In Situ Hybridization (2023)

IN SITU HYBRIDIZATION OF SINGLE-STRANDED RNAPROBES WITH ARABIDOPSIS FLORAL TISSUE

Gary Drews, Tom Jack, Detlef Weigel, Hajime Sakai , BobbyWilliams, Mark Running, Steve Jacobsen, and Beth Krizek.

Meyerowitz Lab

I. Fixation, Embedding, andSectioning

II. Probe Synthesis

III. Hybridization

IV. Wash

V. Autoradiography and Staining

VI. Viewing and Photographingslides

References

Cox et al. (1984) Detection of mRNAs in sea urchin embryos by in situhybridization using asymmetric RNA probes. Develop. Biol. 101, 485-502.

Cox and Goldberg (1988) Analysis of plant gene expression. In Plant MolecularBiology, A Practical Approach. C.H. Shaw, ed. (Oxford: IRL Press), pp 1-34.

Fink (1987) Some new methods for affixing sections to glass slides. I.Aqueous adhesives. Stain Technol. 62, 27-33.

Hayashi et al. (1978) Acetylation of chromosome squashes on Drosophilamelanogaster decreases the background in autoradiographs from hybridizationwith [125I]-labeled RNA. J. Histochem. Cytochem. 26, 677-679.

Huang et al. (1983) Improved section adhesion for immunocytochemistryusing high molecular weight polymers of L-lysine as a slide coating. Histochemistry77, 275-279.

McFadden et al. (1988) A simple fixation and embedding method for usein hybridization histochemistry on plant tissues. Histochem J. 20, 575-586.

Meyerowitz (1987) In situ hybridization to RNA in plant tissue. PlantMolec. Biol. Rep. 5, 242-250

Moench et al. (1985) Efficiency of in situ hybridization as a functionof probe size and fixation technique. J Virol. Methods 11, 119-130.

I. FIXATION,EMBEDDING, AND SECTIONING

A. FAA Fixation:

Materials:

Use bulk 95% or 100% ethanol except for the 100% ethanol steps.

For the 100% ethanol steps, use high quality ethanol (e.g. Gold Seal)

Glacial acetic acid

37% formaldehyde

20 ml glass scintillation vials (e.g. S/P Baxter #R2550-14)

1. Fixative Mix:

% In Mixture Amount

Ethanol 50.0% 50 ml

Acetic Acid 5.0% 5 ml

Formaldehyde 3.7% 10 ml of 37%

Water 41.3% 35 ml

100 ml

2. Place 10-15 ml of fixative into 20 ml scintillation vials.

3. Cut a cluster of flowers at the apex of the floral stem that includesstages 1-14, and immediately immerse in fixative. Cut so that 5 mm or soof floral stem is present--this helps to orient the tissue when sectioning.Cut off older flowers if you are not interested in these stages. Place about20 pieces of tissue in each vial.

4. The tissue will float in the fixative.

5. Place tissue/fixative in a vacuum (with an aspirator) for 15 minutes.Pull the vacuum very slowly. This step pulls the air out of the tissue,allowing the fixative to penetrate better. Usually, air bubbles will comeout of the solution and get trapped in the trichomes, causing the tissueto float.

6. After 15 minutes of slow bubbling, release the vacuum very slowly.Incubate at room temperature, up to a total of 1-2 hours exposure to thefixative, one hour is best for antibody in situ experiments and works finefor RNA in situ experiments.

7. Remove fixative and add 50% ethanol. Incubate at room temperaturefor 30 minutes. Repeat this step.

B. Dehydration:

The tissue must be completely dehydrated when it hits the xylenes; otherwise,the water and xylenes will form a white emulsion. We also stain the tissuewith Eosin Y during these steps to help orient the tissue when sectioning.

Materials:

Use bulk 95% or 100% ethanol except for the 100% ethanol steps.

For the 100% ethanol steps, use high quality ethanol (e.g. Gold Seal)

Xylenes (Fisher)

Eosin Y; Eosin Yellow (Manufacturing Chemists #B286 or Sigma)

1. Remove 50% ethanol and replace with 60% ethanol. Incubate for 30 minutes.

2. Repeat for the following ethanol solutions: 70%, 85%, 95%.

3. Leave overnight in 95% ethanol with 0.1% Eosin (Eosin should be storedwrapped in aluminum foil and should be discarded if no longer bright orange).

4. Next day, remove as much as possible of the Eosin/95% ethanol andreplace with 100% ethanol. Incubate for 1 hour. The tissue destains slowlyin 100% ethanol, so do not incubate too long.

5. Remove as much as possible of the 100% ethanol, replace with fresh100% ethanol, and incubate for 30 minutes. Repeat this step if it is impossibleto remove virtually all of the solution in steps 4 and 5.

C. Clearing:

The tissue must be permeated with xylenes because paraffin is not misciblein ethanol. The tissue does not destain (Eosin) in xylenes.

Materials:

High quality ethanol (e.g. Gold Seal)

Xylenes

1. Remove 100% ethanol and replace with 25% xylenes:75% ethanol. Incubateat room temperature for 30 minutes.

2. Repeat for the following xylene:ethanol solutions:

50% xylenes:50% ethanol

75% xylenes:25% ethanol

3. Remove the 75% xylenes and replace with 100% xylenes. Incubate atroom temperature for 1 hour.

4. Repeat step 3 two times. Fill the vial half full with xylenes thefinal time.

D. Infiltration:

Materials:

Paraplast Plus Chips.

Molten Paraplast Plus. Place a beaker full of paraplast chips in an oven at 60oC well in advance; it will take >12 hours to melt.

1. Add 20 chips of paraplast to each vial. Incubate overnight at roomtemperature.

2. Next morning, the paraplast will be only partially into solution.Place vials in a 42oC incubator. After about 30 minutes, the paraplast chipswill be in solution.

3. Add 20 more chips, and incubate at 42oC until chips are in solution--about30 minutes. Swirl occasionally.

4. Repeat step 3 until the vial is full (4-5 times; total of about 100chips).

5. Pour off xylene/paraplast solution. Add molten paraplast. Swirl tomix. Incubate at 59-62oC for at least 4 hours.

6. Repeat step 5 for a total of 6 changes over 3 days (i.e. change twicea day).

E. Pouring Boats:

Materials:

Molten paraplast plus.

Index cards (3x5).

Variable temperature hot plate (Eberbach, Corp., Ann Arbor, MI)

1. Fold up an index card so that it has raised edges of about 1 cm. Tapethe edges with Scotch tape outside.

2. Place the boat on the hottest part of the variable temperature hotplate.

3. Pour the infiltrated tissue into the boat.

4. Top off the boat with molten paraplast plus.

5. Arrange the tissue into a regular array. Orient tissue pieces withthe stems either straight up or lying on their side. Tissue pieces mustbe at least 5 mm apart.

6. Slowly move the boat to the cooler part of the hot plate. You probablywill need to arrange the tissue again.

7. Label each boat by inserting a flagged string.

8. Let harden completely. This will require overnight at room temperature.If in a hurry, you can float the boat for 2 hours (1 hour per side) in water.

F. Sectioning:

Materials:

Poly-L-lysine-coated slides (Fisher 12-550-15).

TissuePrep Flotation Bath (e.g. Lab-Line # 26103/Baxter # M7655) set to 42oC .

Slide warmer (e.g. Fisher # 12-594) set to 45-50oC.

VWR single edged razors

1. Cut out blocks of embedded tissue and mount onto microtome blocks.

2. Section tissue at 8 um. If the wax is good quality, 5-6 um sectionscan be made, which will give more tissue per sample.

3. Cut ribbons into 1.5 cm pieces. Float ribbon pieces on 42oC waterfor >1 minute. This step takes the compression out of the tissue.

4. Put slide in water just under the floating ribbon.

5. Bring slide up so as to catch the ribbon (don't put sections to closeto any edge of the slide). Use a wooden applicator to position the ribbon.Label the slide with a pencil (ink dissolves in the ethanol/xylene steps).

6. Repeat for as many different ribbon pieces as you want to place ona given slide, frame the sections so that they will fit under the 24 X 50mm cover slips.

7. Incubate slide on a slide warmer overnight to "bake" theribbon piece onto the slide. This is a very important step. This must bedone immediately, and the slide warmer must be set to >45oC (but not>50oC), or the sections may fall off during the hybridization and washsteps. Use the dust cover.

8. Assess quality of slides and choose the worst slides to be used astester slides for estimating exposure length. You may also want to use someof the intermediate quality slides for the sense RNA control probe.

9. After baking, slides can be stored in slide boxes at 4C.

II. PROBE SYNTHESIS

Requires DEPC-treated water and RNAse-free TE, 3M NaOAc, Ethanol, DTT,and tRNA. Also in vitro transcription kit, phenol, chloroform, and radiolabelledUTP.

A. The probes:

Generally two probes are synthesized. The first probe hybridizes withthe RNA of interest, and is generally called antisense, anti-mRNA, or (+)strand probes. You must also synthesize a control probe that will not hybridizewith the RNA of interest. The most convenient thing to do is to synthesizean RNA probe in the opposite orientation as the anti-RNA probe. Probes ofthis type are called sense, mRNA, or (-) strand probes. A second controlprobe that can be used is [3H]-poly(U). This control is used to describethe distribution of total poly(A) RNA in the tissue and to assess whetherthe RNA within a cell is accessible to the in situ hybridization probes.We purchased our [3H]-poly(U) from Amersham, but they no longer sell it.It can probably be purchased from another supplier.

B. Template preparation:

1. Digest 5 ug or more with the appropriate restriction enzyme to linearizethe template so that a "run-off" transcript can be generated.Avoid the use of enzymes that generate 3' overhangs--they can act as promotersfor the polymerases.

2. Phenol/chloroform extract twice, and chloroform extract twice withan equal volume. Use RNAse free eppendorf tubes.

3. Precipitate the DNA with 1/10 volume RNAse free 3M NaOAc and 2.5 volumesEtOH. Wash with RNAse free 70% EtOH, and resuspend in RNAse-free TE at about1 mg/ml. Assess digest extent and concentration of DNA on a gel.

C. Probe synthesis:

Materials:

Transcription kit. We use the Promega Transcription kit (P1460, with SP6 and T7 RNA polymerase), but other kits work equally well.

RNAse-free tRNA (We use BRL # 5401 or Boeringher Manheim #?)

RNAse-free DNase (We use Promega RQ1 DNAse, # M6101 (also available in Promega Transcription Kit))

[35S]-UTP: We purchase our label from Amersham . Generally the 650 Ci/mmole specific activity (catalog number SJ 263) is sufficient.

1. Dry down [35S]-UTP (25 ul of 650 Ci/mmol 250 mmol for a single reaction,50 ul for a double reaction). Resuspend in 20 ul RNase free H2O for a singlereaction, and 40 ul for a double reaction. A half reaction is sufficientfor many slides.

2. Set up transcription reaction:

Mix the following at room temperature for a single reaction:

H2O + [35S]-UTP 20 ul

5x Transcription Buffer 10 ul

100 mM DTT 5 ul

RNasin 2 ul

ATP, GTP, CTP mix (3 ul each, 2.5 mM each) 9 ul

linearized plasmid (1ug/ul) 1 ul

H2O 2 ul

T7 or SP6 polymerase 1 ul

50 ul

3. Incubate at 40°C for 1 hr. Take 1 ul sample for total counts.

4. Add 1 ul RNAse-free DNase per ug of template, and incubate at 37°Cfor 15 min.

From here there are two options:

-- The Beth/Steve approach: This method does not use a column for purification,but could result in higher background in some cases.

5B-S. Add up to 200 ul TE

2 ul 1M DTT

2ul 10mg/ml tRNA

phenol/chloroform extract

chloroform extract

1/10 vol 3M NaOAc, ph 5.2

2.5 vol Ethanol

15 min spin

70 % wash

dry

resuspend in 55 ul DEPC H2O

6B-S. Count 1ul in glass scintillation vial with 5 ml scintillation fluid,Program User 4. Make sure you have enough probe for each slide at 2-4 millioncounts/slide.

7B-S. Take off a sample of 107 cpm to run on a gel (pre-hydrolysis sample).If less than 107 cpm/ 3 ul, just take 3 ul and add to 2 ul of Sequenasestop buffer.

-- The Hajime/Bobby approach:

5H-B. Phenol/chloroform extract twice, chloroform extract once. Run throughG-50 columns (Boeringer Manheim) as instructed. Use one column for eachsingle reaction.

6H-B. Count 1ul in glass scintillation vial with 5 ml scintillation fluid,Program User 4. Make sure you have enough probe for each slide at 2-4 millioncounts/slide.

7H-B. Take off a sample of 107 cpm to run on a gel (pre-hydrolysis sample).If less than 107 cpm/ 3 ul, just take 3 ul and add to 2 ul of Sequenasestop buffer.

8H-B. Precipitate with 1/10 volume 100 mM DTT, 2ul tRNA, 1/10 volume3M NaOAC, and 2.5 volumes of Ethanol. Resuspend in 50 ul H20

D. Probe Hydrolysis:

We reduce the size of our probes to allow better penetration into thetissue. According to Moench et al. (1985), probes of mean length 70 nt givehigher signals than larger probes (150 nt and larger). We chemically degradeour probes to a mean length of 75-100 nt. The RNA probes are chemicallydegraded by incubation in alkali at 60oC. The length of time for incubationis determined by the formula below:

t = (Lo - Lf) / (K)(Lo)(Lf)

Lo = starting length (kb), including all parts of the vector that are transcribed

Lf = final length (kb) = .075-.1 kb

K = .11

Calculated time will be in minutes.

Sample calculation, 1 kb transcript to 100 nt mean size:

(1-.1)/(.11)(1)(.1) = 82 minutes.

1. Hydrolysis buffers:

(a) 200 mM Na2CO3 (Sodium Carbonate) (0.21g in 10 ml)

(b) 200 mM NaHCO3 (Sodium Bicarbonate) (0.17g in 10 ml)

NOTE: Make buffers fresh each time and do not autoclave.

2. Hydrolysis reaction:

(a) Mix the following:

50 ul RNA

30 ul 200 mM Na2CO3

20 ul 200 mM NaHCO3

(b) Incubate at 60oC for the calculated time.

3 ul 3 M Sodium Acetate (pH 6.0)

5 ul 10% Glacial Acetic Acid

3. Take off a sample of 107 cpm to run on a gel (post-hydrolysis sample).

4. Precipitate the probes:

Volume = 108 ul and already has 3 ul of 3 M Sodium Acetate.

Add: 1 ul 1M DTT

1 ul 10 mg/ml tRNA carrier

8 ul 3 M Sodium Acetate

250 ul Ethanol

4. Run pre- and post-hydrolysis samples on a denaturing sequencing gelto determine the size of the probe. Add 3ul to 2 ul of seq loading dye.Heat 75 2min, cool on ice, load 2.5 ul on gel. Just do short run. Seconddye runs at about 75 nt.

5. Store the probe as an ethanol precipitate until needed. After spinningdown the probe, resuspend it in 25-50 ul of TE + 10 mM DTT. Count 1 ul inScintillation fluid.

III. HYBRIDIZATION

Setting up RNAse-Free glassware

Two days before hybridization day, wash with soap and water a minimumof 28 staining dishes and lids (VWR 25445-009) for the first rack of 30slides you plan to do, and a minimum of 6 dishes for each additional rackof 30 slides. (Try to do extra, since dishes tend to break in certain steps).Also wash a minimum of 2 2L glass cylinders and 2 500ml glass cylinders,slide racks, slide rack holders. and one 1L flask for each set of 30 slides.Let dry several hours to overnight. The day before the experiment, bakethe glassware and racks in the Rothenberg oven for 5 hours. (The temperatureis already set.) Turn the oven off and let the glassware cool with the ovendoor closed for at least 8 hours.

A. Solutions (all are DEPC-treated or made with DEPC-treated water):

1. Prepare about 10L of DEPC-water for the first rack of slides, then3-4L for each additional rack.

2. Set up 10 staining dishes containing the following ethanol solutions:100%, 100%, 95%, 85%, 70%, 50%, 30%, 15%, H2O, H2O.

3. 1 M Tris (7.5) RNAse-free 500 ml stock:

Use unopened bottle of tris. Add 60.55 g to about 400 ml DEPC-H2O. Add about 65 ml conc. HCl; confirm with pH paper.

4. Proteinase K Solution:

Need 500 ml for each rack of slides. Prewarm to 37°C.

100 mM Tris (7.5)-- from 10x stock above

50 mM EDTA-- from 10X DEPC-EDTA

5. 2X SSC--500 ml for each rack of slides.

6. 10X PBS-- about 500ml 10X needed for each rack of slides

NaCl 76g

Na2HPO4.2H2O 12.46g (18.76g of Na2HPO4.7H2O)

NaH2PO4.2H2O 4.48 (4.14g of anhydrous NaH2PO4)

H2O to 1 Liter

7. 10x Hybridization Salts:

3 M NaCl

100 mM Tris (7.5)

10 mM EDTA (8)

8. 50% Dextran Sulphate. We use Dextran Sulphate from Pharmacia (# 17-0340-01).

9. 1M Dithiotheitol (DTT)

10. 10 mg/ml RNAse-free yeast tRNA We use tRNA from BRL (# 5401)

11. 50X Denhardt's Solution. We filter it just before use.

12. Other Reagents:

(a) Distilled Formamide (We use BRL # 15515)

(b) Triethanolamine (We use Sigma # T1377)

C. Remove paraplast from sections:

1. Put slides in a staining dish slide holder.

2. Fill a staining dish with xylenes, and place a small stir bar at thebottom. Place slides into this staining dish.

3. Stir for 10 minutes. Stir very slowly--just fast enough to mix thesolution a bit.

4. Repeat with fresh xylenes.

5. If you are processing many racks of slides, you can use the same twoxylenes solutions.

D. Remove xylene from slides:

1. Remove slides from xylenes and place into 100% ethanol (95% ethanolwill not work). Dip up and down about 15 times or until the "streaks"go away.

2. Repeat with the second 100% ethanol.

E. Hydration:

1. Process the slides through the following ethanol solutions: 95%, 85%,70%, 50%, 30%, 15%, H2O, H2O. Once again, dip up and down 15 times or untilthe "streaks" go away. Begin with the 95% ethanol and end withthe H2O, so as to hydrate the tissue gradually.

2. Change the last water for each rack of slides.

3. Save the ethanol solutions for late steps.

F. Denaturation

1. Incubate slides in 0.2N HCl for 20 minutes at room temperature (8.6ml conc. HCl in 500 ml H2O). This step denatures proteins and nicks DNA,but will also partially reverse the fixation step.

2. Incubate in 2X SSC, 70°C, for 15 minutes. NOTE: THIS IS A STEPWHERE DISHES ARE SUSCEPTIBLE TO BREAKING. To avoid breakage, place the dishesin a tupperware container partially filled with water, and place in theRT water bath, then turn on the heat to 70 C. This step denatures RNA andalso removes some of the proteins to make the RNA more accessible to hybridization.

3. Wash slides in 1X PBS for 2 minutes at room temperature.

G. Proteinase K digestion:

Proteinase K is used to partially digest the tissue to allow better probepenetration. This step increases the signal. For Arabidopsis floral tissue,the best incubation time is 30-45 minutes. Less time gives a weaker signal,and greater time tends to destroy the morphology of the tissue. The timingwas calibrated with a Proteinase K solution that was prepared just beforethe incubation.

1. Prewarm the Proteinase K Solution to 37o C in a staining dish, usingthe 37 C incubator.

2. Add Proteinase K to the prewarmed solution to a final concentrationof 1 ug/ml. I make up a fresh 5 mg/ml solution and add 100 ul of this toeach 500 ml of solution.

3. Put slides in solution and incubate for 30 minutes at 37oC. Agitateevery 10 min.

4. After the incubation, remove from the Proteinase K solution and placeinto a staining dish filled with H2O. Dip up and down a few time to rinseoff the Proteinase K.

5. Repeat the H2O wash.

6. Rinse in 1X PBS

7. Incubate in 2mg/ml glycine in 1X PBS for 2 minutes at room temperature(dissolve dry glycine in PBS). This step blocks the protease.

8. Rinse in 1X PBS, 2 times for 2 minutes.

H. Post-Fix

1. Make post-fix solution (4% formaldehyde in PBS), fresh 30 min beforeuse. Dissolve 20g of paraformaldehyde in 450ml water (in a 1L flask whichhas been treated with chloroform or baked), bring pH up to 9-10 with 2mlof 2N NaOH (400 ul of 10N NaOH). Check with pH paper. Microwave to about60°C until the paraformaldehyde goes into solution. Check temperatureof formaldehyde solution with a thermometer that was dipped in chloroform.

2. Bring pH back down to pH 7.0 with 2ml of 2N HCl (345 ul Conc. HCl).Then add 50ml of 10X PBS and cool in ice H2O down to room temperature.

3. Post-fix slides for 20 minutes in staining dish.

4. Rinse in 1X PBS, 2 x 5 minutes at room temperature.

I. Acetylation Reaction:

This reaction acetylates any remaining positive charges in the tissueor on the slides, and is done to reduce background Hayashi et al. (1978).One important point, the half life of acetic anhydride in aqueous solutionis less than 1 minute.

1. Make up 500 ml of 100mM Triethanolamine for each rack of slides (6.7ml Triethanolamine per 500 ml H2O). Adjust pH to 8.0 with about 2 mls ofconcentrated HCl. Check with pH paper.

2. Set up a staining dish that has 500 ml of 100 mM Triethanolamine anda chloroform treated stir bar at the bottom.

3. Stir the bar very hard and add 1.25 ml of acetic anhydride. Wait about5 seconds to mix, turn off stir bar, and add slides.

4. Incubate at room temperature for 5 minutes.

5. Rinse in 1X PBS, 2 x 5 minutes at room temperature.

J. Dehydration:

The purpose of this step is to completely dry the sections so that whenthe hybridization solution is applied, it is sucked into the tissue.

1. Progress through the following ethanol solutions so as to dehydratethe tissue gradually (except for the 100% and 95% ethanols, use the ethanolsolutions from step C): 15%, 30%, 50%, 70%, 85%, 95%, 100%.

2. Dry under a vacuum (will take about 1 hour). Alternatively one canlet sit overnight to dry: wrap in aluminum foil.

K. Pre-Hybridization:

1. Make several humidified boxes. We place several layers of paper towelsthat are soaked with 50% formamide/300 mM NaCl at the bottom of a tupperwarebox. Make sure to use a container with a flat bottom. It is a good ideato elevate the slides so that the probe does not get wicked away. We placethe slides onto a pair of 10 ml plastic pipets.

2. Use 175 ul of pre-hyb solution per slide. Make a little extra in caseyou run out. Keep at RT.

50% Formamide

1X Hybridization salts

1X Denhardt's Solution

70 mM DTT

150 ug/ml tRNA

3. Apply Pre-hyb solution to one end of slide. Slowly place coverslipdown on slide and make sure the pre-hyb is distributed over all the tissue.(Prehyb solution can be kept at room temperature.)

4. Place slides in 45° C incubator for 1-2 hours.

L. Hybridization:

1. Amount of hybridization solution to make:

Use 175 ul per slide for a surface area of 24 x 50 mm. Make 10-20% extra in case you run short.

2. Hybridization Solution for anti-mRNA and mRNA probes:

50% Formamide

1X Hybridization salts

1X Denhardt's Solution

10% Dextran Sulfate

70 mM DTT

150 ug/ml tRNA

500 ug/ml Poly A

2-4 x 10*6 cpm/slide (boil 3 min, quick cool in ice H2O, then 50C block)

3. Hybridization solution for [3H]-poly(U) probe:

300 mM NaCl

10 mM Tris (7.5)

1 mM EDTA

1X Denhardt's Solution

150 ug/ml tRNA

25 units/ml RNasin

1 ng/ul [3H]-poly(U)

4. Remove prehybridization solution and slides by holding the slidesvertically on a paper towel.

5. Apply hybridization solution to slides:

Use appropriate precautions for radioactive work.

(a) Pre-warm the hybridization solution to 50oC in a heat block. This makes it easier to spread.

(b) Apply hybridization solution in a pool to one side of the slide. Spread with a yellow tip. Make sure the tissue is wetted--sometimes the hybridization solution has a tendency to surround but not penetrate the tissue. This step is made easier by using a lot (175 ul) of hybridization solution.

(c) Cover slide with coverslip. When all of the tissue sections are wet, slant the slide briefly, so that a small pool of hybridization solution accumulates to one end. Hold the coverslip at one end with a jeweler's forceps and place the other end on the end of the slide that has the small pool of hybridization solution. Slowly lower the coverslip to completely cover the slide. Avoid bubbles--If you get a lot of bubbles, start over.

6. Place slides in the humidified box and incubate at 45oC (45-48 isfine) overnight.

IV. WASH

These steps do not require RNAse free glassware or solutions.

A. Solutions:

1. RNase Buffer:

Need 500 ml for each set of 30 slides. Preheat to 37°C.

500 mM NaCl

10 mM Tris (7.5)

1 mM EDTA

2. First Wash Buffer-- Need 1.5 L in 3 dishes

4X SSPE

5 mM DTT 1.16 g

3. Low Stringency Wash Buffer-- Need at least 3 L, 3 dishes plus bigcontainer.

2X SSPE

5 mM DTT 2.32 g

4. High Stringency Wash Buffer-- Need 4 L, preheat to 57C

0.1X SSPE

5 mM DTT 1.54 g to each 2L, add just before adding slides.

B. Remove coverslips and hybridization solution:

1. Dip the slides individually in a staining dish filled with First WashBuffer until the coverslip falls off. Dispose in radioactive liquid waste.

2. Place slides back into a rack and into a fresh solution of First WashBuffer. Dip up and down a few times.

3. Repeat so that the slides have been rinsed 3 times with First WashBuffer.

C. RNase Treatment:

This step can reduce background significantly (2-5 fold). In an experimentwith the AGAMOUS probe, I tried to reduce background further by raisingthe RNase concentration to 100 ug/ml and 200 ug/ml. Both concentrationsfailed to reduce background over the reduction achieved with 25 ug/ml RNase.More significantly, the slides incubated with 200 ug/ml RNase had no signal.

1. RNase Precautions:

(a) Making up an RNase stock solution:

Never weigh out RNase!!! You do not need to make up RNase fresh each time. To avoid weighing out RNase, buy a bottle, and add RNase Buffer directly to the bottle to achieve a concentration of 25 mg/ml. Store frozen at -20oC. This is a 1000X stock. This RNase stock will be good for >1 year. We use Ribonuclease A from Sigma (catalog # R-5503). 100 mg size: add 4 ml of RNAse buffer.

(b) RNase glassware:

We have a special set of staining dishes that are used for the RNase incubation and the first 3 washes. Use a plastic 1 ml pipet to measure out the 500 ul of stock solution. Do not put the RNase glassware through the normal wash. Without splashing, rinse off the RNase glassware >10 times to dilute the RNase.

2. Prewarm RNase Buffer to 37oC. You will need 500 ml for each rack of20 slides. The best way to prewarm is to fill the large staining disheswith RNase Buffer and place them into a 37oC water bath. This way, the glassgets warmed up too. Use the RNase staining dishes.

3. Thaw the 25 mg/ml RNase stock, and dilute to 25 ug/ml by adding 500ul to each staining dish filled with 500 ml of RNase Buffer. Mix by stirringwith a plastic pipet. RNase Precaution--Do not use a pipetman to measureout the RNase stock--use a plastic 1 ml pipet.

4. Place slides into the RNase solution, and incubate at 37oC for 30minutes. Dip up and down every 10 minutes during the incubation to removebubbles.

5. Fill 3 staining dishes with Low Stringency Wash Buffer. Use the RNasestaining dishes.

6. After the 30 minute incubation, remove slides from the RNase solutionand place into the first staining dish containing Low Stringency Wash Buffer.Dip up and down 15 times to rinse off the RNase.

7. Repeat with the second and third solutions of Low Stringency WashBuffer. Total of 3 washes.

D. Low Stringency Wash:

1. Fill a large Tupperware container (4 L container) with Low StringencyWash Buffer and place on top of a magnetic stirrer.

2. Place the slides (still in slide holder) in the buffer, and wash withstirring for 30 minutes.

3. At the end of the low stringency wash, remove the poly-U slides andplace in a staining dish containing low stringency wash buffer until theother slides are processed. No high stringency wash for the poly-U slides!

E. High Stringency Wash:

1. Place a 4 L beaker containing 2 L of High Stringency Wash Buffer ina 57oC water bath to preheat. Measure the temperature of the buffer to makesure it is actually at 57oC--sometimes the temperature of the water bathmust be set above 57oC to achieve this temperature in the buffer. Keep another2L warm in the 60oC oven.

2. Place the slides (still in slide holder) in the 57oC High StringencyWash Buffer, and incubate two times for 1 hour each time. Don't forget theDTT. If possible, agitate the buffer by stirring or shaking. Dip slidesup and down occasionally--bubbles accumulate on the slides.

F. Dehydration:

1. Dehydrate the tissue gradually by processing through the followingethanol series: 15%, 30%, 50%, 70%, 85%, 95%, 100%.

2. Dry under a vacuum (requires about 1 hour).

G. Quick Autoradiography:

Optional. Expose slides overnight on Biomax MR X-Ray film to assess background.If there is signal in the larger structures, this will be visible.

V. AUTORADIOGRAPHYAND STAINING

NOTE: Steps A-C should be carried out in complete darkness. Turn offall light sources (i.e. computers) in the darkroom, and carefully coverthe waterbath light and all sides of the waterbath. Minimize entering andleaving, and put a sign on the darkroom door indicating in situs are inprogress.

A. Emulsion:

1. Purchase NTB-2 emulsion from IBI/Kodak (1-800-752-9128). This shouldbe used fresh, or an older batch tested before use by developing a blankslide dipped in the emulsion.

2. Dilute the emulsion 1:1 with H2O.

(a) Prewarm the emulsion to 45°C in a water bath in a 500 ml flask. Place an equal volume of H2O in the flask and prewarm to 45°C.

(b) Pour the emulsion into the flask, swirl to mix, return to the 45°C water bath.

3. Aliquot into slide mailers (S/P Baxter # M6271). Most slide mailerscan be filled by using a 25 ml plastic pipette, filled to maximum volumewith a pink pipette dispenser.

4. Wrap in 2 layers of aluminum foil, place into a dark box, and storeat 4°C.

B. Dipping Slides:

1. Melt emulsion in a 45°C water bath.

2. Dip a blank slide in the emulsion to check the emulsion level, andto insure that the emulsion is well mixed. Look at it outside of the darkroom.

3. Dip each slide into liquid emulsion. Dip slowly, and one time onlyto avoid streaks.

4. Dry slides completely--place in a rack, inside a black box with desiccant,and put in dark room cupboard. Will take 30-60 minutes.

5. Place slides into dark boxes (VWR # 4844-003) with desiccant for exposure.Wrap desiccant in kimwipe, and place in box supported by slides. Wrap with2 layers of aluminum foil. Use separate boxes for slides to test for exposuretime.

6. Expose at 4°C for the appropriate time. For unknown probes, developtesters after 2 and 6 weeks, and develop remaining slides once the signalbecomes visible.

C. Develop slides:

Materials: Available from Lee-Mac or other photography supply stores

Kodak D-19 Developer--to preserve the developer, blow the O2 out of the bottle with N2 after each use. In an emergency, the developer will work when brown. Kodak DEKTOL developer will also work.

Kodak Fixer (Do not use Rapid Fixer).

1. Make fresh developer and fixer:

Developer - while stirring, slowly add contents of packet to 1 gallon of water (3.8 L).

Put in 65°C incubator for several hours, till chemicals are completely dissolved. Makes 2x solution.

Fixer - while stirring, slowly add contents of packet to 3L of water at room temperature.

When powder is dissolved, bring volume up to 1 gallon (3.8 L)

2. For each experiment use freshly diluted developer (1:1 in water).

3. Fill 3 staining dishes with developer, water, and fixer. Put dishesin ice until they cool down to 15oC.

4. Remove the appropriate slides from the darkboxes and place into aslide holder.

5. Place slides into developer, dip up and down 5 times, and let sitin developer for a total of 4 minutes (2 minutes if using DEKTOL developer).Longer developing times preferentially produce grains in the emulsion background.

(note old protocol was D19 full strength for 2.5 min. Hajime. does 1:1 for 2 min in Dektol, Bobby does 1:1 D19 4 min.)

6. Remove slides from the developer and drain briefly.

7. Place slides into the water and dip up and down continuously for 30seconds.

8. Remove slides from the water and drain briefly.

9. Place slides into fixer, dip up and down 5 times, and let sit in fixerfor 5.5 minutes

10. Remove slides from fixer and drain briefly.

11. Place into water until ready to stain. Rinse in running water for30 minutes to remove fixer.

D. Stain sections:

Materials:

Toluidine Blue O (Sigma # T-3260)

0.1% Toluidine Blue in 0.1% sodium borate (filtered)

1. Soak slides in 0.1% Toluidine Blue until tissue is deeply stained.The emulsion and tissue will be stained very deeply. With fresh ToluidineBlue, this takes 15-30 seconds. If using an old Toluidine Blue solution,stain for 1-2 minutes.

2. Rinse off in water to get rid of excess stain.

3. Soak slides in water to destain. The stain will come out of the emulsionmore quickly than the tissue. Soak just until the emulsion is well destained,but the tissue is still darkly stained; generally the emulsion will be faintlystained. This step can take > 1 hour. This step is accelerated by usinga stir bar and rotating the slides every 30 min.

4. Dip 5-10 times in each of the following ethanol solutions: 25%, 50%,75%, 100%, 100%. Move quickly through the ethanols because the tissue becomesdestained in these solutions. If the emulsion is still somewhat stained,move through the ethanols more slowly to control the degree of staining.Note: If the tissue is too deeply stained, the hybridization grains willbe obscured.

5. Dip slides in xylenes 15 times or until the streaks go away. The tissuewill not destain in the xylenes.

6. Place slides into fresh xylenes.

7. Place coverslips on slides:

(a) Remove slide from xylenes and place onto a paper towel.

(b) Quickly add 2 drops of permount to the surface of the slide.

(d) Squeeze out the excess xylene/permount--wipe surface of coverslip while holding down. When dried (next day), wipe surface with xylenes to clean.

8. The coverslips can be removed at any time by soaking in xylenes for1-5 days.

VI. VIEWING AND PHOTOGRAPHINGSLIDES

A. Preparing slides for viewing

1. Scrape off emulsion from back of slide with a razor blade.

2. Rub off excess permount with a cotton-tipped applicator dipped inXylene.

3. Examine slide, and repeat above steps if they are not clean.

B. Setting up microscope for phase contrast

1. Add water to top of condenser lens (directly below the stage). Turncondenser focusing knob until the condenser is all the way up. Turn on theilluminator, place a slide on the stage, and find a good section. Switchto 16x PH (phase contrast) lens.

2. Change the setting on the condenser ring (the ring just below thestage) to 2. The number 2 should be exactly aligned with the short whiteline on the right of the condenser ring. Press in the yellow filter knob(the knob farthest from the front of the microscope). This makes the lightbluish.

3. Close the condenser light aperture (on the bottom of the microscope).A ring of light appears when viewed through the lens. Align this ring withthe cross hairs, using knobs on the condenser ring.

4. Open condenser aperture. Move phase ring (on upper part of microscope)to PH setting. The phase ring contains two rings; the top ring for focusing,and the bottom one for changing the setting . Two donuts should appear,one light and one dark. Align donuts on top of each other with 2 knobs onthe condenser ring.

5. Focus on the rings using the phase contrast focus ring (the upperring). Set bottom of phase ring to 1.25

6. Take care to avoid bumping any of the phase contrast adjustment knobs.Repeat this procedure if this happens.

C. Photographing slides

Generally 3 pictures are advised: One bright field only, and two withboth bright field and dark field (double exposures), with varying exposuretimes for dark field.

1. Use Kodak Ektachrome Elite 200 speed slide film. 100 speed also works.Load film in camera. (The shutter does not advance). Pull out metal shield.

2. Focus on the tissue in bright field. Pull upper knob all the way outto direct light to the camera instead of the eyepieces.

3. Turn on exposure apparatus (directly to the left of the microscope).Set lower left knob for the speed of the film (in most cases 200). The upperleft knob should be on 3.

4. Adjust lower right knob until needle indicator is in the center. Usethe upper right knob for fine adjustment. Press yellow button to take thepicture.

5. Advance the film by pressing button on camera and winding film. ThenTake another picture with the same setting, but do not advance the cameraat this time (to allow for double exposure).

6. Switch to dark field. Switch condenser ring from 2 to D, turn offbottom blue filter (yellow knob out). Place yellow or red plastic platefilter directly on top of the condenser aperture. Push knob in to send lightto the eyepieces.

7. Focus on grains (they are on a slightly different plane as the tissue).Pull knob all the way out to send light to the camera. Adjust exposure timeas before, getting needle to the middle. Once it is in the middle, clickthe lower right knob back one (-1 exposure).

8. Press yellow button to expose film. It will take significantly longerthan the bright field exposure. Once exposed, advance film as before. Takeanother dark field picture at -2 exposure by clicking the lower right knobback one more. Do not advance film after this exposure.

9. Switch back to bright field: Condenser ring from D to 2, turn on bluefilter (yellow knob), remove red or yellow plastic filter, push knob tosend light to eyepieces, and focus on the tissue.

10. Adjust exposure, take another picture, and advance film. This gives3 total pictures per section, including two double exposures.