Method: In-Situ Hybridization with Fluorescently Labeled Probes

Srini Ramachandra

Localization of single-copy clone DNA sequences in interphase nuclear and metaphase chromosomal DNA by non-isotopic in situ hybridization using a highly sensitive fluorescence methodology.

Time required:

A total of 5-8 days for all the steps listed below

Steps involved:

1. Biotin labeling of probe DNA
2. Slot blot analysis of labeled probes
3. Hybridization
4. Detection & Staining
5. Fluorescence microscopy and photography

Procedure:

Day 1

Biotin labeling of the probe DNA

Biotin labeling of probe DNA is achieved by standard nick translation protocols as described below. Ideally, nick translated probes should be approximately 200 bp in size.

Stock solutions required:

• Soln. [A] = 10X Nick-translation buffer
• Soln. [B] = Nucleotide stock combo containing biotin- 11-UTP
• Soln. [C] = 0.1M §-mercaptoethanol made fresh
• Soln. [D] = 1 ng/µl DNAseI dilution made fresh from a 1 mg/ml stock soln.

1. Add the following reagents in order to an eppendorf tube on ice:

        probe DNA                           2.0 µg
        Soln. [A}                           10.0 µl
        Soln. [B]                           10.0 µl
        Soln. [C]                           10.0 µl
   					---------
   add sterile  ddH2O to                   100.0 µl total volume
        Soln. [D]                            4.0 µl
   E.Coli DNA polymerase I                   2.0 µl  (use at 5U 
   per µg of DNA)
      (5 units/µl )
   

2. Mix thoroughly and immediately incubate tubes in 15 degrees C waterbath (in cold room) for approximately 1.5 to 2 hours.
3. Prepare Sephadex G-50 column as described below (also see Maniatis Manual):
   • Sephadex G-50 Spin-Column:
     Weigh out 4 g of Sephadex G-50 (Sigma) and add it to 50 ml of TE buffer in a 100 ml flask. Boil the slurry in boiling water for 10 minutes or heat the slurry in 65 degrees C waterbath for 2 hours. After this the slurry can be stored at 4 degrees C indefinitely and has to be boiled or heated as above before each use.
   • Column preparation:
     Plug the bottom of a 1cc syringe with glass wool and place it in a 15 ml centrifuge tube (blue cap). Add the sephadex slurry with a pasteur pipet to the syringe and spin on the tabletop Beckman TJ-6 centrifuge for 5 minutes at 2000 rpm. The column should pack down with sephadex and the TE should collect in the centrifuge tube. Add more slurry, spin and repeat the above steps until the column is filled to the 0.9 ml mark. Then add 400 ul of TE-SDS buffer and spin as above to equilibrate the column with the buffer. Transfer the filled column to another centrifuge tube with an eppendorf tube in the bottom (without the top) so that the bottom tip of the syringe just sits inside the eppendorf tube. DO NOT ALLOW THE COLUMN GO DRY. If the column does dry out, reequilibrate with the TE-SDS buffer as above before using.
4. Terminate the reaction by adding EDTA to a final concentration of 10-15 mM (= 2.5 µl of 0.5M EDTA, pH 8.0). Add 10% SDS to a final concentration of 0.1% (= 1 µl of 10% SDS to a reaction volume of 100 µl). Incubate at 65-68 degrees C for 15 minutes.
5. Load the sample onto the top of the column and spin at 1500 rpm for 10 minutes. Approximately the same volume of sample that was loaded onto the column should be collected in the eppendorf tube. Transfer the eluted sample to another eppendorf tube and cap it.
6. Take out an aliquot of labeled probe (at least 10 µl), denature it in boiling water for 5 minutes, quick-cool on ice for 2 minutes and run the aliquot on a 2% TA-agarose minigel with 1 kb ladder as standard markers for 30-45 minutes at 80V. The size of the probe should be less than 500 bp, the ideal size being approximately 200 bp. The remaining labeled probe can be stored at -20 degrees C indefinitely in the dark.

Solutions for nick translation:

• Soln. A: 10X nick translation buffer

                                            Final concentration
  1M Tris-HCl, pH 8            5.0 ml               0.5 M
  1M MgCl2                     500 µl               50 mM
  50mg/ml BSA                  100 µl               0.5 mg/ml
  
  Bring the volume to 10 ml with ddH2O, filter sterilize,
  aliquot 1 ml into eppendorf tubes and store at -20 degrees C.
  

• Soln. B: Nucleotide stock
  Prepare 10 mM dilutions each from 100 mM stocks of dATP, dCTP and dGTP (Pharmacia). Then prepare the nucleotide mix as follows:

                                         Final concentration
  10 mM dATP  	           50 µl		0.5 mM
  10 mM dCTP    	           50 µl		0.5 mM
  10 mM dGTP     	           50 µl		0.5 mM
  Biotin-11-dUTP      	   0.5 mg		0.5 mg/ml
  make up volume to     1.0 ml with sterile ddH2O
  
  Mix well, aliquot 50 µl into eppendorf tubes and store frozen 
  at -20 degrees C.
  

• Soln.C: 0.1M ß-mercaptoethanol
  Add 7 µl of the stock (14 M) to 993 µl of ice-cold sterile ddH2O and vortex to mix. Make this solution fresh each time and discard after use.

Soln.D: DNaseI

Make a stock solution of 1 mg/ml DNaseI in 50% glycerol. Add 1µl of 1 mg/ml stock to 999 µl of ice-cold sterile ddH2O to give a 1 ng/µl dilution. Make this dilution fresh every time from the stock and discard after use. Store the 1mg/ml stock solution at -20 degrees C.
• TE-SDS buffer:

   		                          Final concentration
  1M Tris-HCl, pH 8           25 ml               50 mM
  0.5M EDTA, pH 8              1 ml                1 mM
  10% SDS                      5 ml                0.1%
  
  Bring volume to 500 ml with ddH2O, filter sterilize and store 
  at room temperature.

Day 2

Slot blot analysis of biotin-labeled probes

Purpose:

To colorimetrically detect biotin incorporation in nick translated probes.

Time required:

6 hours plus an overnight drying period.

Special reagents required:

• BRL DNA Detection kit (BRL Cat.# 8239SA). All detection reagents for this procedure are supplied with the kit. The buffers that need to be made up are listed at the end of this section.

Procedure:

1. Dilute all biotin-labeled probe DNAs (including the positive & negative controls) to approximately 500 pg/µl in the DNA dilution buffer provided with the kit. Using this as a stock, make serial dilutions of 250 pg, 125 pg, 62.5 pg, 31pg and 16 pg with the DNA dilution buffer.
2. Label the BRL pre-cut nitrocellulose membrane with the respective probe names using a dry-erase marker. Soak the membrane for 10 minutes in a large glass petri plate.
3. Place the middle manifold of the slot-blotter on top of the bottom manifold. With a pair of flat forceps gently place the wet nitrocellulose membrane on the middle manifold centering it between the alignment pin and bolt holes.
4. Place the top manifold (with the wells facing up) on the membrane, place the two alignment pins and the four bolts in their respective holes and tighten the bolts diagonally.
5. Load the DNA sample dilutions starting from 1ng to 16 pg into the wells as shown in the diagram below. The Hybri-slot apparatus can hold 24 samples. Attach the vacuum tubing to the bottom manifold and apply vacuum for 2 minutes.
   
6. With the vacuum still ON loosen & remove the alignment pins and bolts slowly. Remove the top manifold slowly.
7. With the help of flat forceps fold one corner of the nitrocellulose over and remove the blot with one quick motion. Place the wet blot in between Whatman 3MM paper and allow it air dry overnight at room temperature.
8. Turn the vacuum OFF, wash the Hybri-slot apparatus thoroughly with soap and ddH2O only. DO NOT USE ETHANOL OR BLEACH TO CLEAN THE APPARATUS. Dry the manifolds thoroughly taking care not to scratch the surface.

   Note: All reagents for the following steps are supplied with the BRL DNA Detection Kit.

9. Bake the air dried blots in the vacuum oven at 80 degrees C for 2 hours.
10. Rehydrate the blots in 20 ml of Buffer-1 in a large glass petri plate for 1 minute.
11. Incubate the blots at room temperature for 20 minutes on a platform shaker in 20 ml of Buffer-2 that has been prewarmed to 42 degrees C.
12. Drain Buffer-2 and add Streptavidin solution: 10 µl of 1mg/ml Streptavidin stock to 10 ml of Buffer-1. Incubate at room temperature on a platform shaker for 10
13. Drain streptavidin solution and wash the blots 3 times for 5 minutes each wash in 100 ml of Buffer-1 on the platform shaker.
14. Add alkaline phosphatase solution: add 10 µl of 1mg/ml poly(AP) stock to 10 ml of Buffer-1. Incubate at room temperature on shaker for 10 minutes.
15. Drain the AP solution and wash twice for 3 minutes each wash in 100 ml of Buffer-1.
16. Quickly rinse the blots in Buffer-3 and add the dye solution as follows:
    Add 33 µl of NBT solution and 25 µl of BCIP solution to 7.5 ml of Buffer-3 , mix well and incubate the blots in the dye solution for approximately 4 hours in the dark at room temperature on a shaker. Cover the glass petri plate completely with aluminum foil.
17. Check the blots occasionally during the above incubation for development of purple coloration of the DNAs spotted which is an positive indication of biotin incorporation. Visually compare the color intensities of the DNA samples with that of the positive control.
18. Terminate the reaction by incubating the blots in 20 ml of 20mM Tris-HCl pH7.5, 5mM EDTA pH8.0 for 5 minutes.
19. Air dry the blots in the dark between Whatman 3MM filter papers and store them in plastic bags in the dark.

Solutions:

• Buffer-1

                                           Final concentration
  1M Tris-HCl (pH7.5)             50 ml            0.1 M
  5M NaCl                         10 ml            0.1 M
  1M MgCl2                         1 ml             2 mM
  Triton X-100                  0.25 ml             0.05%  
  Bring volume to 500 ml  with ddH2O,  filter sterilize and 
  store at room temperature.
  

• Buffer-2 (3% BSA in Buffer-1)

  Dissolve 3 g of BSA (Sigma, Fraction V) in 80 ml of ddH2O. Bring volume to 100 ml, filter sterilize and store at 4 degrees C.

• Buffer-3

                                  Final concentration
  1M Tris-HCl (pH7.5)      50 ml             0.1 M
  5M NaCl                  10 ml             0.1 M
  1M MgCl2                 25 ml             50 mM
  ddH2O                   415 ml
                          ------       
                          500 ml  -- autoclave  and store at 
                                     room temperature.
  

• 20mM Tris-HCl, 5mM EDTA

  1M Tris-HCl pH7.5         10 ml
  0.5 M EDTA pH8.0           5 ml
  ddH2O                    485 ml
                           ------
                           500 ml  -- autoclave and store at 
                                      room temperature.

Hybridization
1. Add the DNAs as follows:

   biotin-labeled probe DNA                      100 ng
   sonicated human DNA (4 µg)                    8.0 µl            
     (0.5 µg/µl stock)
   sonicated salmon sperm DNA (6 µg)             6.0 µl             
     (1.0 µg/µl stock)                          --------
   Bring the volume to                           40.0 µl  with TE (pH8)
   

2. Mix DNAs and TE, add 1/20th volume (= 2 µl) 3M NaOAc (total volume = 42 µl) and 2X volume cold EtOH (84 µl). Mix well, touch-spin briefly and precipitate the DNAs at -20 degrees C overnight.

Day 4

1. Pellet the DNAs in a microcentrifuge at 12000 rpm, 4 degrees C for 45 minutes. Discard supernatant, add 300 µl of 70% cold EtOH and spin for another 45 minutes at 4 degrees C. Drain supernatant completely and dry the DNA pellets in the vacuum concentrator for 8 minutes.
2. Resuspend DNA pellet in 5 µl of 100% deionized formamide (pure reagent grade from Aldrich, stored at -20 degrees C in 1ml aliquots). Use the pipet tip to resuspend the pellet thoroughly.
3. Add 5 µl of prewarmed (at 37 degreesC) 20% dextran sulfate (made in 2X SSC). Mix well with pipet tip and touch- spin.
4. Denature the probe/competitor DNA mixture for 5 minutes on a 75 degrees C heatblock.

   Incubate at 37 degrees C for 15 minutes.

5. Meanwhile, denature chromosomal DNA on slides as follows:
   • Place slides in a steel pan and pre-warm them in the 50 degrees C incubator.
   • Pre-warm 70% formamide in 2X SSC in a 68-70 degrees C waterbath (for 50ml Coplin jar, mix 35ml deionized formamide, 5ml 20X SSC, 10 ml dH2O).
   • Place the slides in the Coplin jar (8/jar) and denature chromosomes for exactly 1 minute and 30 seconds
   • Transfer the slides in the same order that they were denatured to another coplin jar containing 50 ml of cold 70% EtOH in an ice bucket. Quick rinse and add fresh 70% EtOH.The rest of the EtOH dehydration should be done in coplin jars completely buried in ice.
   • Transfer slides to another coplin jar containing cold 70% EtOH. Let the slides sit for 5 minutes.
   • Transfer slides to cold 90% EtOH for 5 minutes and then to cold 100% EtOH for 5 minutes.
   • Take slides out of the EtOH washes, drain excess EtOH and air dry slides by standing them up against a support on some paper towels.

6. Place the slides on the slide warmer (42 degrees C), remove 2 tubes at a time from the 37 degrees C waterbath and place 10 µl of the hybridization mix on the slide, centered between the two scratch marks which demarcate the area of the metaphase spread. Gently lower a clean 22 mm x 22 mm glass coverslip onto the area between the two marks (without creating any air bubbles). If any air bubbles are seen, gently push them out using a pair of fine pointed forceps.
7. Seal the coverslip edges with rubber cement (loaded in a 10ml syringe). Allow the rubber cement to dry . Incubate the slides at 37 degrees C overnight in a moist chamber. The moist chamber can be prepared by placing wet paper towels in a steel pan or a Tupperware container with 3-5 1ml plastic pipets on top of the wet towels. Place the slides on the pipets and cover the pan with Saran wrap.

Day 5

Detection and Staining
1. Place the slides on the 42 degrees C slide warmer and remove rubber cement carefully with fine forceps.
2. Wash the slides 3 times in 50% deionized formamide (in 2X SSC) for 5 minutes each wash at 42 degrees C, in coplin jars on a platform shaker. The cover slips will come loose in the first wash very easily.The formamide solution should be prewarmed to 42 degrees C in a waterbath at least an hour before. Cover the coplin jars with aluminum foil to protect the formamide and the biotinylated probes from light. Use foil covered coplin jars for all the washing and staining steps.
3. Wash the slides 3 times in 0.1X SSC (high stringency wash) for 5 minutes each at 60 degrees C. (slides can sit in the last wash for a while, do not let them dry out).

Blocking: this step helps avoid non-specific binding of the biotinylated probe thereby reducing background fluorescence.

1. Add 200 µl/slide a solution of 3% BSA, 4X SSC (prewarmed to 37 degrees C) and cover with large coverslips made out of biohazard bags cut to slide size and incubate at 37 degrees C for 30 minutes in the foil covered moist chamber.
2. Drain blocking solution after incubation. There is no need to wash.

Detection: (in the DARK). Avidin-FITC binds to the biotin molecules. FITC emits yellowish-green fluorescence when viewed under the FITC filter through the microscope.

1. Remove coverslips, add 200 µl/slide of Avidin-FITC conjugate (2 mg/ml Avidin-FITC stock diluted 1:400 in 4X SSC, 1% BSA, 0.1% Tween-20, prewarmed to 37 degrees C), cover with biohazard bag coverslip and incubate at 37 degrees C, 30 minutes in the moist chamber covered with foil.
2. Remove coverslips, drain excess avidin-FITC solution and wash slides 3 times in 4X SSC, 0.1% Tween-20 for 5 minutes each wash at 42 degrees C on a platform shaker. The wash buffer should be prewarmed in a 42 degrees C waterbath.

Amplification: Several molecules of anti-avidin D antibodies bind to a single molecule of avidin- FITC thereby amplifying the number of FITC molecules per biotin molecule. This enhances weak FITC signals several fold.

Note: It is not necessary to do the amplification for probes known to contain human DNA inserts of more than 15 kb in size. But it is always good to have duplicate sets of slides for each probe so that one set can be amplified and the other set can be counterstained directly after detection.
1. Add 200 µl/slide of anti-avidin D solution (2.5 µg/ml final concentration) prepared in 4X SSC, 0.1% Tween-20 and 1% BSA (prewarmed to 37 degrees C). Cover with biohazard bag coverslips and incubate in the foil covered moist chamber at 37 degrees C for 30 minutes.
2. Drain excess anti-avidin D solution, remove coverslips and add 200 µl/slide of avidin-FITC as described in the detection step above. Incubate and wash slides as in steps 1 & 2 of detection.

Counterstain: The chromosomes are double stained using DAPI and Propidium Iodide (PI) stains. DAPI stains the chromosomes blue and PI stains them red. The morphology of the chromosomes can be better visualized under DAPI wherein the centromere, the p & q arms can be identified. The PI stain gives the chromosomes a bright red background, enhancing the yellowish-green FITC signals under the microscope with the FITC filters.

1. Immediately after the last wash make up a 50 ml solution of counterstain containing Propidium Iodide (60 µl of 200 µg/ml) and DAPI (40 µl of 200 µg/ml stock) in the last wash buffer (4XSSC, 0.1% Tween-20 maintained at room temperature). Place the slides in the stain solution, cover the coplin jar with foil and incubate at room temperature for 10 minutes on a shaker.

Mount in antifade: Add 30 µl/slide of antifade solution (in glycerol), cover with 24 mm x 60 mm glass coverslips immediately and store slides at 4¡C in a black slide box wrapped in parafilm.

Days 6 through 8

See " Fluorescence microscopy " method by R. Veile for information on microscopy and photography of fluorescently labeled in situ slides.

Solutions:

• Formamide
  Formamide should be of pure reagent grade with the pH around 7.0. If the pH is off, deionize the formamide using BioRad AG 501.X8 resin as described in Maniatis manual (p. 448). For every 100 ml of formamide add 10 g of the resin in a large glass flask covered with foil, place a stir bar and stir overnight. The resin changes color from greenish-blue to yellowish-red, indicating a saturation of the resin with ions. Read the pH. If the pH has reached 7.0, vacuum filter the deionized formamide through Whatman filter paper in a buchner funnel. Make twenty 1 ml aliquots in eppendorf tubes and store in the dark at -20 degrees C. Rinse out the original formamide bottle with ddH2O and store the rest of the deionized formamide in the same bottle at -20 degrees C.
• Dextran sulfate
  Prepare a 50% stock in distilled, deionized water (ddH2O), then autoclave. Prepare a 1:2.5 dilution of the 50% stock in 2X SSC to get a 20% stock.
• Blocking solution
  3%BSA in 4X SSC: First prepare 10 ml of 20% BSA (Sigma Fraction V) solution in ddH2O.

  Filter sterilize and aliquot 1ml into eppendorf tubes.

  			1.5 ml 20% BSA
  			2.0 ml 20X SSC
  			6.5 ml dd water
  			---------------
  			10.0 ml of 3% BSA in 4X SSC
  
  	Filter sterilize and store at -20 degrees C.
  

• Avidin-FITC conjugate

  Avidin-FITC is supplied as a 2 mg/ml solution (Vector Labs). Aliquot 50 µl into sterile eppendorf tubes and store at -20 degrees C in the dark.

• Amplification solution

  Biotin conjugated goat antiavidin-D antibodies are supplied as 0.5 mg powder. Add 1 ml of sterile ddH2O to the 0.5 mg bottle to give a 0.5mg/ml solution. Prepare 50 µl aliquots in eppendorf tubes and store at -20 degrees C.

  For amplification: use this stock solution diluted to 2.5 µg/ml in the 4X SSC, 0.1% Tween-20, 1% BSA solution

• Propidium iodide

  Dissolve 2 mg of PI in 10 ml of sterile 2X SSC to give a 200 µg/ml stock solution.

  Store in 1 ml aliquots in eppendorf tubes covered with foil at 4 degrees C. [PI is light sensitive].

• DAPI
  (4,6-diamidino-2-phenylindole-dihydrochloride)

  Prepare a 200 µg/ml solution as above (for PI), cover with foil and store as above.

• Antifade solution

  	0.233 g  DABCO  (1,4-diazobicyclo(2.2.2)octane)
  	800 µl dd water
  	200 µl 1M Tris-HCl, pH8.0
  	9.0 ml   glycerol
  	--------------------
  	10.0 ml total volume 
  
    Store in 1 ml aliquots in eppendorf tubes at 4 degrees C, 
  covered in aluiminum foil.

Sources of chemical reagents for in situ hybridizations:

Chemical				Company			Catalog#
--------				-------			--------
Bovine serum albumin (BSA), fraction V	Sigma			B 2518
§-mercaptoethanol			Sigma			M 3148
Formamide, pure reagent grade		Aldrich			29587-6
Sephadex G-50				Sigma			G-50-50
Dextran sulphate			Pharmacia		17-0340-03
DNase I	BMB				104 			159
E. Coli DNA polymerase I		BMB			104 485
Biotin-11-dUTP				Sigma			B 5770
Avidin-FITC (fluorescein Avidin DCS)	Vector Labs		A-2011
Biotin-conjugated goat anti-aivdin
 D antibody				Vector Labs		BA-0300
Tween-20 (polyoxyethylene 
 sorbitan monolaurate) 			Sigma			P 1379
Propidium iodide			Sigma			P 4170
DAPI (4,6-diamidino-2-phenylindole-
dihydrochloride)			Sigma			D 1388
DABCO {1,4-diazobicyclo(2.2.2.)octane)	Sigma			D 2522

References:

Lichter, P., Cremer, T., Borden, J., Manuelidis, L., and D. C. Ward. (1988) " Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries." Human Genetics 80: 224-234.

Cremer, T., Lichter, P., Borden, J., Ward, D. C., and L. Manuelidis. (1988) "Detection of chromosome aberrations in metaphase and interphase tumor cells by in situ hybridization using chromosome-specific library probes." Human Genetics 80: 235-246.

Lichter, P., Cremer, T., Tang, C. C., Watkins, P. C., Manuelidis, L., and D. C. Ward. (1988) "Rapid detection of human chromosome 21 aberrations by in situ hybridization." Proc. Natl. Acad. Sci. USA 85: 9664-9668.