�

* -�

‘r,�:;;�. ___________ I .�

-�

�

Figure 1 1 . Electron micrograph of a cell processed for immunoflu-

orescence without prefixation in paraformaldehyde. (a) Cytoplasm has

collapsed into discrete masses (�‘) near the cell wall. (b) Enlargement

of the boxed region in a. Note the aggregated cytoplasm around

bundles of MT (arrowheads). Despite this distortion, bundles of MT

are still oriented transversely to the long axis of the cell (�-�). Bar

= 1.0 jam.

�,:‘ : �: � � .:. ?

j � ,:71.r.�k. � �:�‘ � . � :- -

.,. :.,/ � ,‘.�. .‘ 4 1�� ‘. �

.1�� ‘

-

b� ::t�� � �

Figure 10. Electron micrograph of a cell processed by the standardimmunofluorescence procedure before being prepared for electron

microscopy. Cytoplasmic details closely resemble those in cells fixed

without immunofluorescence processing (cf. Figure 1 ). Bar = 1 .0 jam.

by guest on July 7, 2014jhc.sagepub.comDownloaded from

352 SIMMONDS, SEAGULL, SETTERFIELD

Fowke LC, Bech-Hansen CW, Constabel F, Gamborg OL ( 1974): A

Nonspecific staining both as diffuse background fluores-

cence and staining of organelles, particularly nuclei, was en-

countered using several procedures. In addition, MT often

showed a beaded appearance, particularly in non-prefixed

preparations (Figure 8) where EM sections showed cytoplasm

aggregated around MT (Figure 1 la,b). Nonspecific staining

was common with polyclonal antibodies (Treatments g-j, Ta-

ble I ; Figures 6,8,9), but also occurred with the rat monoclonal

antibody when paraformaldehyde prefixation was omitted or

when extraction of protoplasm was inadequate (Treatments

c-f, Table 1 ; Figure 7). The standard procedure gave very

little non-MT staining (Figures 4,5). Because of variable non-

specific staining, it is impossible to evaluate the distribution

of non-MT tubulin in immunofluorescence preparations.

The results with vetch cells indicate that immunofluores-

cent staining of MT networks comparable to that seen by

electron microscopy can be achieved in plant cells if several

technical variables are carefully controlled. Variations in pro-

cedure, such as prefixation, can, however, lead to differences

in MT arrangements, which have been reported in immuno-

fluorescence studies (Lloyd et al., 1980; Wick et al., 1981).

While the standard method presented here works well for

several cell types other than vetch, it is by no means universally

applicable. With new cell types, the method might serve as a

starting point, but if unsuccessful, systematic variation in wall

permeabilization, prefixation, protoplasmic extraction, and an-

tibodies should be undertaken. In the latter regard it is inter-

esting to note that a monoclonal antibody prepared against the

green alga Polytomella, which readily stains MT ofanimal cells,

failed completely to stain MT ofvetch cells (Treatment j, Table

I).

Acknowledgments

The authors thank K. Rogers and V. Kalnins, Department of Anatomy,University of Toronto, and W. Achison and DL. Brou’n, University of

Ottawa, for the polydonal anti-bovine tubulin and monoclonal anti-Poly-

tomell.a tubulin, respectively, used in these studies.

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Falconer MM, Seagull RW ( 1984): Immunofluorescent and calcofluorwhite staining of developing trachery elements in Zinnia elegans L.

suspension cultures. Protoplasma, in press.

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plasts isolated from a predominantly haploid suspension culture of

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