Using Whole Mount in situ Hybridization to Link Molecular and Organismal Biology

1. Plasmid Transformation of Target cDNA

Part I: Transformation of Plasmid

(Time Required: 3 hours plus overnight incubation)

1. Warm SOC nutrient broth in a 42°C water bath (500 μL per reaction)
2. Thaw competent cells on ice
3. Add 1 μL plasmid to 25 μL competent cells
4. Place on ice for 20 minutes
5. Heat shock cells in 42°C water bath for 45 seconds
6. Immediately place cells on ice for 2 minutes
7. Add 500 μL of 42°C nutrient broth to each vial of cells
8. Shake at 37°C for 2 hours at 255 rotations per minute (rpm)
9. Plate 75 μL transformation mix on Luria Bertani (LB) agar plates
10. Incubate plates inverted at 37°C overnight

Part II: E. coli Culture

(Time Required: 15 minutes plus overnight incubation)

1. For each reaction, aliquot 3 mL LB broth and 3 μL of 50 mg/ mL ampicillin into a culture tube
2. Scrape 1 bacteria colony from the agar plate and add to each culture tube
3. Shake culture tubes at 37°C at 255 rpm overnight

Part III: Plasmid Prep

(Time Required: 1.5 hours)

1. Isolate plasmid from overnight cultures using the 5 Prime FastPlasmid Mini Kit (Catalog #2300000)
2. To check for the presence of prepared plasmid, run the DNA eluted from the kit on a 1% Tris-Acetate-EDTA (TAE) gel stained with ethidium bromide

2. In Situ DIG-labeled RNA Probe Synthesis

Part I: Linearization of Plasmid

(Time Required: 2.5 hours)

1. In a 1.5 mL microfuge tube, combine:
   

   Prepared Plasmid	20 mL
   Restriction Enzyme*	2 mL
   Buffer**	10 mL
   10X Bovine Serum Albumin (BSA)	10 mL
   Diethyl Pyrocarbonate (DEPC) Water	58 mL
   	100 mL

2. Incubate at 37°C for 2 hours
   *Varies with plasmid
   **Varies with restriction enzyme

Part II: Transcription

(Time Required: 3 hours)

1. In a 1.5 mL microfuge tube, combine:
   

   Linear Plasmid	4 mL
   10X Transcription Buffer**	4 mL
   Digoxigenin Label Mix	4 mL
   Polymerase*	2 mL
   RNase Inhibitor	2 mL
   DEPC Water	24 mL
   	40 mL

2. Incubate at 37°C for 1 hour
3. Add 2 μL of polymerase*
4. Incubate at 37°C for 1 hour
5. Add 2 μL of DNase
6. Incubate at 37°C for 20 minutes
   *Varies with plasmid
   **Varies with polymerase

Part III: Precipitation

(Time Required: 5 minutes plus 2 hour to overnight incubation, 1 hour to centrifuge and re-suspend)

1. Add 4 μL of 0.2M EDTA
2. Add 5 μL of 4M lithium chloride
3. Add 150 μL of ice cold 100% ethanol
4. Incubate at -80°C for 2 hours to overnight
5. Centrifuge at 14,000 rpm for 30 minutes at 4°C, decant away supernatant
6. Dry pellet for 7 minutes
7. Re-suspend in 20 μL DEPC water
8. Incubate at 37°C for 5 minutes

Part IV: Fractionation – Perform only if probe size is greater than 0.6 kb

(Time Required: Varies based on probe size, usually no more than 20 minutes)

1. In a 1.5 mL microfuge tube, combine:
   

   RNA Probe	20 mL
   DEPC Water	12 mL
   Sodium Bicarbonate	4 mL
   Sodium Carbonate	4 mL
   	40 mL

2. Incubate in a 60°C water bath. Base the incubation time on the equation:
   Time (min.) = (starting kb – desired kb) / (.11 x starting kb x desired kb)
   Average desired size = 0.6 kb

Part V: Final Precipitation

(Time Required: 5 minutes plus 2 hour to overnight incubation, 1 hour to centrifuge and re-suspend, 1 hour for gel electrophoresis)

1. Add 40 μL DEPC water
2. Add 8 μL Sodium Acetate
3. Add 1.04 μL Glacial Acetic Acid
4. Add 240 μL ice cold 100% ethanol
5. Incubate at -80°C for 2 hours to overnight
6. Centrifuge at 14,000 rpm for 30 minutes at 4°C, decant away supernatant
7. Dry pellet for 7 minutes
8. Re-suspend in 20 μL DEPC water
9. Vortex to mix
10. To check for the presence of riboprobe, run the re-suspended solution on a 1% TAE gel stained with ethidium bromide

3. Whole Mount In Situ Hybridization

Part I: Fixation of Embryos and Proteinase K Digestion

(Time Required: Fix overnight plus 4 hours the next day for storage, 2.5 hours from storage to Part II)

1. Collect staged zebrafish embryos and fix in 4% paraformaldehyde (PFA) overnight at 4°C
2. Wash fixed embryos in phosphate buffered saline containing 0.1% Tween-20 (PBSt) 3 times for 10 minutes each
3. To ensure exposure of the embryos to the experimental reagents, manually dechorionate embryos (if necessary) using fine-tipped forceps
4. Dehydrate embryos by washing in a graded series of 25% and 50% methanol in PBSt for one hour each wash, then store in 100% methanol at -20°C
5. When ready to use, rehydrate embryos in PBSt by washing in 50% (2 times) and 25% (1 time) methanol in PBSt for 10 minutes each. Finally wash 2 times for 10 minutes each in 100% PBSt. Exact timing is not important for PBSt/methanol washes
6. Bleach embryos in a 10% hydrogen peroxide solution in PBSt, if necessary, to remove dark pigments. Embryos should incubate in hydrogen peroxide solution for 10-20 minutes, depending on the age of the embryo, and the cap of the microfuge tube should remain open to prevent build-up of air pressure
7. Digest embryos with 50 mg/ mL Proteinase K diluted 1:5000 in PBSt for 3-15 minutes, depending on the age of the embryo
8. Re-fix embryos in 4% PFA for 30 minutes, and then wash 3 times in PBSt for 5 minutes each

Part II: Hybridization of Riboprobes

(Time Required: 3 hours plus overnight for hybridization, 1.5 hours the next day to Part III)

9. Prehybridize embryos with prehybridization solution (PHS) in a preheated 70°C water bath for 2-3 hours
10. Remove PHS, add 0.5 mL hybridization solution and 1.5 μL previously synthesized Digoxigenin labeled riboprobes, incubate at 70°C overnight. Temperature can fluctuate within a few degrees depending on the riboprobe target
11. The next day, wash embryos at 70°C in graded solutions of 75%, 50%, and 25% PHS in 2X saline-sodium citrate (SSC) for 10 minutes each, then wash in 0.2X SSC for 30 minutes at 68°C
12. Wash in Maleic Acid Buffer (MAB) 2 times for 10 minutes each at room temperature

Part III: Anti-Digoxigenin (α-DIG) Antibody Incubation

(Time Required: 3 hours plus overnight to block, 2.5 hours the next day to Part IV)

13. Transfer embryos to a 12 well plate
14. Pre-block embryos in 1-2 mL blocking solution for at least 3 hours at room temperature
15. Simultaneously, pre-block the antibody by preparing a second volume of blocking solution and diluting the anti-digoxigenin antibody 1:2000 in this solution
16. Remove pre-block and add 1-2 mL pre-blocked α-DIG solution, incubate overnight at 4°C
17. The next day, wash embryos in MAB. Allow the embryos to incubate in MAB for 5 minutes first, then perform buffer changes and incubate for two 10 minute, one 30 minute and one 60 minute interval. Exact timing is not necessary
18. Wash embryos 3 times for 5 minutes each in Alkaline Phosphatase (AP) buffer

Part IV: Staining and Final Processing

(Time Required: 1 hour to overnight for staining, depending on the riboprobe used, 4 hours to beginning of glycerol washes, 6-10 hours per glycerol wash, may be stored in glycerol)

19. Add 1-2 mL staining solution to the embryos, wrap the plate in foil, and check staining at regular intervals (about every 20 minutes) until staining is sufficient
20. Wash embryos with PBSt 2 times for 5 minutes each to stop the staining reaction
21. Dehydrate embryos using 10 minute washes in 25% (1 time) and 50% (2 times) methanol in PBSt, then in 100% methanol, to remove background staining
22. Allow embryos to incubate in 100% methanol for at least 2 hours at room temperature
23. Rehydrate in PBSt using 10 minute washes in 50% (2 times) and 25% (1 time) methanol in PBSt, then wash in 100% PBSt 2 times
24. Transfer stained embryos to an 80% glycerol solution in PBSt, using a graded series of washes, and store at 4°C.

Recipes:

• LB agar plates- 10 g LB agar + 250 mL distilled water, autoclave, when cool to the touch add 250 μL ampicillin, pour 15-20 mL warm solution into each petri dish, allow agar to solidify
• LB broth- 12.5 g LB Broth + 200 mL distilled water, autoclave, allow to cool before use
• 1% TAE gel- 0.4 g agarose, 40 mL 1X TAE, 2 μL ethidium bromide; load 7 μL plasmid (Plasmid Transformation of Target cDNA) or 3 μL riboprobe and 4 μL DEPC water (In situ DIG-labeled RNA Probe Synthesis) + 1 μL loading dye, 5 μL DNA ladder
• Prehybridization solution- 50% formamide, 5X SSC, 9.2mM citric acid, 1% Tween-20
• Hybridization solution- prehybridization solution plus 500 μg/ mL tRNA and 50 μg/ mL Heparin
• MAB- 100mM Maleic Acid, 150mM NaCl, 0.2M NaOH, 0.1% Tween-20, pH to 7.5
• Blocking Solution- 3 parts MAB, 1 part 10% Boehringer blocking reagent in MAB, 1 part heat deactivated lamb serum
• AP buffer- 60mM Tris-HCl pH to 9.5, 60mM NaCl, 30mM MgCl₂, 0.1% Tween-20
• Staining Solution- 5-Bromo-4-chloro-3-indolyl phosphate (BCIP) and Nitro blue tetrazolium (NBT) in AP buffer

4. REPRESENTATIVE RESULTS

When performed correctly, the reaction between the NBT, BCIP, and alkaline phosphatase will form a purple precipitate that should appear on the zebrafish embryo as a purple stain. Riboprobes should be previously synthesized from cDNA corresponding to the gene of interest. Therefore, it can be concluded any stain visualized represents areas of the zebrafish in which the gene of interest has been transcribed at that particular developmental stage. For the purposes of this course, riboprobes were synthesized from aldh1a2 (previously raldh2; Begemann et al., 2001; Figure 1); fgf8a (Reifers et al., 1998; Figure 2); deltaC (Oates et al., 2005); myod1 (Weinberg et al., 1996); shha (Krauss et al., 1993), pax2a (Brand et al., 1996; Figure 3) and myl7 (previously cmlc2; Yelon et al., 1999) cDNA. Staining was expected in the midline and in anatomical structures including the somites, tailbud, myotome, brain, and heart. Ace/fgf8a mutants were expected to have defects in many of these structures. Staining was easily visualized using a standard dissecting microscope. Additional sources contain more information and troubleshooting on WISH techniques similar to those described here (Clark, 2003; D’Costa et al., 2009; Schmoldt et al., 2009; Schoenwolf, 2009).

Figure 1. A zebrafish embryo 24 hours post fertilization, which has been hybridized with riboprobes specific for aldh1a2. Specific staining can be seen in the eyes, hindbrain, pectoral fin bud primordia, and somites. Anterior is to the top, posterior is to the bottom.

Figure 2. A zebrafish embryo at the 13 somite stage of development that has been hybridized with a probe specific for fgf8a. Specific staining is seen in the telencephalon, dorsal diencephalon, midbrain-hindbrain boundary, somites, and tailbud. Ventral is to the left, dorsal is to the right.

Figure 3. A 22 hours post fertilization zebrafish embryo that has been hybridized with a riboprobe specific for pax2a, a robust marker useful for visualizing the nervous system. Specific staining can be seen in the choroid fissure, midbrain-hindbrain boundary, otic vesicle, and spinal cord neurons. Dorsal view with anterior to the left.