Microsc. Microanal.

4 , 4 9 1 4 9 6 , 1999
Microscopy ...
Microanalvsis 4

O MICROSCOPY SOCIETY OF AMERICA 1999

Microwave Protocols for Paraffin Microtechnique a n d In

Situ Localization in Plants

Denise Schichnes,* Jeff Nemson, Lorraine Sohlberg, and Steven E. Ruzin

C N R Biological Inzagitlg Facility, Department of Plant and Microbial Biology, University of California at Berkeley, 1 I 1
Koshland Hall, Berkeley, CA 94720

Abstract: We have developed a microwave protocol for a paraffin-embedding microtechnique of the shoot
apical meristem of Zea mays and have successfully applied this protocol to other plant tissues. This protocol
decreases the time required for all aspects of microtechnique tissue processing, including fixation (24 hr to 15
min), dehydration (73 hr to 10 min), and infiltration (96 hr to 3 hr). Additionally, the time required to adhere
paraffin ribbons to gelatin-coated slides and for the Johanson's safranin 0,fast green FCF staining protocol has
been significantly decreased. Using this technique, the quality of tissue preservation and subsequent in situ
localization of knotted mRNA was increased by using microwaves.

Key words: in situ, microtechnique, microwave, paraffin, plants, staining

probe for in situ analysis because of its well-characterized

In situ localization of nucleic acids has caused a resurgence
of interest in plant microtechnique. However, long periods
of time-1 to 2 weeks-have been required for proper pro-
cessing (fixation, dehydration, and infiltration) of the plant
tissues for parafin embedding. During this time, the nucleic
acids of interest degrade, especially under the temperature
conditions required for paraffin processing (Jackson, 1991).
There are many protocols available that allow paraffin pro-
cessing of tissue in a minimum amount of time (Jackson,
1991; Kouchi and Hata, 1993; Ruzin, 1999), but these pro-
tocols require the sacrifice of good anatomical preservation
to maintain nucleic or protein integrity. We set out to find
a technique that offered nucleic acid preservation as well as
anatomical preservation. We chose the knotted mRNA
Received May 5 , 1998; accepted November 6, 1998
*Corrnpond~ngauthor
differential hybridization pattern to portions of the maize
shoot apical meristem (Smith and Hake, 1992). We used
this well-defined probe to evaluate the differences between
traditional and microwave paraffin-embedding microtech-
niques.
Electron microscopists have used microwaves to sig-
nificantly reduce the time required for tissue processing of
samples in resin (Giberson et al., 1997; Kok and Boon, 1898;
Login and Dvorak, 1994). By adapting these processes to
light microscopy, we have developed a microwave tech-
nique for plant paraffin embedding that requires an average
of 5 hr. This technique works well for preserving anatomy
as well as nucleic acids. We used a Pelco microwave oven
with the magnetron regulated by a temperature probe, and
were able to decrease the time required for fixation, dehy-
dration, and infiltration of Zea mays shoot apical meristeins
as well as all other plant materials tested.
Initially, we chose to develop this protocol for the Zea
492 Denise Schichnes et al.

mays shoot apical meristem because it is a scientifically

Table 1. Traditional Protocol for Paraffin-Embedding
valuable yet difficult tissue to process for paraffin sectioning
Microtechnique
(Freeling and Lane, 1994). The leaves surrounding the Zea
mays meristem are at different developmental stages, with Temperature Time
cells ranging from small, densely cytoplasmic cells to large, Step ("(3 (hr)
vacuolate cells (Esau, 1943). This dramatic variation makes 1. Fixation in FAA 4 24
proper fixation of the sample difficult. The leaves also tend 2. 5096 EtOH:40% H,O:lOO/o TBA 24 4
to trap air pockets between each other and the meristem, 3. 50% EtOH:30% H,O:20% TBA 24 20
increasing the difficulty involved in fixation and infiltration. 4. 50% EtOH:10% H,O:40% TBA 24 4
We achieved better or equivalent results using the micro- 5. 50% EtOH:50% TBA 24 20
wave protocol than when using the traditional paraffin- 6. 25% EtOH:75% TBA
with 0.05% wlv safranin 0 24 1
processing protocol.
7. 25% EtOH:75% TBA 24 4
8. 100% TBA 58 4
9. 100% TBA 58 16
10. Add ?/ivolume molten paraffin 58 24
11. Remove ?hvolume, replace with
Generation of Materials molten paraffin, repeat 2x 58 4
We used the shoot apical meristem and 6 surrounding 12. Remove '15 volume, replace with
leaves of the Zea mays (Pioneer Hybrid International, USA) molten paraffin 58 16
13. Fresh molten paraffin,
inbred line B73 for all paraffin processing discussed in this
repeat 3x 58 8
report. For each experiment, 40 kernels of B73 were planted
in genetics mix soil (50% sand, 50% peat), and a 0.5-inch
FAA: 50% ethaliol, 10% formalin, 5% acetic acid; TRA, tert-buy1 alcohol;
layer of vermiculite was placed on top of the soil. The plants
wlv, weight per volume.
were grown under greenhouse conditions and watered daily
until 14 days after planting. The seedlings were then har-
vested and dissected so that the tissue to be fixed included
the shoot apical meristem, the 6 leaves surrounding the Traditional Tissue Processing for In Situ
meristem, and 2 m m of the stem. The resulting sample Localization Studies
tissue was approximately 7 x 4 x 3 m m in size. The tissues
Samples for in situ localization were fixed in PFA (4% para-
were immediately placed into 20-ml glass vials of fucative on
formaldehyde in phosphate-buffered saline) overnight (16
ice (10 samples per vial) until all tissues were dissected.
hr) at 4°C. Samples were processed through the dehydration
Twenty samples were processed using the traditional pro-
and infiltration series shown in Table 2. We used TBA as an
tocol and 20 samples were processed using the microwave
intermediate solvent, and Paraplast Extra for infiltration
protocol. This procedure was performed twice for anatomi-
and embedding. The complete protocol, from futation to
cal studies and twice for in situ localization experiments.
embedding, required 7 days.

Traditional Tissue Processing for

Anatomical Studies Microwave Tissue Processing

Samples for anatomical studies were fixed in FAA (50%
ethanol, 10% formalin, 5% acetic acid) overnight (16 hr) at
4°C. Samples were processed through the dehydration and
infiltration steps listed in Table I. We used tert-butyl alco-
hol (TBA) as an intermediate solvent, considered the best
choice for intermediate solvent (Ruzin, 1999), and Paraplast
Extra (Fisher Scientific, Pittsburgh, PA) for infiltration and
embedding. The complete protocol, from fucation to em-
bedding, required 9 days.
Samples for anatomical studies were fixed in FAA, and
samples for in situ localization were fixed in PFA. Table 3
summarizes the times and temperatures of each stage of the
protocol. We used isopropanol as an intermediate solvent
because our microwave oven is not completely enclosed in
a fume hood, and isopropanol is the least toxic solvent
available. We used Paraplast Extra for infiltration and em-
bedding. The complete protocol, from fxation to embed-
ding, required 4 hr. Control samples were exposed to the
 Microwave Paraffin Protocols 493

Table 2. Traditional Protocol for Paraffin-Embedding Micro- Table 3. Microwave Protocol for Paraffin-Embedding Micro-
technique and In Situ Localization technique and In Situ Localization

Temperature Time Temperature Time

Step ("C) -
.
(hr) Step ("C) (min)
1. Fixation in PFA 4 24 1. Fixative, repeat 2x 37 15
2. 0.85% NaCl 4 1.4 2. 0.85% NaCl for in situ only 67 1.25
3. 50Y0 EtOH, 0.85% NaCl 4 1.4 3. 50% EtOH 67 1.25
4. 70% EtOH, 0.85% NaCl 4 1.4 4. 70% EtOH 67 1.25
5. 85% EtOH, 0.85% NaCl 4 1.4 5. 70% EtOH with 0.05%
6. 95% EtOH 4 1.4 wiv safranin 0 67 1.25
7. 100% EtOH 4 1.5 6. 100% EtOH, repeat l x 67 1.25
8. 100% EtOH 4 15.5 7. 50% EtOH:50% isopropanol 77 1.5
9. 25% TBA:75% EtOH 27 0.75 8. 100% isopropanol 77 1.5
10. 50% TBA:50% EtOH 27 0.75 9. 50% isopropanol:50% molten
11. 75% TBA:25% EtOH 27 0.75 paraffin 77 10
12. 100% TBA, repeat 2x 40 0.75 10. Molten paraffin 67 10
13. TBA with 2 paraffin chips 40 15.5 11. Molten paraffin, repeat 4x 67 30
11. Increase temperature 58 4
15. Remove l/2 volume, replace
with molten paraffin
16. Fresh molten paraffin,
in the static water load was changed between every step in
repeat 5x
the protocol.

PFA, 4% paraformaldehyde in phosphate-buffered saline.

In Situ Localization of knotted mRNA

We used DIG-labeled (digoxigenin-UTP) knotted (kn)

same temperatures for the same length of time, but not to
microwaves.
For all microwave procedures, we used a Pelco 3440
MAX laboratory microwave oven (Ted Pella, Inc., Redding,
CA) with a PolyTemp Polysciences load cooling water bath
(Polysciences, Inc., Warrington, PA). Before each experi-
ment, we checked for areas of high micro~~ave flux using a
Pelco 36140 microwave bulb array (Ted Pella). We avoided
placing samples in areas having high microwave flux, as
indicated by illuminated bulbs. The sample vials were
placed in a water bath (8.5 x 12 x 5 cm, holding 50 ml of
water) that was preheated to the desired temperature. The
temperature was regulated by placing the microwave's tem-
perature probe into a vial of the same solution that the
samples occupied. The microwave oven's built-in tempera-
ture probe continuously determined and regulated the tem-
perature of the sample, which was displayed continuously
on the microwave oven front panel. The wire that attaches
the probe to the microwave was submerged in the water to
decrease the antennae effect. An additional 400 ml static
water load was placed in the oven at an optimal position
determined by using the microwave bulb array. The water
mRNA as'a probe that hybridizes to mRNA from the knot-
ted gene in the meristem and procambial tissue (Smith and
Hake, 1992). The probe was labeled as suggested by Boeh-
ringer-Mannheim (1996). In situ localization was carried
out as described in Jackson (1991). Probe was detected via
a colormetric nitroblue tetrazolium (NBT) reaction as de-
scribed in Kouchi and Hata (1993) except that glass-
distilled, autoclaved water was substituted for diethyl pyro-
carbonate-treated (DEPC) water throughout the protocol.
Coverslips were mounted with Merkoglas (Bryant Labs,
Berkeley, CA).
Section Mounting
Parafin-embedded samples for anatomy comparisons were
sectioned at 8 pm, and the ribbons were adhered to gelatin-
coated slides. The slides were coated with a modified ver-
sion of Haupt's gelatin (Haupt, 1930), but with no phenol
added. Paraffin ribbons were floated on 4% formalin on a
42°C warming plate for 2-3 min to relax ribbon compres-
sion. The formalin was then removed and the slides were
loaded into a slotted glass staining dish placed on its side (so
494 Denise Schichnes et al.

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Figure 1. Comparison of microwave- and traditionally processed
Zea mays shoot apical meristems and the surrounding developing
leaves in longitudinal section. A: Microwave-processed tissue ac-
cording to the protocol in Table 3 for anatomical studies. Inset
shows a close-up of the meristematic region. These sections were
fixed to the slide and stained using the microwave procedure de-
scribed in Materials and Methods. B: Traditionally processed tis-
sue according to the protocol in Table 1 for anatomical studies.
Inset shows a close-up of the meristematic region. These sections
were fixed to the slide and stained following the protocols outlined
in Ruzin (1999). C: Microwave-processed in situ experiment fol-
lowing the protocol outlined in Table 3. D: Traditionally processed
in situ experiment following the protocol outlined in Table 2.
Bars = 50 pm.
+ RESULTSA N D DISCUSSION
the sections would not slide off), and put in the microwave
oven.
Samples for in situ experiments were sectioned at 8 pm
and adhered to Probe-on Plus slides (Fisher), and the rib-
bons were floated on autoclaved water on a 42°C warming
plate for 2-3 min to relax ribbon compression. The remain-
ing water was removed with a paper towel, and the slides
were placed in a slotted glass staining dish on its side in the
microwave oven.
The tip of the oven temperature probe was placed in a
300-p1 drop of water on a clean glass slide. Droplet tem-
perature was determined by the probe. We used a PAP pen
(Ted Pella, Inc.) to draw a hydrophobic ring on the glass
slide to contain the drop. To adhere the sections, the slides
were microwave-heated 30 min with the temperature limit
set to 43"C, and then stained. As a control, ribbons on slides
were heated to 43°C for 30 min but were not exposed to
microwaves. All microwave-processed tissue was adhered to
the slide using the microwave method, whereas all tradi-
tionally processed tissue was adhered to the slide by incu-
bation at 42'C for 16 hr (Ruzin, 1998).
Staining
For all anatomical tissue comparisons, we used a modified
version of Johansen's safranin and fast green FCF staining
(Johansen, 1940). We deparaffinized, rehydrated, and pre-
pared stains as previously described by Johanson. The stain-
ing dish was filled with the safranin stain and placed in a
water bath in the microwave, and loosely covered with plas-
tic wrap to prevent splattering (a tight-fitting cover or lid on
hlicro~vaveParafin Protocols 495
the staining dish will cause it to explode). The temperature
probe was inserted into the staining jar through the plastic
~h~ slides were stained for 45 min in the microwave
at 60°C. we followed ~ ~method for
h the ~ ~ ~
of the staining series. The slides were mounted in Merko-
glas. Controls were stained for 45 min at 60°C, but were
exposed to no microwaves. All microwave-processed tissue
was stained using the microwave staining protocol (45 min
in safranin 01, whereas all traditional staining (48 hr ill
safranin 0 ) was carried out according to oha an sen's method
(Johansen, 1940).
A comparison of microwave- and traditionally processed
Zea mays tissue is shown in Figure 1. Tissues were processed
using either the microwave or traditional protocol, then
mounted and stained for anatomical studies, or used for in
situ experiments. A comparison of the tissues prepared for
anatomical studies (see Fig. lA,B) showed the level of ana-
tomical preservation and staining to be equivalent, even
though a less desirable intermediate solvent (Johansen,
1940; Berlyn and ~Miksche,1976) was used in the microwave
protocol because it was less toxic. The major advantage of
the microwave protocol in this comparison is speed; the
microwave protocol required 4 hr, whereas the traditional
protocol required 7 days. Regardless of whether the paraffin
ribbons were adhered to the slides through the traditional
or the microwave method, no sections were ever lost during
staining or coverslip mounting. The primary advantage of
the microwave method again is speed-30 min vs. 16 hr.
Other plant tissues (maize root tips, radish roots, Arabidop-
sis roots, shoot apical regions and inflorescences) were com-
pared using traditional and microwave methods and were
seen to be of equivalent quality (data not shown).
Controls were run for furation, dehydration, embed-
ding, adhering ribbons to slides, and staining as described in
Materials and Methods. In each case, the controls were
subjected to the same time period and temperature as the
microwave samples but were not exposed to microwaves.
Controls for fixation, dehydration, and embedding were so
poor that they could not be sectioned on the microtome.
Controls for adhering ribbons to slides were so poor that
the sections fell off the slides during depdraffinization. Con-
trols for staining were not visibly stained by safranin O once
the staining procedure was complete. Data for controls were
of such poor quality that they are not reported here.
496 Denise Schichnes et al.
A comparison of microwave- and traditionally pro-
cessed tissue for in situ experiments yielded dramatic dif-
ferences. The degree of anatomical preservation in the mi-
crowave-processed tissue (Fig. 1C) was superior to the tra-
ditionally prepared material (Fig. ID). Although we used
identical fixatives, the level of fixation shown in Figure 1 C
is better than that shown in Figure 1D. This difference is
clearly evident in the shoot meristem (Fig. l C , D ) , a delicate,
easily damaged region of the plant. The meristem in the
traditionally prepared sample (Fig. I D ) is crumpled and
sunken, making it difficult to determine in which cell layers
the probe is localizing. However, in Figure l C , it is clear that
the probe is localizing to the central zone of the meristem,
and not the outer two layers of cells or the developing leaf
primordia, as is known to occur with this probe (Smith et
al., 1995).
The faster fixation due to increased rate of penetration
caused by the microwaves (Kok and Boon, 1989) yields
better preservation than the standard 16 hr for normal dif-
fusion of this weak fixative. The major benefit of the mi-
crowave protocol for in situ tissue processing is the preser-
vation of anatomy, making localization of the mRNA probe
more precise. Finally, the reduction in time spent for tissue
processing is also a benefit.
Microwave paraffin processing of plant tissue is a valuable
technique that decreases the overall time required for tissue
processing. It yields an equivalent or superior degree of
anatomical preservation for light-microscopy research pur-
poses. Additionally, we have shown that compounds sensi-
tive to degradation in traditional tissue-processing tech-
niques, such as RNA, are more precisely detected when the
microwave processing protocol is used.
The authors thank the following people for their help with
this study: Richard Schneeberger, Keisuke Namoto, Barbara
Kloeckener-Gruissem, Justine ZYalsh, Lew Feldman, and
Randall Tyers. This research was supported by NSF grant
DIR-8719933. We dedicate this paper to the memory of
Wally Porter.
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