In Situ Hybridization with Embryonic Sections

In situ Hybridization with Embryonic Sections

This section describes the in situ hybridization histochemical method to detect expression of mRNA encoding proteins that are critical for endothelial or smooth muscle cell differentiation and/or vascular morphogenesis.

Solutions:

* ³⁵S-CTP (70Ci/ml, NEN Cat# NEG-064C)
* NICK column (Pharmacia, Cat# 17-0855-02)
* Proteinase K (stored at 20mg/ml in 0.5ml aliquot in DEPC treated dH₂O at -20 C)
* 5X TE: 50mM Tris-HCl pH8.0, 5mM EDTA
* TEA/Acetic Anhydride: 0.625 ml acetic anhydride in 250 ml 0.1M TEA pH8.0, immediately before use
* Hybridization Mix:

50% formamide
0.3M NaCl
20mM Tris-HCl pH8.0
5mM EDTA
10mM Na-Pyrophosphate pH 8.0
1X Denhardt's
10% Dextran Sulfate
0.5 mg/ml yeast RNA (Store 0.9 ml/tube at -80 C).

Procedure:

A. Linearization of a plasmid DNA

1. The template DNA can be prepared by a plasmid prep kit (e.g. Qiagen kit). Assemble the following:
               20|ìl plasmid DNA (=20 |ìg)
               10|ìl 10X Buffer
               10|ìl 1mg/ml BSA
               55|ìl dH₂O
                 5|ìl restriction enzyme
                Total volume=100|ìl
2. Incubate at 37??C for 1.5hr. Check 1|ìl on a mini-gel for completion of the digest.
3. Extract twice with phenol/chloroform/IAA, saturated with TE pH8.0, then extract once with Chloroform/IAA.
4. Add 10|ìl 3M NaOAC pH5.5 and 300|ìl absolute EtOH. Precipitate DNA on dry ice for 15min.
5. Microfuge at 4??C for 15min and pour off EtOH. Add cold 70% EtOH gently and re-spin for 2min at 4??C.
6. Pour off EtOH and re-spin for 5sec. Remove EtOH completely using a micropipette tip, and dry the DNA pellet in a speed vac for 2min.
7. Dissolve the pellet into 10|ìl TE^1/10 prepared with DEPC dH₂0. Place in 37??C for 30 min. to completely dissolve the pellet.
8. Take 1|ìl and mix with 499|ìl dH₂O (i.e. 1:500 dilution) and measure the OD260/280.
9. Adjust the concentration to 1|ìg/|ìl with TE^1/10 prepared with DEPC dH₂0, and store the DNA at -20??C.

B Riboprobe Synthesis and Purification

probe=tie1

A. Transcription reaction

1. Assemble in the following order:
        4.75|ìl DEPC treated dH₂O
        5|ìl 5X buffer
        1|ìl linearized template DNA (=1|ìg)
        1.25|ìl10mM ATP
        1.25|ìl 10mM UTP
        1.25|ìl 10mM GTP
        2.5|ìl 100mM DTT
        1|ìl RNAsin (40u/|ìl, Promega, Cat# N2111)
        5ul ³⁵S-CTP (70mCi/ml, NEN)
       2|ìl RNA polymerase (e.g. T3, T7, or SP6 polymerase, 10-20u/|ìl)
        Total volume = 25|ìl
2. Incubate at 37??C for 2 hr
3. Dilute 1|ìl with 99|ìl dH₂O. (i.e. 1:100 dilution) Calculate the incorporation of ³⁵S-CTP and the amount of the synthesis by TCA precipitation.

*Usually the incorporation is about 60% or more. If it is less than 50%, something is wrong with either the template DNA or the transcription reaction.
*Calculation of the amount of riboprobe synthesized: (The amount of RNA) in grams = 58.3X^10-12 X 5 X 330 X 4 X incorporation (e.g. 0.6 for 60% incorporation) We usually obtain 200-300ng riboprobe per above reaction.

B. Termination Reaction:

1. Add 1|ìl RNase-free DNase (2u/|ìl, Ambion) to the rest of the transcription reaction mix. Incubate at 37??C for 15 min.
2. Add the following: Alkaline hydrolysis: 60|ìl TES (10mM EDTA, Tris-HCl pH7.5, 0.2% SDS) 1.67|ìl 5M NaCl 1|ìl 1M DTT Mix well and leave on ice for at least 30 min.
3. Make 2N NaOH from 10N NaOH stock (stored at 4??C) and pre-cool on ice. Make sure the sample is ice-cold before adding NaOH. Otherwise, the RNA will be too hydrolyzed.
4. Add 10|ìl of ice-cold 2N NaOH and mix well. Briefly spin at 4??C and incubate the tube on ice for 45 min.

C. Neutralization:

1. Add 20|ìl 2M HEPES, mix, vortex, and spin briefly at room temperature.

D. Purification of the RNA probe:

1. Add 100|ìl TE saturated Phenol/chloroform/IAA (25:24:1) and vortex well.
2. Spin at RT for 5 min. and carefully transfer the upper phase to an empty microfuge tube.
3. Prepare the "Nickcolumn" (Pharmacia) as follows:

Wash the column first by pouring-in-and-out the freshly prepared equilibration buffer (10mM EDTA, 10mM Tris-HCl pH7.5, 10 mM DTT). Then, equilibrate the column by washing with 3ml of equilibration buffer.
* preparation of the equilibration buffer(20ml):
         19.2ml DEPC treated dH₂O
          0.2mlTris-HCl, pH7.5
           0.2ml DEPC treated 0.5M EDTA
           0.4ml 1M DTT

4. Load the extracted probe (100|ìl) to the pre-equilibrated NICK column.
5. Wash the column with 400|ìl equilibration buffer. Discard this first wash, and elute the probe in a second wash of 400|ìl equilibration buffer. Do not discard the second wash.
6. To this eluate (400|ìl), add 40|ìl DEPC treated 3M NaOAC pH5.5, 2|ìl of 10 mg/ml tRNA, and 1000|ìl absolute EtOH. Mix well and precipitate RNA at -80??C overnight or longer (The probe can be stored in this condition at -80°C until use).
7. Spin at 4??C for 15 min. Then wash the RNA ppt with 1000|ìl of cold 70% EtOH by spinning at 4??C for 2min.
8. Pipette out the liquid and re-spin briefly. Remove the residual EtOH completely by pipetting.
9. Dry the pellet completely in the speed vac for 2 min. (Do not dry it too much or it will be difficult to dissolve completely)
10. Dissolve the pellet into 50-100|ìl TE^1/10 and save 1|ìl for quantitation. * Dilute this 1|ìl with 99|ìl dH₂O (i.e. 1:100) and count the radioactivity of the diluted sample.
11. Use the probe for hybridization immediately after dissolving in TE.

B. Pretreatment of the paraffin-embedded sections:

1. Xylene 10min x 2
2. 100% EtOH 5min x 2
3. 95%, 85%, 70%, 50%, 30% EtOH, Saline, PBS 30sec (5 min. for 70% EtOH, Saline and PBS)
4. 4% Paraformaldehyde 20min
5. PBS 5min x 2
6. 20|ìg/ml protease K in 5X TE 7.5min
7. PBS 5min
8. Re-use #4 (4% paraformaldehyde in PBS) 5min
9. H2O dip
10. TEA/Acetic Anhydride 10min
11. PBS 5min
12. Saline 5min
13. Follow #3 in reverse order (PBS-30%-95%, etc.) 30sec (5min. for 70%, Saline and PBS)
14. Air dry for about 2hr

1. Mix probe at 0.2 ng/|ìl/kb in 10% 1M DTT/TE^1/10 and heat it at 85°C for 5 min. Then mix with H.X. by the ratio of 1 to 9 (e.g. 90|ìl probe in TE + 10ul 1M DTT is heated and mixed with 900ul H.X.)
2. Hybridize (use 50 - 70|ìl per slide) at 55°C overnight in 5X SSC/50% formamide

probe=Tie2

1. 5X SSC/10 mM DTT remove coverslip
2. 5X SSC/10mM DTT 20min
3. 5X SSC/10mM DTT 55°C, 30min
4. 50% Formamide/2X SSC/ 100mM DTT 65°C, 25min
5. 1X TE/0.5M NaCl room temperature, 10min
6. 1X TE/0.5M NaCl 37°C 10 mi. x 3
7. 20 |ìg/ml RNase in above buffer 37°C, 30min
8. 1 X TE/0.5M NaCl 37°C, 15 min.
9. 50% formamide/2X SSC/100mM DTT 65°C, 25min
10. 2X SSC 65°C, 15min
11. 0.1X SSC 65°C, 15min
12. 30%, 60%, 80%, 95% EtOH/30mM Ammonium Acetate 30sec each
13. 100% EtOH 30sec
14. Air dry
15. Dried slides are ready for emulsion coating and exposure:

1. Set the water bath to 42??C. Make sure that the water level reaches the upper level of the emulsion.
2. Make sure that all of the glassware used for emulsion preparation are very clean and free of traces from old emulsion.
3. Prepare the "slide cover box" by using a hard paper box cover wrapped with aluminum foil to loosely cover the drying slides.

1. Add 10ml of 300mM ammonium acetate to the coplin jar.
2. Turn off all the lights (including the safelights) and transfer about 10ml of solid emulsion to a small beaker. Transfer remains to the coplin jar containing ammonium acetate solution.
3. Cover the jar with a glass lid and let the emulsion melt for 20-30min.
4. Stir the emulsion very gently with a clean slide, then slowly dip two glass test slides into the emulsion and let them set in the vertical position (use a test tube rack to hold the slides).
5. Take the test slides out of the dark room to check the level of emulsion solution. Make sure the emulsion is mixed well. (i.e. emulsion should coat the glass slide uniformly).

1. Put the slides in order before going into the dark room.
2. Dip each slide slowly into the emulsion at a constant speed (counting helps to keep a constant speed) to obtain a uniform monolayer of emulsion coating.
3. To dry the dipped slides, lean the slides against the test tube rack in a vertical position and cover with the "slide cover box" as prepared above.
4. Dry in the box for 2-3hr at room temperature in the dark room.
5. Do not unplug the water bath, as this may generate an electrical spark.

1. Clean the small black slide boxes.(free of dust) (Fisher Cat# 22-167-403).
2. Wrap a fair amount of desiccant in Kimwipes and place the wrapped desiccant in between the blank slides to hold it in the box. Close the slide box until ready to use emulsion-coated slides.
3. Prepare one sheet of plastic wrap and one sheet of aluminum foil for each slide box and set aside.

1. After 2-3hr of drying, place about 5 slides in each black slide box in the dark room.
2. Close the boxes completely and wrap the boxes with plastic wrap, then with foil.
3. Now it is O.K. to turn on the safelight and check the box, then turn on the room light.
4. Clean the emulsion jar and the room.
5. Put the slide boxes in the desiccator with enough desiccants in the cold room for exposure. The required exposure time for each probe:
                  TIE1, TIE2, VEGF-A: Three to four weeks
                  Angiopoietin-1, Angiopoietin-2: Two to three weeks
                  FLK1, FLT1, FLT4, SM22a : Two weeks

1. Take the slide boxes out from the desiccator and warm them up to room temperature for at least 30min.
2. Set Kodak D-19 and Rapid Fix solution on ice, and monitor the temperature until it reaches down to 14??C.
3. Once it reaches 14??C, develop the slides immediately in the dark room as follows: Dip a slide for 3.5min in Kodak D-19 solution, then briefly dip in H₂O twice. In complete darkness, fix for 5 min. in Rapid Fixer. (No Safelight!). Now you can turn on the room light!
4. Rinse the slides under running tap water for 20 min or longer.
5. Counterstain sections with hematoxyline and eosin, or other histological staining methods.