Microsc. Microanal. 4 ,491496 , 1999 Microscopy ... Microanalvsis

4

O MICROSCOPY SOCIETY OF AMERICA 1999

Microwave Protocols for Paraffin Microtechnique and In Situ Localization in Plants

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

CNR Biological Inzagitlg Facility, Department of Plant and Microbial Biology, University of California a t 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

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~ng author

probe for in situ analysis because of its well-characterized

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

valuable yet difficult tissue to process for paraffin sectioning

(Freeling and Lane, 1994). The leaves surrounding the Zea mays meristem are at different developmental stages, with

cells ranging from small, densely cytoplasmic cells to large,

vacuolate cells (Esau, 1943). This dramatic variation makes

proper fixation of the sample difficult. The leaves also tend

to trap air pockets between each other and the meristem,

increasing the difficulty involved in fixation and infiltration.

We achieved better or equivalent results using the micro-

wave protocol than when using the traditional paraffin-

processing protocol.

Generation of Materials

We used the shoot apical meristem and 6 surrounding

leaves of the Zea mays (Pioneer Hybrid International, USA)

inbred line B73 for all paraffin processing discussed in this

report. For each experiment, 40 kernels of B73 were planted

in genetics mix soil (50% sand, 50% peat), and a 0.5-inch

layer of vermiculite was placed on top of the soil. The plants

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

meristem, and 2 m m of the stem. The resulting sample tissue was approximately 7 x 4 x 3 m m in size. The tissues

were immediately placed into 20-ml glass vials of fucative on

ice (10 samples per vial) until all tissues were dissected.

Twenty samples were processed using the traditional pro-

tocol and 20 samples were processed using the microwave

protocol. This procedure was performed twice for anatomi-

cal studies and twice for in situ localization experiments.

Traditional Tissue Processing for Anatomical Studies

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.

Table 1. Traditional Protocol for Paraffin-Embedding

Microtechnique

Temperature Time

Step ("(3 (hr)

1. Fixation in FAA 4 24

2. 5096 EtOH:40% H,O:lOO/o TBA 24 4

3. 50% EtOH:30% H,O:20% TBA 24 20

4. 50% EtOH:10% H,O:40% TBA 24 4

5. 50% EtOH:50% TBA 24 20

6. 25% EtOH:75% TBA

with 0.05% wlv safranin 0 24 1

7. 25% EtOH:75% TBA 24 4

8. 100% TBA 58 4

9. 100% TBA 58 16

10. Add ?/i volume molten paraffin 58 24

11. Remove ?h volume, replace with

molten paraffin, repeat 2x 58 4

12. Remove '15 volume, replace with

molten paraffin 58 16

13. Fresh molten paraffin,

repeat 3x 58 8

FAA: 50% ethaliol, 10% formalin, 5% acetic acid; TRA, tert-buy1 alcohol; wlv, weight per volume.

Traditional Tissue Processing for In Situ Localization Studies

Samples for in situ localization were fixed in PFA (4% para-

formaldehyde in phosphate-buffered saline) overnight (16 hr) at 4°C. Samples were processed through the dehydration

and infiltration series shown in Table 2. We used TBA as an

intermediate solvent, and Paraplast Extra for infiltration

and embedding. The complete protocol, from futation to

embedding, required 7 days.

Microwave Tissue Processing

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-

technique and In Situ Localization

Step

1. Fixation in PFA

2. 0.85% NaCl

3. 50Y0 EtOH, 0.85% NaCl

4. 70% EtOH, 0.85% NaCl

5. 85% EtOH, 0.85% NaCl

6. 95% EtOH

7. 100% EtOH

8. 100% EtOH

9. 25% TBA:75% EtOH

10. 50% TBA:50% EtOH

11. 75% TBA:25% EtOH

12. 100% TBA, repeat 2x

13. TBA with 2 paraffin chips

11. Increase temperature

15. Remove l/2 volume, replace

with molten paraffin

16. Fresh molten paraffin,

repeat 5x

Temperature Time

("C) (hr) -.

4 24

4 1.4

4 1.4

4 1.4

4 1.4

4 1.4

4 1.5

4 15.5

27 0.75

2 7 0.75

27 0.75

40 0.75

40 15.5

58 4

PFA, 4% paraformaldehyde in phosphate-buffered saline.

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

Table 3. Microwave Protocol for Paraffin-Embedding Micro-

technique and In Situ Localization

Temperature Time

Step ("C) (min)

1. Fixative, repeat 2x 3 7 15

2. 0.85% NaCl for in situ only 67 1.25

3. 50% EtOH 67 1.25

4. 70% EtOH 67 1.25

5. 70% EtOH with 0.05%

wiv safranin 0 67 1.25

6. 100% EtOH, repeat l x 67 1.25

7. 50% EtOH:50% isopropanol 77 1.5

8. 100% isopropanol 77 1.5

9. 50% isopropanol:50% molten

paraffin 77 10

10. Molten paraffin 67 10

11. Molten paraffin, repeat 4x 67 30

in the static water load was changed between every step in

the protocol.

In Situ Localization of knotted mRNA

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

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.

':%: Ti\, I;'

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hlicro~vave Parafin Protocols 495

Figure 1. Comparison of microwave- and traditionally processed the staining dish will cause it to explode). The temperature Zea mays shoot apical meristems and the surrounding developing probe was inserted into the staining jar through the plastic leaves in longitudinal section. A: Microwave-processed tissue ac- ~h~ slides were stained for 45 min in the microwave cording to the protocol in Table 3 for anatomical studies. Inset at 60°C. we followed ~ ~ h ~ ~ ~ ~ ~ ' ~ method for the shows a close-up of the meristematic region. These sections were of the staining series. The slides were mounted in Merko- fixed to the slide and stained using the microwave procedure de-

glas. Controls were stained for 45 min at 60°C, but were scribed in Materials and Methods. B: Traditionally processed tis-

exposed to no microwaves. All microwave-processed tissue sue according to the protocol in Table 1 for anatomical studies.

was stained using the microwave staining protocol (45 min Inset shows a close-up of the meristematic region. These sections

were fixed to the slide and stained following the protocols outlined in safranin 01, whereas all traditional staining (48 hr ill

in Ruzin (1999). C: Microwave-processed in situ experiment fol- safranin 0 ) was carried out according to oha an sen's method lowing the protocol outlined in Table 3. D: Traditionally processed (Johansen, 1940).

in situ experiment following the protocol outlined in Table 2.

Bars = 50 pm. + RESULTS A 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

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. lC ,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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