Hybridization to mRNA of Tissue Sections

METHODS 23, 325–334 (2001)doi:10.1006/meth.2000.1144, available online at http://www.idealibrary.com on

In Situ Hybridization to mRNA of ArabidopsisTissue Sections

Janice de Almeida Engler,* Ruth De Groodt,* Marc Van Montagu,*,1

and Gilbert Engler†

*Vakgroep Moleculaire Genetica, Departement Plantengenetica, Vlaams Interuniversitair Instituut voorBiotechnologie, Universiteit Gent, B-9000 Gent, Belgium; and †Laboratoire Associe de l’InstitutNational de la Recherche Agronomique (France), Universiteit Gent, B-9000 Gent, Belgium

In situ hybridization detection of mRNA is an essential tool for under-standing regulation of gene expression in cells and tissues of differentorganisms. Over the years, numerous in situ protocols have been devel-oped ranging from whole-mount techniques that allow fast transcriptlocalization in intact organs to high-resolution methods based on theelectron microscopic detection of mRNAs at the subcellular level. Here,we present a detailed protocol for the detection of mRNAs in planttissues using radiolabeled single-stranded RNA probes. Hybridizationsare carried out on tissue sections of paraffin- and plastic-embeddedplant tissues. Although this in situ protocol is appropriate for planttissues in general, it has been optimized for Arabidopsis thaliana.Variations on the procedure, required to obtain optimal results withdifferent Arabidopsis tissues, are described. q 2001 Academic Press

Messenger RNA in situ hybridization (ISH) is apowerful technique that can be used for analysis oftemporal and spatial patterns of gene expression intissues from a great variety of organisms, includingplants (1–3). Many aspects of an in situ procedurehave an empirical basis and, therefore, result in anarray of different protocols. However, most protocolsinclude the same basic steps: fixation of the tissue,preparation of tissue sections, pretreatment of thetissue, probe synthesis, hybridization, washing, anddetection of the signal. During the course of our in

1 To whom correspondence should be addressed at VakgroepMoleculaire Genetica, Universiteit Gent, K.L. Ledeganckstraat35, B-9000 Gent, Belgium. Fax: 32 9 2645349. E-mail: [email protected].

1046-2023/01 $35.00Copyright q 2001 by Academic PressAll rights of reproduction in any form reserved.

situ work, we have found that fixation conditionscan strongly influence the sensitivity of an in situexperiment, especially in Arabidopsis tissues. Also,DNA amplified by polymerase chain reaction (PCR)proved to be a much more efficient template for probesynthesis. Minor variations in other basic steps ofthe protocol did not significantly affect the outcomeof the in situ results. Aspects of ISH procedures thatare not directly related to Arabidopsis thaliana arenot discussed.

Recently, whole-mount in situ hybridization(WISH) techniques have been developed for animaltissues, mainly to study the three-dimensional distri-bution of transcripts within small organs such as anembryo (4, 5). Although whole-mount procedures arenot presented here, it is worth mentioning that animproved WISH procedure for plants, adapted in ourlaboratory, proved to be very valuable for the morerapid detection of gene expression patterns in Arabi-dopsis and other plantlets (6). The rationale of thisprocedure is that not only a large number of samplescan be processed, but also an intact root system canbe analyzed. Although most WISH procedures onlyuse nonradioactive probes, the resolution remainsinferior when compared with sectioned material. Thedevelopment of a WISH protocol based on fluores-cence-marked probes combined with a confocal mi-croscopic analysis of the data should significantlyincrease the resolution of a whole-mount method (7).

Here, we provide protocols that have proven usefulfor detection of transcripts in various tissues of Ara-bidopsis. The RNA ISH techniques described make

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DE ALMEIDA ENGLER ET AL.326

use of sectioned material. The main reason to per-form ISHs on sectioned material is because it pro-vides more detail on the tissue structure and on thecell type specificity of gene expression. In addition,ISHs on tissue sections always complement data ob-tained by WISH procedures.

Both radioactive (8–11) and nonradioactive meth-ods (2, 12, 13) have been used for ISH of Arabidopsis.We, however, believe that the reliability and use-fulness of the nonradioactive method strongly de-pend on the type of tissue to be analyzed. It is gener-ally accepted that nonradioactive methods are safe,fast, and offer the possibility of obtaining increasedcellular resolution. Indeed, several papers presentbeautiful nonradioactive in situ results in Arabi-dopsis tissues, but the target organs usually consistof small and poorly vacuolated cells as encounteredin meristems, primordia, and embryonic cells (2, 12,13). However, we find that nonradioactive methodsare not as sensitive as the more traditional radioac-tive technique. The enormous decrease in transcriptdensity in more voluminous cells may also be respon-sible for negative in situ results when using the non-radioactive method. In our experience, when lessabundant mRNAs have to be localized in a heteroge-nous cellular background, the radioactive procedureremains the method of choice.

A protocol for preparation of nonradioactiveMRNA in situ hybridization is presented in the arti-cle by Abraham in this issue (14).

Although the protocol presented here is optimizedfor Arabidopsis, it is also applicable to most otherplant species. However, no universal ISH protocolexists that meets all the standards for resolution andsensitivity for all biological specimens. Therefore, theparticular protocol will need to be adapted to theneeds of the individual experimenter.

REAGENTS AND STOCK SOLUTIONS

Note: Prepare all solutions necessary for in situhybridization, up to the RNase treatment step, withdouble-distilled water (ddH2O), add 0.1% diethyl pyr-ocarbonate (DEPC), and mix vigorously. Autoclavesolutions the next day. During all steps of the in situprocedure gloves should be used until RNase treat-ment.

Slide Preparation

Poly(L-lysine) (100 mg/ml) in 10 mM tris(hydroxy-methyl)aminomethane (Tris), pH 8.0 (No. P-1399;Sigma, St. Louis, MO); Vectabond (SP-1800; Vector,Burlingame, CA, USA); or 3-aminopropyltriethoxy-silane (Sigma).

Fixation of Plant Material for Paraffin orMethacrylate Embedding

Six-well tissue culture plate (Falcon 3046).Cell strainers (Falcon V5) or tubes (Eppendorf).10, 30, 50, 70, 85, 90 and 100% ethanol series di-

luted in DEPC–water. Note: For methacrylate em-bedding, add 1 mM dithiothreitol (DTT) to ethanoldilutions ranging from 10 to 95% and 10 mM DTTto 100% ethanol.

0.2 M Cacodylate buffer (23 stock solution) inDEPC–water (for paraffin embedding); adjust pH to7.2 (may be aliquoted and stored in the freezer); or 0.5M piperazine-N,N8-bis(ethanesulfonic acid) (Pipes)buffer (103 stock solution) in DEPC–water (formethacrylate embedding); adjust pH to 6.9 (may bealiquoted and stored in the freezer).

10% Paraformaldehyde: Dissolve 10 g of paraform-aldehyde in 50 ml of 0.2 M cacodylate buffer (or inPipes buffer for methacrylate embedding) with stir-ring and heating to 608C. Cool to room temperature,adjust to pH 7.2 with H2SO4, and adjust the volumeto 100 ml with DEPC–water. Make this solutionfresh before each use.

Fixatives: 2.5% glutaraldehyde (1 to 3% glutaral-dehyde can be used) in 0.05 to 0.1 M cacodylate buffer(pH 7.2) prepared in DEPC–water; formaldehyde 1glutaraldehyde: 4% formaldehyde, 1% glutaralde-hyde (0.25% is better for some tissues) in 0.05 to 0.1M cacodylate buffer (pH 7.2) prepared in DEPC–water; or FAA: 50% ethanol, 5% acetic acid, 4% form-aldehyde in DEPC–water.

Embedding and Sectioning

Paraplast chips.Methyl methacrylate.Butyl methacrylate.Benzoin ethyl ether.

Probe Preparation, Hybridization, and Washing

103 Transcription buffer: 400 mM Tris-HCl, pH7.5, 60 mM MgCl2, 100 mM DTT, 50 mM NaCl, 20mM spermidine hydrochloride.

Arabidopsis mRNA IN SITU HYBRIDIZATION 327

10, 30, 50, 70, 85, 90 and 100% ethanol series di-luted in DEPC–water

103 Phosphate-buffered saline (PBS): 1.4 M NaCl,0.02 M KCl, 0.1 M Na2HPO4, 0.02 M KH2PO4 (pH7.4).

2% Glycine in 103 PBS.Proteinase K buffer: 100 mM Tris (pH 7.5) and 50

mM EDTA in DEPC–water.Proteinase K: 1 mg/ml in proteinase K buffer;

aliquot the solution, preincubate for 1 h at 378C(to digest potential RNases) and keep at 2208C.

0.1 M Triethanolamine (TEA) in DEPC–water, pH8.0 (adjusted with HCl and freshly prepared).

53 NTE: 2.5 M NaCl, 50 mM Tris, 5 mM EDTA,pH 7.5.

203 SSC: 3.0 M NaCl, 0.3 M sodium citrate, pH 7.0.

Equipment

In addition to standard equipment, it is also neces-sary to prepare humidified boxes for use during thehybridization reaction to the sectioned material. Adescription of suitable boxes is contained in the arti-cle by Butler et al. (15).

METHODS

A. Slide Preparation

1. Place clean slides in slide racks and place theracks in a large glass tray. Wash thoroughly in hottap water (608C) containing detergent for 2 h or over-night at room temperature. Rinse with hot ddH2Oand then air-dry.

2. For aminopropyltriethoxysilane-coated slides,incubate the slides for at least 2 h at 1808C to elimi-nate RNases. Wash with 45% ethanol containing 1%acetic acid and air-dry. Incubate slides for 5 to 10min in 2% aminopropyltriethoxysilane in acetoneand then rinse with 100% acetone. Rinse the slideswith 10% acetone and finally in DEPC-treated water.Air-dry the slides and keep them at room tempera-ture. Note: It is best to use slides within 3 months.

3. For poly(L-lysine) and Vectabond-coated slides,place the slides in 0.8% HCl in water overnight. Rinsethree times with ddH2O and place in acetone for 60min. Air-dry the slides and incubate for at least 2 h at1808C to eliminate RNases. The slides can be coatedusing either of following procedures. (A) Immerse theslides in 100 mg/ml poly(L-lysine)/10 mM Tris and

incubate at room temperature for 5 to 10 min. Re-move from poly(L-lysine) solution and air-dry. (B) Im-merse the slides for 5 min in Vectabond diluted inacetone. Rinse the slides by dipping them severaltimes into DEPC-treated water and air-dry. Note: Itis best to use the slides within 3 months.

B. Fixing, Embedding, and Sectioning Plant Material

1. Fixation of plant material for the paraffin-em-bedding method. Cut the tissue into small pieces witha razor blade in a Petri dish containing fixative (glu-taraldehyde, glutaraldehye plus formaldehyde, orFAA). Note: To allow proper fixation, one of the di-mensions should not exceed 1 mm (e.g., 1 3 2 3 10mm). Transfer the tissue samples to plastic tubes orglass vials containing fresh fixative. Apply a vacuumto the plant tissue samples for 10 to 30 min to degas,which improves infiltration of fixative. Then replacewith fresh fixative and incubate overnight at 48Cwith gentle shaking. If the samples are fragile, trans-fer them to cell strainers in six-well tissue cultureplates to avoid damage during handling. Otherwise,proceed in the plastic tubes. Rinse the fixed tissue,with gentle shaking, in 0.1 M cacodylate buffer atroom temperature twice for 30 min or longer at 48C.Next, dehydrate the fixed samples in an ethanol se-ries from 10 to 100% at room temperature, allowingat least 30 min in each of the ethanol dilutions. (Note:If using FAA-fixed tissue rinse in 50% ethanol andthen proceed with the dehydration series.) As anoption the 95% ethanol step can include 0.1% eosin(stains pink) for better tissue visualization. Finally,repeat the 100% ethanol incubation twice for 30 minto ensure complete dehydration.

2. Embedding plant material in paraffin. (Note:After fixation and dehydration, the samples must betransferred to glass vials because clearing agentsdissolve plastic.) Replace the 100% ethanol with 25%xylene (or Histoclear or toluene) and incubate atroom temperature for 30 min with shaking. Replacewith 50% xylene:50% ethanol, then 75% xylene:25%ethanol, allowing at least 30 min in each solution.Replace the last solution with 100% xylene and incu-bate at room temperature for 60 min, with shaking.Repeat the 100% xylene incubation 2 3 60 min, tocompletely remove ethanol. Slowly add Paraplastchips (a few chips per hour) to the vial containingthe plant tissue in xylene until about half-full andthen shake gently overnight at room temperature.After the Paraplast is partially dissolved, place theglass vials in a 608C oven for a few hours and then


replace the solution with freshly molten Paraplast.Replace with fresh molten Paraplast twice a day forat least 3 days to obtain perfect infiltration. (Note:Arabidopsis tissue usually takes 4 days for optimalinfiltration.) For the final embedding, put the embed-ding molds containing molten Paraplast on a hotplate (608C); place and orient the sample withwarmed tweezers. Finally, float the embedding moldson ice water and let the Paraplast solidify. Labeleach paraffin block. (Note: The paraffin blocks canbe stored at 48C or at room temperature for sev-eral years.)

3. Sectioning paraffin-embedded plant material.Mount the paraffin-embedded tissue onto woodenblocks and shape the edges with a razor blade tocreate a pyramid. The tip of the pyramid should con-tain about 2 mm of paraffin around the embeddedsample to facilitate sectioning and to fit as manysections in one slide as possible. Cut 10-mm ribbonsusing a metal knife on a microtome and store theribbons on paper or aluminum foil at 48C. Now, placethe ribbons on DEPC–water-flooded slides (coated)with the shiny side of the ribbon toward the watersurface. Remove the excess water and incubate over-night at 428C. (Note: Maintaining the slides at 428Cis essential for optimal tissue adhesion to the slide.)Store the slides in a dry place.

4. Fixation of plant material for methacrylate em-bedding method. (This procedure should be per-formed on ice or at 48C.) The first steps of this proce-dure are essentially identical to those described forparaffin embedding, except that the fixative shouldcontain 1 mM DTT. After complete fixation, rinse thefixed tissue in 0.05 M Pipes buffer, 2 3 30 min atroom temperature or longer at 48C, with shaking.Dehydrate fixed samples gradually in an ethanol se-ries from 10 to 95% containing 1 mM DTT and 100%ethanol containing 10 mM DTT. For Arabidopsis tis-sues 30 min in each ethanol dilution is sufficient.Repeat the incubation with 100% ethanol 2 3 30 minto ensure complete dehydration.

5. Embedding plant material in methacrylate.After dehydration, pass samples through a 25, 50,75% ethanol–methacrylate series (4:1 butylmetha-crylate:methylmethacrylate) containing 1 mM DTT,with occasional gentle agitation. Repeat with fourchanges of 100% methacrylate containing 10 mMDTT with gentle agitation. Replace with fresh em-bedding medium containing 0.5% benzoin ethylether. It may help to bubble nitrogen through themedium to displace dissolved oxygen. Orient the

samples in embedding capsules containing the me-dium and allow to polymerize for 18 h at 48C underUV light.

6. Sectioning methacrylate-embedded plant mate-rial. Remove the polymerized sample from the cap-sule and cut 5-mm sections (or thicker) with a glassknife on a microtome. Place as many methacrylatesections as possible on drops of DEPC–water oncoated slides and dry on a hot plate at 608C. Incubatethe slides overnight at 428C. Store in a dry placeuntil use.

C. Synthesis of Radioactive 35S-Labeled Probe

1. DNA template may be prepared either by lin-earization of a plasmid using standard techniques,or by PCR amplification of the DNA of interest con-taining appropriate phage RNA polymerase promot-ers. The latter method usually gives better probes.In general we prepare both an antisense probe anda sense probe, to serve as a control. To ensure efficientprobe synthesis, the transcription reaction should beperformed at a final UTP concentration of at least10 mM. Therefore, depending on the amount of probeto be prepared, dry down an appropriate volume of[35S]UTP (between 10 and 100 ml of 1000 Ci/mmol,10 mCi/ml), that will result in a final [35S]UTP con-centration between 10 and 20 mM in the reactionmix. For a 20-ml reaction volume, resuspend the[35S]UTP at room temperature in the following tran-scription mixture:

Dried [35S]UTP103 transcription buffer 2 ml10 mM DTT 2 mlRNase inhibitor (25 U/ml) 1 mlATP, GTP, CTP mix (2.5 mM each) 4 mlUnlabeled UTP (100 nM) 1.6 mlLinearized plasmid DNA (1 mg/ml) 1 ml

or PCR DNA (0.5 mg/ml)Transcription enzyme (10 U/ml) 1 mlDEPC–water 7.4 ml

Final volume 20 ml

2. Incubate the reaction mixture for 2 h at 378C.For shorter transcripts (, 500 nucleotides) longerincubation times (e.g., overnight) will increase theyield.

3. Add 2 ml of DNase I and incubate for 15 minat 378C to digest the DNA template.

4. Probe hydrolysis (if desired; see Note 8). Theobjective is to hydrolyze the probe down to fragments


approximately 150 to 300 bp long. The hydrolysistime in minutes can be estimated using the equationt 5 (Lo 2 Lf)/(K )(Lo)(Lf), where Lo is the startinglength (kb), Lf is the final length (kb), and K is 0.11kb21 min 21. Hydrolyze the RNA by mixing 50 ml ofRNA in DEPC-treated water with 30 ml 0.2 MNa2CO3 and 20 ml 0.2 M NaHCO3 at 608C for thecalculated time. Stop the hydrolysis by adding 5 ml10% acetic acid. The size of the hydrolyzed fragmentscan be determined by standard formaldehyde gelelectrophoresis followed by detection of the labeledfragments.

5. The efficiency of label incorporation can be esti-mated as follows. Take a 1-ml sample of the hy-drolyzed reaction mix (i.e., 1/100th total volume) be-fore probe purification and determine the totalnumber of counts.

6. Remove unincorporated label with a spin col-umn or a gravity flow column or by ethanol precipita-tion. For ethanol precipitation, add 0.13 vol of 3 Msodium acetate (pH 6.0), 5 mg of carrier tRNA, and2.5 vol of ethanol.

7. After probe purification, determine the totalvolume and then determine the number of counts in1/100th of the total volume. The percentage incorpo-ration is simply (total counts incorporated)/(totalcounts in the reaction mix) 3 100.

D. Removal of Embedding Medium

For paraffin-embedded sections, insert the slidesinto slide racks and then place into staining dishescontaining Histoclear (or toluene or xylene) to re-move the paraffin. Stir very slowly for 15 min andreplace with fresh Histoclear, stirring for another 15min. Remove slides from Histoclear and rinse in twochanges of 100% ethanol. Dip the slides up and downuntil any visible “streaks” disappear. For methacry-late-embedded sections, place the slides into acetoneand stir gently for 20 to 30 min. Remove the slidesfrom the acetone and rinse twice in two changesof 100% ethanol. Dip up and down until any“streaks” disappear.

E. Hybridization and Washing

1. Treatments prior to hybridization. Hydrate thesections gradually in 90, 85, 70, 50, and 30% ethanoland finally in PBS for at least 1 min in each solution.Check one slide (mounted in water with a coverslip)under the microscope to confirm that the embedding

medium has been properly dissolved. Equilibrate theslides in proteinase K buffer for a few minutes at378C and then digest in 1–2 mg/ml proteinase K for30 min at 378C. Stop digestion by incubating theslides with 0.2% glycine in PBS for 5 min at roomtemperature and rinse in PBS. Equilibrate slides in0.1 M TEA at room temperature and then incubatewith TEA containing 0.25% acetic anhydride for 10min at room temperature. Wash slides twice in PBS.Dehydrate gradually by going through the ethanoldilution series and then dry the slides (under vacuumif desired).

2. Hybridization. Prepare the following hybridiza-tion solution allowing 100 ml per slide. It is advisableto make up a slightly larger volume than neededto allow for losses during handling due to its highviscosity. For every 1 ml of hybridization solution,assemble the following:

50% Formamide 0.5 ml300 mM NaCl 60 ml of 5 M stock10 mM Tris (pH 7.5) 10 ml of 1 M stock1 mM EDTA 2 ml of 0.5 M

stock25 units/ml RNase inhibitor 1 ml of 25 U/ml

stock13 Denhardt’s solution 20 ml of 503

stock10% Dextran sulfate 200 ml of 50%

stock70 mM DTT 70 ml of 1 M stock150 mg/ml tRNA (denatured 15 ml of 10 mg/

at 858C for 5 min) ml stock500 mg/ml poly(A) (denatured 50 ml of 10 mg/

at 858C for 5 min) ml stock

In the remaining 72 ml volume, include 5–300 ng/ml/kb probe (denatured at 858C for 5 min and quicklycooled just before use). In practice, use about two-thirds of the slides to hybridize with antisense RNAprobe and one-third for the sense or control RNAprobe. Add prewarmed (428C) hybridization solutionto the slides and spread evenly with the help of abaked coverslip. Make sure not to touch the sections.Cover the slide with a coverslip, avoiding the forma-tion of bubbles. Place the slides in a humidified box(place paper towels soaked in 50% formamide and50% PBS on the bottom of the boxes) and hybridizeovernight at 428C.

3. Posthybridization treatments and washing.After hybridization, remove the coverslips by dippingthe slides for 5–10 min in 43 SSC containing 10 mMDTT. The coverslips must slide off on their own. Do


not be tempted to pull them off because this willdamage the sections. After the coverslips are re-moved, dip the slides up and down a few times infresh 43 SSC containing 10 mM DTT. Equilibratethe slides at 378C in prewarmed NTE buffer. Placethe slides into fresh NTE containing 25 mg/ml RNaseA and incubate at 378C for 30 min with occasionalgentle mixing. Wash slides with NTE containing 10mM DTT for 30 min at 378C. Repeat the final NTEwash four times at room temperature.

To wash the sections, place the slides in a largeglass container or beaker containing 23 SSC and10 mM DTT. Wash the slides for 60 min at roomtemperature with gentle stirring. To increase thestringency of the washing, place the slides in 0.13SSC, 50% formamide, and 10 mM DTT. Wash for 60min (paraffin-embedded sections) or 10 min (methac-rylate-embedded sections) at 42 to 478C (accordingto desired stringency) with gentle agitation. Finally,rinse the slides in 0.13 SSC containing 10 mM DTTwithout formamide at room temperature with gen-tle agitation.

F. Probe Detection by Autoradiography

Dehydrate the tissue gradually through the etha-nol series (30, 50, 70, 85, 90, and 100%) and thenair-dry, using a vacuum if desired. Dried slides maybe stored for a few days prior to autoradiography ifnecessary. Heat the photographic emulsion (KodakNTB-2 or Ilford K2) to 428C and dilute 1/1 with 0.6 Mammonium acetate prewarmed at 428C, under safetylight (Ilford 904 safety filter). Aliquot the dilutedemulsion into 50-ml plastic tubes, wrap in severallayers of aluminum foil, and store at 48C. Dip theslides gently into one of the aliquots to produce aneven layer of emulsion, free from bubbles. Dry thedipped slides in plastic-coated test tube racks in avertical position in a humidified environment (e.g.,box containing wet paper towels) for at least 60 min.Remove the humidifying source and replace with sil-ica gel, and let the slides dry for at least 60 min.Place the slides into dark slide boxes with desiccant,wrap in aluminum foil, and expose at 48C for theappropriate time.

After exposure, develop, fix, and wash the slidesaccording to the manufacturer’s recommendations.After the final wash, dip the slides into filtered 0.05%toluidine blue solution for approximately 30 to 60 s.This will stain the emulsion and tissue deeply butnot strongly. Rinse four times in changes of distilledwater. Dehydrate the slides by dipping 5 to 10 times

in each of the following ethanol solutions: 30, 50, 70,85, and 100% (twice); then rinse twice in xylene (ortoluene) to remove ethanol. Briefly drain off the ex-cess xylene (do not let slides dry) and mount slideswith DePex and a coverslip. Allow the mounted slidesto dry and then carefully scrape the dried emulsionfrom the back of the slide with a clean razor blade.The location of the silver grains can now be visualizedand photographed using brightfield or darkfield mi-croscopy techniques. Digital images presented in Fig.1 were generated by a Zeiss CLSM confocal micro-scope by overlaying three RGB nonconfocal images.

DISCUSSION OF THE METHOD

Fixation of Plant Material

The role of a fixation step in an ISH protocol is topreserve tissue morphology and to retain mRNA inthe cytoplasm of the cells. The final fixation proce-dure is generally a compromise between the methodthat gives optimal morphology and RNA retentionbut allows probe to penetrate into the tissue to hy-bridize with target mRNAs. Since Arabidopsis thali-ana seedlings are very fragile, fixative combinationswere tested to obtain acceptable morphology com-bined with good retention of the mRNA. In our hands,aldehyde fixatives gave better results than alcohol-based fixatives. For A. thaliana seedlings, optimaltissue preservation and signal detection were ob-tained using a straight glutaraldehyde fixative(2.5%) for most tissues, except flowers and siliques.However, a combination of glutaraldehyde (1%) withformaldehyde (4%) gave better results for flowersand siliques. Aldehyde fixatives penetrate the tissuerapidly, but the rate falls with time. Therefore, toguarantee complete fixative infiltration, fixationshould be performed at low temperature (4–108C)overnight. FAA-fixed tissues often require longer ex-posure times for signal detection.

Embedding Media

Once tissue is fixed, dehydrated, and cleared, par-affin is the most convenient embedding medium. Par-affin is easy to cut to different thicknesses, it can bestored for long periods, and the morphology is farbetter than with cryosections. However, superior res-olution can be obtained by embedding tissues in adissolvable plastic medium consisting of a mixtureof butyl methacrylate and methyl methacrylate (16).


Tissue morphology is far superior when plastic mediaare used, resulting in a more precise localization oftranscripts (17). A limitation of this method is thatsections of maximum approximately 5 mm can beproduced, requiring longer exposure times for sig-nal detection.

Probes for ISH

Three types of probes are commonly used for ISH:double-stranded DNAs, single-stranded antisenseRNAs, and synthetic DNA oligonucleotides. Double-stranded DNA probes can be prepared by nick trans-lation, random priming, or PCR with a labeled nucle-otide. They are denatured before use, but can par-tially reassociate during the ISH procedure, therebydecreasing the sensitivity. DNA oligonucleotides (ingeneral ,50 nucleotides) can be labeled by incorpo-rating the label during chemical synthesis or by add-ing a tail of labeled nucleotides. Oligonucleotidespenetrate tissue very readily, but hybrids are less

FIG. 1. Micrographs representing in situ hybridizations on sec-tions of different Arabidopsis thaliana organs. Red dots representhybridization signals (silver grains) on toluidine blue-stained tis-sues hybridized with radioactive probes (A–H). (A) Cross sectionof a root hybridized with a 35S-labeled Cdc2aAt antisense RNA.

Signal is observed in the vascular cylinder. (B) Longitudinal sec-tion of a root hybridized with the Arath;CycA2;1 antisense probe.The mRNA was localized in specific cells of the vascular cylinder,resulting in a patchy pattern. (C) Longitudinal section of a rootmeristem hybridized with Cks1At antisense 35S-labeled probe. Thetranscripts are distributed throughout the meristem except withincells of the quiescent center. (D) Longitudinal section of an embryohybridized with a 35S-labeled Cdc2aAt antisense RNA. Signal wasobserved throughout the embryo at the late torpedo stage. (E)Cross section of an anther hybridized with atgrp-7 antisense 35S-labeled probe. In this example the flower was embedded in adissolvable plastic medium that gives a higher resolution at thecellular level. (F) Longitudinal section of a flower hybridized withthe AGAMOUS antisense probe (17). At that stage transcript isstrongly expressed throughout the flower. (G) Cross sectionthrough a leaf hybridized with the Cdc2bAt antisense probe. Thehybridization signal was localized in leaf cells, resulting in apatchy pattern. (H) Shoot apical meristem hybridized with a 35S-labeled Arath;CycB1;1 antisense probe. The mRNA was localizedin cells of the shoot apex and tissues of young leaves. The patchysignal pattern suggests that these cells are mitotically active.Images were recorded using a confocal microscope (LSM 510)equipped with three visual lasers. By using the transmissionsetup, three nonconfocal transmission images were recorded foreach sample: one image with the blue laser line (488), anotherwith the green laser line (543), and the third with the red laser(633). The three single-channel images were overlaid on a truecolor multichannel image and electronically adjusted to create acorrect color balance. Samples that contain silver grains wereadditionally scanned in reflection mode using the 543 laser toimage silver particles. These images were overlaid on their corres-ponding three-color multichannel image. Images were croppedand adjusted using LSM 510 software, MS Photo Editor, andMS Word. Reproductions were generated using a LasergraphicsPersonal LFR plus (BLL) slide maker.


stable and the signal may be much weaker, de-pending on the probe size. The most commonly usedprobes are single-stranded RNAs synthesized usingan RNA polymerase (T7, T3, or SP6). Linearizedplasmids containing the DNA of interest and theRNA polymerase promoter sequences serve as goodtranscription templates, but PCR-amplified DNAfragments produce the best probes. The great stabil-ity of RNA/RNA hybrids allows high stringencyduring washes. Sense-strand probes can be used ascontrols to monitor nonspecific hybridization back-ground.

Several isotopes, including 32P, 3H, and 35S, havesuccessfully been used for radioactive ISH. The highenergy of 32P results in poor resolution, whereas thelow energy of tritium gives superior resolution butrequires very long exposure times. 33P and 35S offera compromise between 32P and 3H and provide goodresolution in a reasonable time. When using 35S-la-beled nucleotides, DTT must be added to all solutionsto avoid oxidation of the thiol group. Probes used fornonisotopic ISH generally contain haptenized nucle-otides. These probes are stable, sensitive, and safeand yield single-cell resolution. In general, enzyme-linked antibodies are more sensitive than fluores-cent-labeled ones. Alkaline phosphatase is the en-zyme of choice for plant material and the color reac-tion can proceed for extended periods with significantproblems of background. In general, the decision be-tween radioactive and nonradioactive ISH is a per-sonal choice. While a radioactive procedure often pro-vides greater sensitivity, single-cell resolution isbetter using nonradioactive probes.

Hybridization and Washing

During hybridization, labeled probe forms a duplexwith the target mRNA. To achieve maximum sensi-tivity, optimal conditions must be determined empiri-cally, depending on probe concentration, tissue typeand fixation, and abundance of the target RNA. Simi-larly, increasing the RNase concentration may im-prove signal detection and decrease the background.The suggested concentration of RNase is based onour experience with various Arabidopsis tissues.During washes, increasing stringency (higher wash-ing temperatures or reducing the salt concentration)can be introduced. Optimal stringency can be deter-mined empirically and will depend on the length ofthe probe and the degree of sequence homology be-tween probe and target RNA (18).

Autoradiography

Once slides are dipped in photographic emulsion,exposure times must be determined empirically. Ex-posure time may vary from a few days to approxi-mately 9 months, depending on the abundance of thetarget message. The diameter of a silver grain isapproximately 0.26 mm. We find that weaker signalsare better visualized by darkfield microscopy (visibleas white dots) and a strong signal is best analyzedusing brightfield optics (signal is seen as black dots).

NOTES ON THE METHOD

1. While all the described methods for coatingslides efficiently stick paraffin sections to the slidesurface, we find that aminopropyltriethoxysilaneholds tissue sections better during the complete ISHprocedure. We have also tried several “ready-to-use”coated slides and find that SuperFrost Plus (BDH,Poole, UK) gives good adhesion of tissue sections.

2. Some researchers suggest that strong fixationincreases background signal but we have not ob-served this to be a problem with Arabidopsis. Poorlyfixed tissues may cause sectioning difficulties, how-ever. This is especially true for fragile root meristemsand embryos within siliques. In the case of siliques,cutting their edges or making superficial perfora-tions with a needle helps fixative penetration.

3. Vacuum infiltration is also important for fixa-tive penetration in harder plant tissues. To preventcell collapse, the vacuum should be released veryslowly.

4. Arabidopsis tissues should not be dehydratedlonger than 30 min (usually 10 min is enough) be-cause tissues can become brittle. Manipulation ofsmall plant samples is difficult and the loss of plantmaterial can be prevented by placing them on a sieveand performing the fixation and dehydration stepsin six-well plates. After dehydration, samples mustbe transferred to glass vials because clearing agentsdissolve plastic.

5. It is very difficult to orient Arabidopsis roots toobtain optimal longitudinal sections of root meri-stems. We suggest fixing and embedding longitudi-nally placed clusters of roots (or other tissues) packedin lens paper so that paraffin ribbons will containthe majority of sections in longitudinal orientation.For flowers, we collect branches containing severalflowers at different developmental stages and orientthem with warmed needles.


6. We find that paraffin solidified in ice water hasa smoother texture and produces tissue sections ofhigher quality. After sectioning, store the slides at428C overnight to ensure that sections will not falloff the slide during the in situ procedure.

7. After calculating the incorporation efficiency ofa probe, we notice that certain sequences tend toincorporate labeled nucleotides less efficiently thanothers. In practice, probes with an incorporation effi-ciency as low as 15% have been used with success.

8. It is often recommended that RNA probeslonger than 500 bp should be hydrolyzed to allowbetter tissue penetration. Although the optimalprobe length is reported to be approximately 150 bp(19), we often do not observe significant differencesbetween intact and hydrolyzed probes.

9. If tissue sections become damaged during theprehybridization procedure, it may be useful to in-clude one or two postfixations steps with 4% formal-dehyde for 15 min after proteinase K treatment and/or after acetylation steps. This is especially relevantfor fragile tissues such as roots.

10. The half-life of acetic anhydride in water isless than 1 min. Never prepare it in advance.

11. For the hybridization step, we use closed plas-tic boxes containing paper towels moistened withPBS containing 50% formamide, and glass bars atthe edges to support the slides. This prevents thehybridization solution from drying out.

12. The ideal concentration of radioactive probefor obtaining good signals will depend on the localiza-tion and abundance of the transcript. Usually, 300ng/ml/kb of probe sequence complexity is applied inseveral protocols and should be more than enoughto saturate the target mRNA. We apply 15 3 106

cpm/slide (7.5 ng RNA probe/100 ml hybridizationsolution) or less for all tissues, except siliques (5 3106 cpm). Higher concentrations can give back-ground. Because methacrylate sections are thinnerthan paraffin ones and generate very low back-ground, we recommend hybridizing most of the slideswith higher counts (up to 50 3 106 cpm).

13. To determine the optimum temperature forthe high-stringency wash, individual slides can bewashed separately at progressively higher tempera-tures. This approach is particularly useful whenbackground problems are encountered with a partic-ular probe.

14. Do not overstain developed slides with tolu-idine blue because this will interfere with visualiza-tion of silver grains. We recommend filtration of thetoluidine blue stock solutions and then leaving them

exposed to daylight. Older solutions give a nicerstaining with various tones of blue because ofstain oxidation.

ACKNOWLEDGMENTS

The authors thank Dirk Inze for the cell cycle genes, MarcioAlves Ferreira for the slide containing the sections imaged in Fig.1E, Rosa Maria de Pinho Barroco for the slide imaged in Fig. 1Hand for critical reading of the manuscript, and Martine De Cockfor help preparing the manuscript. G.E. is a Research Engineerof the Laboratoire Associe de l’Institut National de la RechercheAgronomique (France).

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Hybridization to mRNA of Tissue Sections