Triple Fluorescent In Situ

Tyr-Fluor/Cy3/Cy5 Triple RNA in situ Protocol

Conceptualized protocol:

Permeabilize embryos, hybridize probes, destroy native peroxidase activity,bind anti-Fluorescein-peroxidase, develop Tyr-Fluorescein, destroy peroxidase activity, bind anti-DIG-peroxidase, develop Tyr-Cy3, destroy peroxidase activity, bind anti-DNP-peroxidase, develop Tyr-Cy5, H2O2 treat to reduce background.

Embryo preparation:

1. Fix embryos overnight at 4˚c in 4% PFA/1X PBS

2. Wash twice in PBST

3. Dechorionate embryos using a pair of watchmakers forceps

4. Dehydrate with a series of methanol/PBST solutions (25%, 50%, 75% methanol mixed with PBST), then twice with 100% methanol. Shake 3-5 minutes in each solution.

5. Store the embryos in 100% methanol at -20˚C

Day 1:

1. Rehydrate the embryos through a methanol/PBST series (75%, 50%, 25% methanol mixed with PBST) 3-5 minutes per wash.

2. Wash five minutes in PBST, four times.

3. Treat the embryos with proteinase K in PBST to increase the permeability of the membrane. Stagger start times, so all fish end treatment concurrently.

                        Embryonic stage                        Length of ProK            ProK concentration

                        24 hpf                                                10 min                                    1µg/ml

                        30 hpf                                                20 min                                    1µg/ml

                        36 hpf                                                30 min                                    1µg/ml

                        48 hpf                                                45 min                                    1µg/ml

                        55 hpf                                                1 hour                                    1µg/ml

                        60 hpf                                                25 min                                    10µg/ml

                        72 hpf                                                30 min                                    10µg/ml

4. Fix the embryos in 4% PFA/1XPBS for 20 minutes to ensure the ProK has stopped.

5. Wash 5 X 5 mins in PBST

6. Replace with 1ml Pre-Hyb solution

7.  Incubate at 68-70˚C for at least 4 hours.

8. Replace with 200µl fresh Pre-Hyb.

9. Add 2µl of each probe (no more than 1µg of each probe) to the tubes.

10. Incubate overnight @ 68-70˚C

Day 2:

11. Remove probes, and save them for reuse. (store @ -80˚C)

12. Wash 5 mins in “5X” @ 68-70˚C

13. Wash 5 mins in 3:1  5X:2X @ 68-70˚C

14. Wash 5 mins in 1:1  5X:2X @ 68-70˚C

15. Wash 5 mins in 1:3  5X:2X @ 68-70˚C

16. Wash 5 mins in “2X” @ 68-70˚C

17. Wash 3 X 20 mins in 0.2X SSC, 0.25% tween-20 @ 68-70˚C

18. Wash 2 X10 mins in PBST  @ room temp

19. Replace with freshly prepared 2% H202 in PBST

20. Shake 60 mins @ room temp

21. Wash 4 X 5 mins in TNT

22. Block at least four hours in 400µl TBSTB

23. Replace with 400µl preabsorbed 1:5000 anti-Fluorescein-POD in TBSTB.

24. Rock overnight @ 4˚C

Day 3:

25. Wash 8 times in TNT at room temp over the course of 1-2 hours

26. Wash five minutes in 50µl Perkin Elmer Amplification Diluent

27. Prepare 1:50 Tyr-Fluorescein in Amplification Diluent for 50µl*(n+1) samples.

28. Replace amplification diluent with 50µl 1:50 Tyr-Fluorescein

29. Shake one hour in the dark, tubes upright. Don’t exceed one hour. From here on out longer steps are done in the dark (ie- a covered box), though the dyes are quite photo-stable, so I do the shorter washes under normal lighting.

30. Wash 2 X 5 mins in TNT

31. Wash one hour in 2%H2O2/TNT

32. Wash 4 X 5 mins in TNT

33. Block with 400µl TBSTB 1-4 hours

34. Replace with preabsorbed 1:1000 anti-DIG-peroxidase in TBSTB.

35. Rock overnight at 4˚C.

Day 4:

36. Wash 8 times in TNT at room temp over the course of 1-2 hours

37. Wash five minutes in 50µl Amplification Diluent

38. Prepare 1:50 Tyr-Cy3 in Amplification Diluent for 50µl*(n+1) samples.

39. Replace amplification diluent with 50µl 1:50 Tyr-Cy3

40. Shake one hour, tubes upright.

41. Wash 2 X 5 mins in TNT

42. Wash one hour in 2%H2O2/TNT

43. Wash 4 times in TNT

44. Block with 400µl TBSTB 1-4 hours

45. Replace with preabsorbed 1:500 anti-DNP-peroxidase in TBSTB.

46. Rock overnight at 4˚C.

Day 5:

47. Wash 8 times in TNT at room temp over the course of 1-2 hours

48. Wash five minutes in 50µl Amplification Diluent

49. Prepare 1:50 Tyr-Cy5 in Amplification Diluent for 50µl*(n+1) samples.

50. Replace amplification diluent with 50µl 1:50 Tyr-Cy5

51. Shake one hour, tubes upright.

52. Wash 2 X 5 mins in TNT

53. Wash one hour in 2%H2O2/TNT

54. Wash 4 times in TNT

55. Store fish in TNT @4˚C.

Pertinent Materials:

Anti-DNP-POD: Perkin-Elmer Cat#NEL747A001KT. Comes with blocking powder.

Anti-DIG-POD: Roche cat# 1207733

Anti Fluor-POD: Invitrogen cat# A-21253

The DIG probe sysnthesis materials can be obtained from Roche in a kit (Cat# 11175025910), or individually. With the exception of the DIG mix, this kit can also be used for Fluor probe synthesis, or DNP probe synthesis.

Fluorescein Labeling Mix: Roche, cat# 11685619910

20XNTP mix: 100mM NTP’s obtained from Amersham Bioscience (cat# 27202501).

Mix: 10µl each of ATP, GTP, CTP with 6.5µl UTP in 13.5µl nuclease free H2O

Resulting in: 20mM each ATP, GTP, CTP, 13mM UTP stock.

20X DNP-11-UTP stock: Obtain a 250nmol/25µl stock of DNP-11-UTP from Perkin-Elmer (cat# NEL555001EA). Add 10.7µl nuclease free H2O for a 7mM stock

TSA Cyanine 3 and Fluorescein system: Perkin Elmer Cat# NEL753001KT

TSA Cyanine 5 system: Perkin Elmer Cat# NEL745001KT

Antibody preabsorption (Optional- if desired.):

1. Prepare100 embryos of mixed stages with in situ (Day 1) protocol steps 1-7, ending in pre-hyb on day one of the in situ. These can be stored at -20˚c for quite a while. On in-situ Day 2, Day 3, and Day 4, prepare embryos respectively for µFluor µDig, µDNP preabsorption.

2. Wash five times in TNT

3. Wash once in TBSTB

4. Add TBSTB, 400µl times (number of samples plus 1)

5. Add anti-DIG-peroxidase (1:1000) or anti-DNP peroxidase (1:500) or anti-Fluorescein peroxidase (1:5000) to the tubes.

6. Shake several hours at room temp. After this wash, the pre-absorbed antibodies are ready for use.

7. After use, store the embryos in pre-hyb solution. They can be reused several times. Be sure to label what antibody was used on those embryos, and only reuse them for that antibody. To reuse, remove fish from freezer and enter pre-absorption protocol on step 2.


PBST: (1X PBS, 0.25% tween-20)

100ml 10X PBS            12.5ml 20% tween-20                        sterile H2O to 1L

store at room temp

TNT: (0.1 M Tris-Hcl pH 7.5; 0.15 M NaCl; 0.5% Tween20)

100ml Tris pH 7.5            30ml 5M NaCl            25ml 20% tween-20            sterile H2O to 1L

store at room temp

TBSTB: (TNT with 0.5% Perkin-Elmer blocking powder)

50ml TNT                        0.25g Perkin Elmer Blocking Powder

Mix well, and heat @ 68˚ for one hour to get powder into solution.

Store at -20˚C, and once thawed, never refreeze.

Pre-Hyb: (50% formamide, 5X SSC, 100µg/ml yeast RNA, 50µg/ml Heparin, 0.25% tween-20, Citric acid to pH 6.0 [appx 0.02M citric acid final])

100ml formamide            50ml 20X SSC             2.5ml 20% tween-20   sterile H2O to 200ml

200µl 50mg/ml Heparin            400µl  250mg/ml yeast tRNA             1.9ml 1M citric acid

Test pH, should be 6.0-6.5

store @ 4˚c

“5X”: (5X SSC, 50% formamide, 0.25% tween-20)

100ml formamide            50ml 20X SSC            2.5ml 20%tween            47ml sterile H2O

store at 4˚C

“2X”: (2x SSC, 0.25% tween-20)

40ml 20X SSC            5ml 20% tween-20            sterile H2O to 400ml

store at room temp

“0.2X”: (0.2X SSC, 0.25% tween-20)

4ml 20X SSC                        5ml 20% tween-20            sterile H20 to 400ml

store at room temp

Probe synthesis:

DNP Probes:                                    DIG probes:                                    Fluor Probes:

1-2µg linear plasmid                       1-2µg linear plasmid                    1-2µg linear plasmid

1µl 20X NTP                                    2µl DIG NTP mix                           2µl Fluor NTP mix

1µl 20X DNP-11-UTP

2µl 10X TXN buffer                        2µl 10X TXN buffer                      2µl 10X TXN buffer

1µl RNAse inhibitor                       1µl RNAse inhibitor                      1µl RNAse inhibitor

2µl T7/SP6/T3                                 2µl T7/SP6/T3                                2µl T7/SP6/T3                       

Nuclease free H2O to 20µl            Nuclease free H2O to 20µl           Nuclease free H2O to 20µl

1. Prepare the above mixtures

2.Incubate 2 hours @ 37˚C

3. Mix in 2µl DNAse I

4. Incubate 15 minutes at 37˚C

5. Add 0.8µl 0.5M EDTA pH 8.0

6. Add 2µl 5M LiCl

7. Add 75µl Prechilled 100% Etoh

8. Place @ -80˚C for 45 mins to overnight

9. Centrifuge 10 mins @ 4˚C

10. Remove liquid

11. Rinse with 200µl Prechilled 80% Etoh

12. Centrifuge 5 mins @ 4˚C

13. Remove as much liquid as possible

14. Air dry 5-10 mins at room temp

15. Resuspend in 60µl nuclease free H2O

16. Mix in 1µl RNAse inhibitor

16. Store at --20˚C ASAP.

Diagnostic gel:

1. Mix 2µl probe with 5µl formamide, 3µl nuclease free H2O.

2. Heat this aliquot 3 mins @ 68-70˚C

3. Run products 20 mins on a 1.5% gel @ 130V


The largest factor that dictates whether the in situ will work well is the quality of probes used. When I’ve had problems with this protocol, I’ve generally been able to solve them with new, cleaner, probes. When a probe has worked well once, it has continued to work well when reused several times. In my hands, DNP works as well, if not better, than DIG for probe labeling, though Fluorescein labeled probes have the reputation for giving background. Tyr-Fluor and Tyr-Cy3, and Tyr-Cy5 produce comparable signal to noise ratios and good labeling when developed under similar conditions. Cy5 (far red) wont show up under many fluorescent dissecting microscopes, so we generally use this probe primarily to give context to the other two probes. As per the norm with in situ, it is critical that the pH of pre-hyb is around 6-6.5, and it is also critical that the fish never dry out. The dyes show very little photobleaching, even if left in the light overnight. It is critical that the antibodies are not left at room temperature for too long, as this destroys peroxidase activity. Some of the antibodies are shipped at room temperature- they should work, but must be stored at 4 degrees immediately upon receipt.


This protocol combined procedures developed in a number of labs, and contains modifiers from Isabelle Manfroid, Macie Walker, and Yi-Lin Yan’s working protocols, in addition to our own (Jared Coffin Talbot). See also:

MCM Welten et al. 2006. ZebraFISH: Fluorescent in situ hybridization protocol and three dimensional imaging of gene expression patterns. Zebrafish v3 (4) 465-476 

I Manfroid et al. 2007. Reciprocal endoderm-mesoderm interactions mediated by fgf24 and fgf10 govern pancreas development_. Development_ v134 (22) 4011-4021

M Lopez et al. 2006. Expression of the somatolactin beta gene during zebrafish embryonic development. Gene Expression Patterns v6 (2) 156-161

T Jowett, YL Yan. 1996. Double fluorescent in situ hybridization to zebrafish embryos. Trends In Genetics v12 (10) 387-389