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Immunoelectron Microscopy of Cryofixed and Freeze-Substituted Plant Tissues

Article  in  Methods in molecular biology (Clifton, N.J.) · June 2010

DOI: 10.1007/978-1-60761-783-9_12 · Source: PubMed

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ChapterTitle Immunoelectron Microscopy of Cryofixed and Freeze-Substituted Plant Tissues

Chapter Sub-Title
Chapter CopyRight - Year Springer Science+Business Media, LLC 2010
(This will be the copyright line in the final PDF)
Book Name Immunoelectron Microscopy
Corresponding Author Family Name Takeuchi
Particle
Given Name Miyuki
Suffix
Division Department of Life Science
Organization Graduate School of Life Science, University of Hyogo
Address 2167 Shosha, Himeji, Hyogo, 671-2280, Japan
Email takeuchi@sci.u-hyogo.ac.jp

Author Family Name Takabe

Particle
Given Name Keiji
Suffix
Division Graduate School of Agriculture
Organization Kyoto University
Address Kyoto, Japan
Email

Author Family Name Mineyuki

Particle
Given Name Yoshinobu
Suffix
Division Department of Life Science
Organization Graduate School of Life Science, University of Hyogo
Address 2167 Shosha, Himeji, Hyogo, 671-2280, Japan
Email

Abstract
Keywords (separated by '-')
Cryofixation and freeze-substitution techniques preserve plant ultrastructure much better than conventional chemical
fixation techniques. The advantage of cryofixation is not only in structural preservation, as seen in the smooth
plasma membrane, but also in the speed in arresting cell activity. Immunoelectron microscopy reveals the subcellular
localization of molecules within cells. Immunolabeling in combination with cryofixation and freeze-substitution
techniques provides more detailed information on the immunoelectron-microscopic localization of molecules in the
plant cell than can be obtained from chemically fixed tissues. Here, we introduce methods for immunoelectron
microscopy of post-embedded, cryofixed plant tissues by applying an antibody to a thin plastic resin-embedded
section prepared by cryofixation followed by freeze-substitution.
Immunoelectron microscopy - plant tissue - cryofixation - freeze-substitution - high-pressure freezing
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Chapter 12
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Immunoelectron Microscopy of Cryofixed
10 and Freeze-Substituted Plant Tissues
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12
Miyuki Takeuchi, Keiji Takabe, and Yoshinobu Mineyuki
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16
Abstract
17
Cryofixation and freeze-substitution techniques preserve plant ultrastructure much better than conven-
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19
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tional chemical fixation techniques. The advantage of cryofixation is not only in structural preservation,
as seen in the smooth plasma membrane, but also in the speed in arresting cell activity. Immunoelectron
20 microscopy reveals the subcellular localization of molecules within cells. Immunolabeling in combina-
21 tion with cryofixation and freeze-substitution techniques provides more detailed information on the
22 immunoelectron-microscopic localization of molecules in the plant cell than can be obtained from chem-
23
ically fixed tissues. Here, we introduce methods for immunoelectron microscopy of post-embedded,
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cryofixed plant tissues by applying an antibody to a thin plastic resin-embedded section prepared by
24
cryofixation followed by freeze-substitution.
25

26 Key words: Immunoelectron microscopy, plant tissue, cryofixation, freeze-substitution, high-

27 pressure freezing.
28
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32
1. Introduction
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Immunoelectron microscopy enables us to study the in situ
36
localization of specific molecules within cells. While conven-
37
tional electron microscopy provides no information about spe-
38
cific molecules, immunoelectron microscopy can help to con-
39
nect a visible structure with a specific molecule. Colloidal gold
40
particles are often used for labeling antibodies. Among sev-
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41
eral techniques for immunolabeling are labeling methods using
42
plastic resin-embedded samples, i.e., post-embedding and pre-
43
embedding immunolabeling. Post-embedding labeling (antibody
44

45

46 S.D. Schwartzbach, T. Osafune (eds.), Immunoelectron Microscopy, Methods in Molecular Biology 657,
DOI 10.1007/978-1-60761-783-9_12, © Springer Science+Business Media, LLC 2010
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49 labeling on a section of a resin-embedded specimen) is the most
50 widely employed technique. In this method, the most impor-
51 tant aspect for success is the balance between preservation of
52 cell structure and retention of the antigenicity against the anti-
53 bodies applied. Ultrastructural preservation in immunoelectron

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54 microscopy is usually inferior compared to that in conventional
55 transmission electron microscopy because fixatives used for struc-
56 tural preservation may prevent the antibody–epitope reaction.
57 Hence, high concentrations of glutaraldehyde and/or osmium
58 tetroxide are avoided in immunoelectron microscopy. In addition,
59 one of the drawbacks of chemical fixation is the speed of diffusion

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60 of the fixative into the specimen, and dramatic structural changes
61 occur inside the cell during the penetration and fixation process
62 with chemicals (see Fig. 12.1 in Mineyuki and Gunning (1)).
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74 Fig. 12.1. Comparison of cross-sectional images of onion root tip cells fixed by glu-
75
taraldehyde/osmium tetroxide (A) and by high-pressure freezing and freeze-substitution
76
(B) showing the superior ultrastructure preservation of the cryofixed sample. The plasma
membrane (arrows) is smooth in the cryofixed cell (B), although it is wavy in the chemi-
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cally fixed cell (A). These are prophase cells and the preprophase band of microtubules
78
(arrowheads) is visible. CW, cell wall. Bar, 200 nm. (Reproduced from (11) with permis-
79 sion from Plant Morphology.)
80

81 Cryofixation is an alternative to chemical fixation. This tech-

82 nique immobilizes molecules in the cell and immediately stops all
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83 cell activities. It has been applied in immunoelectron microscopy

84 for plant tissues and good results have been reported, with
85 excellent structural preservation with both the minimum use
86 of fixatives and without fixatives (2–5). Cryofixation is usually
87 followed by freeze-substitution, and during this procedure, water
88 within the frozen samples is gradually substituted with pure ace-
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89 tone or acetone containing a low concentration of fixatives at

90 −80◦ C. After freeze-substitution, specimens are gradually
91 warmed, embedded in resin, and then an immunoreaction using
92 gold particles is performed on ultrathin sections.
93 Several cryofixation techniques have been used for biological
94 specimens, such as slam freezing (or cold metal block freezing),
95 plunge freezing, propane jet freezing, and high-pressure freez-
96 ing. Details of these techniques are summarized in the review by
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97 Gilkey and Staehelin (6). The freezing must achieve very rapid
98 cooling rates so as to minimize damage to the sample caused
99 by ice crystal formation. As the cell wall in plant tissues prevents
100 rapid cooling of the sample, the well-preserved region of the spec-
101 imen is relatively less than that in animal tissues. To preserve plant

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102 tissues in good condition, high-pressure freezing is often used
103 (4, 7–9). In high-pressure freezing, specimens are frozen in a liq-
104 uid nitrogen jet at 2,100 bar to prevent ice crystal nucleation and
105 growth in the specimens. The advantages of high-pressure freez-
106 ing include high efficiency in achieving good preservation and
107 a well-preserved area that can reach 0.2 mm below the sample

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108 surface.
109 Preparation of ultrathin sections requires that samples be
110 embedded in plastic resins. LR-White and Lowicryl resins are
111 often used for immunolabeling. A good antibody–epitope reac-
112 tion is expected with LR-White. This is a hydrophilic acryl resin
113 having a low viscosity that is an advantage in infiltrating into plant
114 tissue. Lowicryl resins such as HM20 are also widely used. HM20
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115 is a low-temperature cured resin that can be polymerized at –60◦ C
116 under UV irradiation minimizing antigenicity loss. Epoxy resins,
117 such as Epon or Spurr, are not often used for immunomicroscopy
118 because heating specimens are required for resin polymerization
119 and heating can compromise the epitope-reducing immunolabel-
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120 ing. However, some antibodies do remain active in these resins

121 presumably because the epitope is not heat sensitive.
122 In this chapter, we present a procedure for high-pressure
123 freezing. Propane plunge freezing, a conventional method, is also
124 described because it is easy to apply and works well, although it is
applicable only to very thin specimens. Procedures for embedding
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126 in both LR-White and Lowicryl HM20 are also presented.

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2. Materials
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2.1. Cryofixation 1. High-pressure freezer, BAL-TEC HPM 010 (BAL-TEC
133
AG, Balzers, Liechtenstein).
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2. Specimen carrier: Freezer hats for high-pressure freezing
136
(Brass Planchet “A” and “B”; Ted Pella, Inc., Redding, CA,
USA).
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138 3. Plunge freezer, VFZ-1 (Vacuum Devices Inc., Tokyo,

139 Japan).
140
4. Propane.
141
5. Liquid nitrogen.
142

143 6. Onion seeds (Allium cepa L. cv. Highgold Nigou, Sakata

144 Seed Co., Yokohama, Japan).
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2.2. Freeze- 1. 0.5% glutaraldehyde in acetone: dilute a 70% aqueous solu-
146
Substitution tion of glutaraldehyde (Electron Microscopy Science, Hat-
147
field, PA, USA) with acetone for a final concentration of
148
0.5% and chill in liquid nitrogen prior to use.
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2. Cryotube vials (Nunc A/S, Roskilde, Denmark).
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152
2.3. Embedding 1. LR-White (Hard) (London Resin, Berkshire, UK).
153 2. Lowicryl HM20 (Polysciences, Warrington, PA, USA): Mix
154 2.98 g cross-linker D and 17.02 g monomer E by gently
155 stirring with a glass rod (see Note 1).

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3. Lowicryl HM20 with initiator C: Mix 2.98 g cross-linker D
157
and 17.02 g monomer E by gently stirring with a glass rod.
158
Add 0.1 g initiator C until it is completely dissolved in the
159
resin.
160

161
4. Rotator TYPE N (TAAB, Berkshire, UK).
AQ1 162 5. Gelatin capsule, 8 mm in diameter (No. 00, Lilly Co.,
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163 Indianapolis, Indiana, USA).
164 6. Ultraviolet polymerizer, TUV-200 (Dosaka EM Co., Kyoto,
165 Japan).
166

167 2.4. On-Grid Section 1. Formvar-coated nickel grid (SPI supplies, West Chester,
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168 Immunolabeling PA, USA).

169
2. Ultra microtome (Leica, Wetzlar, Germany).
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3. Phosphate-buffered saline (PBS): 8 mM disodium hydro-
172
gen phosphate, 1.5 mM potassium dihydrogen phosphate,
137 mM sodium chloride, 2.7 mM potassium chloride.
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174 4. PBS-T: PBS containing 0.1% Tween 20.

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5. Blocking buffer: PBS containing 0.8% bovine serum albu-
176
min (BSA), 0.1% gelatin from cold water fish skin (Sigma-
177
Aldrich), 5% normal goat serum (same species as secondary
178
antibody), and 2 mM NaN3 .
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6. Washing buffer: PBS containing 0.8% BSA, 0.1% gelatin
180
from cold water fish skin, and 2 mM NaN3 .
181

182 7. Primary antibody to molecule of interest.

183 8. Colloidal gold (5–15 nm)-conjugated secondary antibody
184 that is appropriate for the primary antibody such as Goat
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185 anti-mouse IgG antibody, 10-nm gold conjugate (British

186 Biocell International, Cardiff, UK).
187
9. 2% glutaraldehyde in PBS.
188

189
10. 2% (w/v) uranyl acetate in 60% ethanol. Store at 4◦ C in the
190
dark.
191 11. Reynolds’ lead citrate (10): dissolve 1.33 g lead nitrate and
192 1.76 g sodium citrate in 30 mL distilled water. The distilled
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193 water is boiled to remove carbon dioxide before use. Clarify
194 the solution by adding 8 mL 1 N NaOH solution. Add
195 distilled water for a final volume of 50 mL seal in a syringe
196 and store at 4◦ C in the dark.
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199

200

201 3. Methods
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3.1. Cryofixation 1. Germinate onion seeds in 0.05 M sucrose for 2 days and
205 3.1.1. High-Pressure transfer to 0.1 M sucrose 1 day before freezing (9) (see
206 Freezing of Onion Note 2).
207 Cotyledon 2. Set up the high-pressure freezing apparatus according to the
208 manufacturer’s instructions.
209
3. Excise a piece of onion cotyledon that will fit in the sam-
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ple carrier. The sample carrier, Planchet A and B are both
2.0-mm internal diameter, and the cavity depths are 0.1, 0.2,
and 0.3 mm. Using various combinations of Planchet A and
213
B, the cavity thickness can be varied from 0.1 to 0.6 mm. A
214
thickness of 0.2 or 0.3 mm is often used. The free space of
215
the carrier cavity is filled with 0.1 M sucrose (see Note 3).
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4. Place the sample in the high-pressure freezing apparatus and
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freeze.
219 5. Immediately after freezing, transfer the sample into liquid
220 nitrogen (see Note 4). Under liquid nitrogen, open the sam-
ple carrier sets and remove a carrier (see Note 5). Store the
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222 frozen sample in liquid nitrogen until freeze-substitution.

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224 3.1.2. Plunge Freezing 1. A plunge freezer VFZ-1 device is composed of containers
225 with Liquid Propane of of liquid propane and liquid nitrogen, and a sample plunger
226
Poplar Xylem equipped forceps (see Note 6). Place liquid nitrogen in the
bottom container.
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2. Flush the propane gas slowly and place liquid propane in the
229
cryogen holder (see Note 7).
3. Excise a small piece of plant tissue (about 5 mm × 2 mm ×
230

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0.5 mm).
232
4. Pick up the specimen with the forceps of the sample plunger
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and plunge the specimen into the liquid propane.
235 5. Immediately transfer the frozen specimen into liquid nitro-
236 gen and release it. Store the frozen sample in liquid nitrogen
237 until freeze-substitution.
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239 3.2. Transfer the cryofixed samples into a small screw cap vial con-
240 Freeze-Substitution taining 0.5% glutaraldehyde in acetone chilled in liquid nitrogen
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241 (see Note 8). Keep the sample at –80◦ C for more than 72 h to
242 complete the freeze-substitution.
243 Cryofixation provides excellent ultrastructure preservation
244 as exemplified by the remarkable difference in smooth plasma
245 membrane ultrastructure compared to chemically fixed tissue

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246 (Fig. 12.1). However, organelle-dependent artifacts can arise
247 during cryofixation and freeze-substitution. For example, micro-
248 tubules which are 25-nm diameter hollow, rod-like structures,
249 easily observed by conventional electron microscopy (Fig. 12.1A
250 arrowheads) in chemically fixed tissue and in well-preserved
251 cyrofixed samples (Fig. 12.1B arrowheads) are susceptible to

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252 cryodamage. If specimens are not preserved well under cryofixa-
253 tion, their cylindrical structure collapses. Sometimes we find only
254 a few microtubules, or damaged microtubules, in cells whose
255 plasma membrane and other organelles seem to be successfully
256 fixed (11).
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258 3.3. Embedding 1. Gradually transfer the freeze-substituted samples in screw

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259 in Resin capped vials to room temperature (RT) by incubation at
260
3.3.1. Embedding –20◦ C for 2 h, 4◦ C for 2 h, then RT for 2 h.
261 in LR-White 2. When the temperature of the sample reaches RT, remove
262
the 0.5% glutaraldehyde in acetone from the vial and wash
263
the specimens with acetone three times for 15 min.
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3. After the third wash, remove the acetone from the vial and
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wash the specimens with ethanol, three times for 15 min.
267 4. After the third wash, remove the ethanol and add LR-
268 White: Ethanol = 1:2 to the vial. Incubate for 2 h at RT
under gentle agitation with a rotary shaker (see Note 9).
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5. Incubate in LR-White: Ethanol = 1:1 overnight.
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6. Incubate in LR-White: Ethanol = 2:1 for 8 h.
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7. Incubate in 100% LR-White overnight.
274 8. Incubate in 100% LR-White for 8 h.
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9. Remove the specimen from the screw cap vial and place it at
276
the bottom of a gelatin capsule. Fill the capsule with fresh
277
LR-White resin and close.
278

279
10. Seal capsules in a container under nitrogen gas. Polymerize
280
at 50◦ C for 16 h.
11. Store the polymerized samples in silica gel.
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283 3.3.2. Embedding in 1. Warm freeze-substituted samples in screw capped vials to

284
Lowicryl HM20 –60◦ C (see Note 10).
285 2. Remove the 0.5% glutaraldehyde in acetone from the vial
286 and wash specimens with acetone at –60◦ C, three times for
287 1 h.
288
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289 3. After the third wash, remove the acetone and incubate in
290 12.5% HM20 in acetone for 3 h at –60◦ C.
291
4. Incubate in 25% HM20 in acetone overnight at –60◦ C.
5. Incubate in 50% HM20 in acetone for 4 h at –60◦ C.
292

293

6. Incubate in 75% HM20 in acetone for 4 h at –60◦ C.

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295 7. Incubate in 100% HM20 in acetone overnight at –60◦ C.

296
8. Incubate in 100% HM20 in acetone for 8 h at –60◦ C.
297

298
9. Incubate in 100% HM20 in acetone for 3 days at –60◦ C.
299 10. Incubate in 100% HM20 with initiator C in acetone for 2

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300 days at –60◦ C.
301
11. Place a specimen in a gelatin capsule. Fill the capsule with
302
fresh HM20 with initiator C and close the cap.
12. Polymerize at –60◦ C under UV for 2 days using the UV
303

304
polymerizer.
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307

308
3.4. On-Grid Section
Immunolabeling
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1. Prepare the embedded specimen block for sectioning. If it
is necessary, fix the specimen block in the desired orienta-
tion on a support for the microtome. Trim the specimen
309
surface about 0.5 mm × 0.5 mm and cut ultrathin sections
310
(50–90 nm thick) using an ultra microtome. Pick up the
311
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sections on Formvar-coated nickel grids (see Note 11).
312

313
2. Incubate the sections with 50 mM glycine in PBS for
314
15 min (see Note 12).
315 3. Wash the sections for 5 min with PBS.
316
4. Incubate the sections with blocking buffer for 30 min to
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317
block nonspecific binding sites on the sections.
318

319
5. Wash the sections for 5 min with PBS.
320 6. Incubate the sections in the primary antibody diluted with
321 antibody dilution buffer for 1–2 h at 37◦ C (see Note 13).
322 7. Wash the sections three times for 5 min with PBS-T.
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8. Incubate the sections in the colloidal gold-conjugated sec-
324
ondary antibody diluted with antibody dilution buffer for
1–2 h at 37◦ C.
325

326

327
9. Wash the sections three times for 5 min PBS-T.
328 10. Post-fix the sections with 2% glutaraldehyde in PBS for
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329 5 min.
330
11. Wash the sections with distilled water with a stream of run-
331
ning water from a bottle.
332

333
12. Counterstain the sections with 2% uranyl acetate in 60%
334
ethanol for 10 min followed by Reynolds’ lead citrate (10)
335
for 2 min.
336
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337 13. Examine samples under the transmission electron micro-
338 scope. An example of a high-pressure frozen HM20-
339 embedded onion cotyledon epidermal cell immunolabeled
340 with a monoclonal anti-α-tubulin antibody is shown in
341 Fig. 12.2, while a plunge-frozen LR-White-embedded sec-

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342 ondary wall-forming fiber cell of poplar secondary xylem
343 immunolabeled with anti-PRX3 antibody is shown in
344 Fig. 12.3. Note that both the cotyledon (Fig. 12.2) and
345 the difficult to fix relatively hard woody tissue (Fig. 12.3)
346 exhibit excellent ultrastructure preservation and immuno-
347 labeling.

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349

350

351

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359
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361

362

363
Fig. 12.2. Immunolabeling of an onion cotyledon epidermal cell with the monoclonal
364
anti-α-tubulin antibody. A high-pressure frozen onion cotyledon was freeze-substituted
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365 with 0.25% glutaraldehyde/0.1% uranyl acetate in acetone and embedded in HM20
366 resin. The sample was immunolabeled with a 1/50 dilution of mouse monoclonal anti-
367 α-tubulin primary antibody and a 1/50 dilution of goat anti-mouse IgG secondary anti-
368 body conjugated to 10-nm gold particles. Microtubules are clearly visible, but are not
369
labeled because they are within the resin. In contrast, gold particles (10 nm diameter)
appear at the end of microtubules (arrowheads) and on faintly contrasted linear struc-
370
tures (arrows). Microtubules may be exposed on the surface of the section at these
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371
positions. MT, microtubule. Bar, 200 nm.
372

373

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375

376

4. Notes
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378

379

380
1. Use well-ventilated fume hood for mixing resins. Avoid
381
contact with skin and eyes and avoid inhalation of resin
382
vapor.
383 2. In the case of onion cotyledon, feeding sucrose prior to
384 freezing decreases cryodamage (9). This pretreatment is
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406 Fig. 12.3. Immunolocalization of a peroxidase with anti-PRX3 antibody in a secondary

407 wall-forming fiber cell of poplar secondary xylem. Plunge-frozen samples were freeze-
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408
substituted with 0.5% glutaraldehyde in acetone and embedded in LR-White resin. The
sample was immunolabeled with a 1/50 dilution of anti-PRX3 primary antibody and
409
a 1/25 dilution of goat anti-rabbit IgG secondary antibody conjugated to 15-nm gold
410
particles (5). The peroxidase is involved in woody cell wall formation and gold par-
411
ticles (15 nm diameter) are observed in the vicinity of the plasma membrane in the
412 cross section (A). In a longitudinal section of a fiber (B), the gold particles are also seen
in the plasma membrane area, where cortical microtubules are also visible. Note that
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413

414 organelles are well preserved in this method although the woody tissues are relatively
415
hard and not easy to fix. CW, cell wall; V, vacuole; MT, microtubule. Bar, 500 nm. (Repro-
416
duced from (5) with permission from the Journal of Wood Science.)
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418
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419
not necessary for all plant materials and un-treated mate-
420
rials are generally supplied for freezing.
421
3. The carrier cavity is filled with a cryoprotectant containing
422
solution. We use 0.1 M sucrose for onion cotyledon. Cry-
423
oprotectants, e.g., hexadecane, dextran, polyethylene gly-
424
col, and hydroxyethyl starch, have been used for various
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samples.
426

427
4. Work carefully with liquid nitrogen and frozen samples.
428
Forceps and any other tools must be pre-chilled before
429
touching the frozen sample. Wear eyeglasses and gloves to
430
protect yourself.
431 5. For infiltration of fixatives into the sample, the cover car-
432 rier should be removed. The sample can remain on the
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433 carrier for freeze-substitution and the following washing
434 steps until the step where resin is applied.
435
6. Alternatively, small containers and forceps can be used.
436
7. Use propane under ventilation.
437

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438 8. A very weak fixative, e.g., 0.25% (or 0.5%) glutaraldehyde,
439 is used for immunoelectron microscopy. For better con-
440 trast, low concentrations of osmium tetroxide (e.g., 0.01%)
441 or 0.1% uranyl acetate can be used but they might inter-
442 fere with the antibody–antigen reaction. Even a very low
443 concentration of glutaraldehyde can inhibit the antigen–

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444 antibody reaction. In such cases, pure acetone can be used
445 for freeze-substitution (2).
446 9. LR-White is a low-viscosity resin and easily infiltrates into
447 the specimens. The specimens should sink into the resin
448 solution. If samples float on top of the solution at the end
449 of an infiltration step, resin has not penetrated into the
450 specimens. If resin infiltration is problematic, try a lower
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451 concentration of resin and/or lengthen the time of each
452 step.
453
10. For temperature control, an automatic freeze-substitution
454
system is sold by Leica. Alternatively, a −80◦ C deep freezer
455
can be reset to –60◦ C.
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456

457
11. A nickel grid is used to avoid a reaction between grid metal
458
and the solutions.
459 12. For immunostaining the sections, a grid is incubated in
460 solutions by floating section side down on a drop of solu-
tion and transferred sequentially from drop to drop to
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461

462 complete the immunostaining. To cover a grid, 5–50 µL

463 drops of solution are placed on a piece of Parafilm in a
464 Petri dish kept in a moist chamber to avoid drying of
465 the solution. The section must never dry out during the
466 procedure.
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467
13. Optimal conditions for labeling with the primary and the
468
secondary antibodies should be found through preliminary
469
trials. The antibody dilution is often much less than that
470
used for non-embedded samples.
471

472
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474

475

476 Acknowledgments
477

478

479
This work was supported by JSPS Grant-in-Aid for Scientific
480
Research (A) 17207006 to YM.
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481 References
482
1. Mineyuki, Y. and Gunning, B. E. S. (1988) 7. Craig, S. and Staehelin, L. A. (1988) High
483
Streak time-lapse video microscopy: analy- pressure freezing of intact plant tissues. Eval-
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