CHROMOSOME IN SITU HYBRIDIZATION USING BIOTIN LABELED PROBES

Preparation of Chromosome Squashes

This method of preparing polytene chromosomes; for use with in situ hybridization; is very similar to the protocol described in the previous chapter. It is an easily learned technique and with a little practice will yield consistent results.

  1. For the best results larvae should be grown in well yeasted media at 18C and care should be taken not to overcrowd the bottles.

  2. On a clean siliconized slide place two drops of 45% acetic acid and a clean siliconized 18 mm square coverslip. On the coverslip place a small drop (2-3 mm) of fixative( 1/2/3 mixture of lactic acid/water/glacial acetic acid).

  3. In the first drop of 45% acetic acid dissect out the salivary glands from a third instar larva.

  4. Transfer the glands to the second drop of 45% acetic acid and cut off the anterior ends of the glands. Quite often this will allow the glands to separate from the membrane and the attached fat body. If this does not occur, carefully dissect away the fat body.

  5. Transfer the "fat free" glands into the fixative and incubate for 4-5 minutes.

  6. Pick up the coverslip and glands with a clean slide by carefully touching the slide to the drop of fixative.

  7. Place the slide, coverslip up, on several sheets of paper towel. With another sheet of paper towel hold the edge of the coverslip to prevent it from moving.

  8. Using a stiff, pointed probe, gently press once over the glands. Then gently tap the coverslip, starting over the glands, working your way toward the edge in a spiral pattern.

  9. Turn the slide over so that the coverslip is between the paper towel and the slide. Press gently on one edge of the slide so that the fixative flows to the opposite edge.

  10. Turn the slide back over and again hold the edge of the coverslip with a paper towel.

  11. Using the probe, gently streak the coverslip with a rapid back and forth motion at one edge and then gradually move to the opposite edge.

  12. Blot off excess fixative and examine under the microscope using phase contrast. If spreading is not sufficient, gently restreak the coverslip. On a good slide the chromosomes should appear low contrast, cytoplasmic debris should be vague, and no fixative should be flowing between the slide and the coverslip.

  13. Incubate the slide over night at a temperature between 4 and 18C. Leaving the slides at room temperature for a couple of hours before incubating them will help when incubating at or near 4C.

  14. Place the slides in a dry ice/ethanol bath for 60 minutes. During or before this time cool down some 100% ethanol in a staining dish to -60 to -80C.

  15. Remove the slides individually from the dry ice/ethanol bath and quickly flip off the coverslip with a razor blade and place the slide immediately into the chilled ethanol.

  16. Allow the ethanol to warm up to room temperature, then air dry the slides. Chromosome squashes can be stored at this stage for several months before hybridization.

Pretreatment of Chromosomes for Hybridization

The chromosomes used for hybridization must be non-refractile after ethanol dehydration (A [16]). The chromosomes on the slide should look the same at this point as they did when the slide was made or it is not worth using. In general one slide is enough for a single probe if the chromosomes have good morphology. After the pretreatment the slides should be hybridized within 24 hours.

  1. Place the slides in 2X SSC at 65C for 30 minutes.

  2. Wash for 2 minutes in 2X SSC at room temperature.

  3. Acetylate the slides as follows: Prepare 500 ml of 0.1 M triethanolamine-HCl (1M stock pH 8.0) in a staining dish containing a magnetic stirring bar. Rapidly agitate the solution and add 0.625 ml of acetic anhydride. Turn the stirrer off and immediately place the slides into the solution. Incubate for 10 minutes. (Some people claim this step is unnecessary, but it has not been tested in this lab.)

  4. Wash the slides in 2X SSC two times for 5 minutes each.

  5. Ethanol dehydrate two times 5 minutes in 70% ethanol and 5 minutes in 95% ethanol. Air dry.

  6. Denature the chromosomes by incubation in freshly prepared 0.07 N NaOH for 3 minutes.

  7. Repeat steps 4 and 5.

Biotinylation of DNA

The following protocol is for 0.5 ug of DNA or less. The quality of the DNA is not important. Quick prep DNA and DNA fragments isolated by gel purification work equally well as CsCl purified DNA. Probes can be stored at -20C for several months.

  1. Prepare a 1:400 dilution of a 1 mg/ml stock of DNAse I (Worthington, DPFF grade) in 10 mM Tris, pH 7.5/10 mM MgCl2 (TM10). The DNAse I stock should be prepared in TM10 and should be stored in 20ul aliquots at -20C.

  2. Mix:

    add 10 U of DNA polymerase I and incubate at room temperature for 60 minutes.

  3. Stop the reaction by adding 25 ul of 50 mM EDTA, pH 8.0.

  4. Remove unincorporated nucleotides by separation over a P 10 spin column.

  5. Check the percentage of incorporation by comparing the signals of the column and the flow through on a Geiger counter. 5 - 10% incorporation is sufficient. Probes that incorporated more than 40 - 50% might not do so well in the hybridization, since oversubstitution of the Bio-16-dUTP renders the forming hybrids less stable.

  6. Add 20 ug of carrier DNA (Herring Sperm or equivalent) and 5 ul of 3 M sodium acetate. Precipitate the DNA by adding 2.5 volumes of ethanol and incubate in dry ice/ethanol for 20 minutes or at -20C overnight.

  7. Recover the DNA by centrifugation and dry under vacuum for 2 minutes.

  8. Resuspend the pellet in 75 ul of hybridization buffer.

Hybridization and Washes

  1. Denature the hybridization probe by boiling for 3 minutes and chill on ice.

  2. Apply 5 ul of hybridization solution on the slides and distribute the solution over the chromosomes with a 18 x 18 mm coverslip. Try to avoid trapping air bubbles.

  3. To prevent evaporation of the probe seal the edges of the coverslip with rubber cement.

  4. Incubate in a moist chamber at 58C for 12-18 hours.

  5. Place the slide in a beaker containing 2X SSC and pry off the coverslip and rubber cement with a scalpel blade.

  6. Wash the slides 3 times 15 minutes in 2X SSC at 53C. Proceed directly with the signal detection. It is important not to let the slide dry out during the signal detection procedure.

Signal Detection

The age or storage conditions of the Detek I-hrp kit can have a dramatic effect on the kit's proficiency to detect signals. Kits that are over six months old or that have not been kept cold (2-8C) can give weak or inconsistent signals. On the other hand, under ideal conditions probes can be diluted 10-fold and still yield strong signals.

  1. Wash the slides in 1X PBS 2 times for 5 minutes each at room temperature.

  2. Wash for 2 minutes in 1X PBS/0.1 % Triton-X-100.

  3. Rinse in 1X PBS.

  4. Place the slides horizontally on two 5ml pipettes in a tray lined with moist paper towels. Apply 100ul of a 1:250 dilution of the Streptavidin-biotinylated peroxidase complex in 1X dilution buffer (all the components are supplied in the Detek I-hrp kit by ENZO Diagnostics, Inc.) Distribute the solution by placing a 22 x 40 mm coverslip on the slide.

  5. Cover the tray with plastic wrap and incubate at 37C for 30 minutes.

  6. Float the coverslip off in a beaker of 1X PBS and repeat steps 1 through 3.

  7. Prepare a solution containing 0.5 mg/ml of diaminobenzidine in 1X PBS. Add 1/100 volume of 1% hydrogen peroxide (prepared from a 30 % stock in distilled water). The staining solution should always be prepared fresh. Diaminobenzidine is a carcinogen. Wear gloves when handling the solution. Diaminobenzidine can be inactivated by bleach which should be used liberally when disposing of unused diaminobenzidine and contaminated materials.

  8. Place the slides horizontally in a tray and add 50 ul of staining solution onto each slide, cover with a 22 x 40 mm coverslip and incubate at room temperature for 1 - 10 minutes. The staining reaction can be observed by blotting off excess solution and examining the slide under the microscope using a low magnification phase contrast lens (e.g. 16x).

  9. Stop the reaction by dipping the slides in distilled water three times and then place them in distilled water until staining.

  10. Prepare a 4% dilution of Giemsa stain in 10 mM sodium phosphate buffer, pH 6.8. Stain the slides for 30 seconds, rinse in water and allow the slides to air dry. It is important not to overstain the chromosomes for this may obscure the diaminobenzidine precipitate.

  11. Place a drop of mounting media over the chromosomes and cover with a 22 mm square coverslip.

  12. Examine the chromosomes using phase-contrast optics. Hybridization signals appear as black bands. Weaker signals are brown.

Solutions

1) 10X PBS 
	1.3 M NaCl 		
	0.07 M Na2HPO4	  
	0.03 M NaH2PO4 	   		 

2) 10X Nick-Translation Buffer
	0.5  M Tris-HCl, pH 7.5
	0.1  M MgSO4
	1 mM dithiothreitol (DTT)
	500 ug/ml bovine serum albumin (BSA Pentax Fraction V)

3) Hybridization Buffer
	0.6 M NaCl
	50 mM Sodium phosphate buffer, pH 6.8
	1X  Denhardt's  (0.02% BSA / 0.02% Ficoll / 0.02% polyvinylpyrrolidone)
	5 mM MgCl2

Reagents and Suppliers

1) Bio-16-dUTP
	Cat. No. 42811 
	ENZO Diagnostics, Inc.
	325 Hudson St.
	New York, NY  10013
	1-800-221-7705

2) Detection Kit
	Detek I-hrp     
	Cat. No. 43820       
	contains: Streptavidin-biotinylated peroxidase complex / 10X dilution buffer 
	ENZO Diagnostics, Inc.
	325 Hudson St.
	New York, NY  10013
	1-800-221-7705

3) Diaminobenzidine 
	3',3' diaminobenzidine tetrahydrochloride
	Cat. No. D 9015
	Sigma Chemical Company
	P.O. Box 14805
	St. Louis, MO  63187
	1-800-352-3010

4) DNAse I (Grade DPFF)
	Cat. No. LS 6330
	Worthington Biochemicals
	Halls Mill Road
	Freehold, NJ  07728
	1-800-445-9603

original text by Todd Laverty