3 color Fluorescent in situ on sections

Tyr-Fluor/Cy3/Cy5 Triple Fluorescent in situ Protocol on  Cryostat Sections of Zebrafish


Yi-Lin Yan, Jared Coffin Talbot, and Ruth BreMiller




.                                                                  Protocol

Fixation and sectioning

1. Fix fish in 4% paraformaldehyde in PBS (see appendix) over night at  4o  C. 

2. Rinse fish 3X in PBS.

3. Embed fish in embedding medium (see appendix). 

4. Store blocks in 30% sucrose at 4oC until they sink.  Usually this takes overnight.

5. Cut 16 µm cryostat sections of embedded fish and transfer to Fisher brand Superfrost /Plus microscope slides  (Fisher Scientific Cat #12-550-15).

6. Allow sections to air-dry overnight at room temperature.

7. Store slides in a box in a -20o C freezer for up to one year.



Day 1

8. Defrost sections at room temperature.   Allow for at least an hour before opening box.   Ring the sections with a PAP pen.

9. Dilute probes (dig labeled, fluorescent labeled and DNP labeled probes)  in hybridization buffer (usually 0.1-1 µg / ml).  Denature the probe mix for 5-10 minute. at 70o C.  Vortex thoroughly.

10. Add ~250µl probe mix to sections. Keep the probe mix warm (~70˚ C) while adding probes on slides.

11. Cover slide with a coverslip. Choose sizes of the coverslips which are appropriate for the number of sections on the slide.

12. Hybridize overnight at 68o C in a covered Perspex box with 2 sheets of Whatman paper wetted with 1X SSC/50% formamide.  Use wood applicator sticks or appropriate material to support the slides so that they don't sit on the filter paper.



Day 2

13. Pre-warm wash solution (1XSSC, 50% formamide, 0.1% Tween-20) to 70o C. Transfer slides to polypropylene Coplin jars containing the warm solution.  As many as 9 slides angled (as opposed to back to back) can be accommodated in one jar.

14. Wash for 15 minutes at 68-70o C to allow coverslips to fall off.  Transfer slides to fresh wash solution.  (Count the coverslips to be sure all have fallen off).

15. Wash 3X in wash solution at 68-70o C, 30 minutes each.

16. Transfer slides to TNT (0.1 M Tris-HCl pH 7.5; 0.15 M NaCl; 0.5% Tween 20) (see appendix) at room temperature.

17. Wash 2X in TNT, 10 minutes each at room temperature.

18. Add 2%  H2O2 in TNT and place on a shaker for 15 minutes, to quench native peroxidase activity.

19. Wash 3X in TNT, 10 minutes each at room temperature.

20. Drain slides, shake off excess fluid and recircle the section with a PAP pen.  (Don't allow sections to dry.)

21. Transfer slides to Perspex box containing filter paper wetted with PBS.   Add 500 µl of blocking solution (TBSTB) to slide.  Incubate at room temperature for a minimum of an hour.  Longer incubation (2 to 3 hours) is desirable.

22.  Drain slides and renew PAP pen circles if necessary. Return slides to Perspex box and add 1:2000 anti-Fluorescein-POD in TBSTB.

23. Incubate overnight at room temperature. 



Day 3

24. Transfer slides to Coplin jars containing 40 ml of TNT.

25. Wash in TNT 4 X for 10-15 minutes at room temperature.

26. Put slides back to Perspex box.

27. Incubate with Perkin Elmer Amplification Diluent for 5 minutes.

28. Prepare 1:50 Tyr-Fluorescein in Amplification Diluent for 100-200 µl*(n+1) samples.

29. Replace amplification diluent with 100-200 µl of 1:50 Tyr-Fluorescein. Let reaction develop for 45 minutes in the dark. Don’t exceed one hour. All steps from here on out are done in the dark.

30. Wash 4 X 5 minutes in TNT.

31. Quench development reaction by washing in 2% H2O2 in TNT for 15 minutes.

32. Wash 4 X 5 minutes in TNT.

33. Block with 500 µl TBSTB 1-4 hours.

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

35. Incubate overnight at room temperature.



Day 4

36. Wash slides 4X in TNT at room temperature over 1 hour total.

37. Wash five minutes in 100-200 µl Amplification Diluent.

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

39. Replace amplification diluent with 100-200 µl of 1:50 Tyr-Cy3.

40. Incubate for 45 minutes.

41. Wash 2 X 5 minutes in TNT.

42. Quench development reaction by washing in 2% H2O2 in TNT for 15 minutes.

43. Wash 4 X 5 minutes in TNT.

44. Block with 500 µl TBSTB for 1-4 hours.

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

46. Incubate overnight at room temperature.



Day 5 

47. Wash slides for 4 X in TNT at room temperature for over 1 hour total.

48. Wash five minutes in 100-200 µl Amplification Diluent.

49. Prepare 1:50 Tyr-Cy5 in Amplification Diluent for 100-200 µl*(n+1) samples.

50. Replace amplification diluent with 100-200 µl of 1:50 Tyr-Cy5.

51. Incubate for 45 minutes.

52. Wash 2 X 5 minutes in TNT.

53. Quench development reaction by washing for 15 minutes in 2% H2O2 in TNT.

54. Wash 4 times in TNT.

55. Drain slides and apply with 50 µl of SlowFade Antifade reagent (Invitrogen Cat# S36936).


Slides are ready for imaging.








Probe synthesis:

DNP Probes:                              DIG probes:                          Fluor Probes:

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

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

1 µl 20X DNP-11-UTP

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

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

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

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


1. Prepare the above mixtures

2.Incubate for 2 hours @ 37˚C

3. Mix in 2 µl DNAse I

4. Incubate for 30 minutes at 37˚C

5. Add 0.8 µl 0.5 M EDTA pH 8.0

6. Add 2 µl 5M LiCl

7. Add 75 µl prechilled 100% ethanol

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

9. Centrifuge for 10 minutes at 12,000 rpm @ 4˚C

10. Remove liquid

11. Rinse with 200 µl prechilled 80% ethanol

12. Centrifuge for 5 minutes at 12,000 rpm@ 4˚C

13. Remove as much liquid as possible

14. Air dry for 5-10 minutes at room temp

15. Resuspend in 60 µl nuclease free H2O

16. Mix in 1 µl RNAse inhibitor

note: one could use RNA clean up kit to purify RNA probes instead of ethanol precipitation. RNA clean up kit: ZYMO CAT# R1015. Elute RNA in  60 µl H2O (using with 65˚C H2O could help to elute more RNA, optional).

17. Check the probe by heating 1 or 2 µl probe in 50% formamide 3 minutes at 68˚ C and running this heated mixture on an 1.5% agarose, 1x TBE or TAE gel @ 130V

18. Store probes at --20˚C or -80˚C ASAP.




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.

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

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

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

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

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

TSA Cyanine 5 system: Perkin Elmer Cat# NEL745001KT.


Embryo preparation:

1. Fix embryos overnight at 4˚C in 4% PF in1X PBS.

2. Wash twice in PBST (1X PBS, 0.25% tween-20).

3. Dechorionate embryos using a pair of  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 up to one year before sectioning.




anti-Fluorescein peroxidase (1:2000) in TBSTB

Add anti-DIG-peroxidase ( 1:1000) in TBSTB

Anti-DNP peroxidase (1:500) in TBSTB



10X PBS:

NaCl 74 g, Na2HPO 10.2 g, NaH2PO4   3.86 g , and H2O to 1 L .Then autoclave.


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

100 ml 10X PBS,   12.5 ml 20% tween-20,   Sterile H2O to 1 L.

Store at room temp


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

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

Store at room temp


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

0.25 g Perkin Elmer Blocking Powder in 50 ml TNT,                       

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

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


20X SSC:

NaCl 140 g , NaCitrate  88 g, EDTA-Di  7.4 g,  H2O less 1 L , adjust pH with 4% NaOH to pH 7.4 and add H2O to  1 L.


50% formamide, 1X SSC, 0.25% tween-20

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


4% PFA

40 g paraformaldehyde in 1 L of 1x PBS.  Heat to dissolve.  Filter the solution and  aliquot in 40 ml tubes. Keep an aliquot of fresh made 4% PFA at 4˚C for no more than one week. Keep the rest aliquots in -20˚C for long term storage.


Alternative Fix

8 g paraformaldehyde                                    4% (w/v)

8 g sucrose                                                    4% (w/v)

24 µl 1M CaCl2                                              0.12 µM

77 ml 0.2 M Na2HPO4                                              0.1 M, pH 7.4

23 ml 0.2 M NaH2PO4

Make up to 200 ml with ddH2O. 

Warm buffer and add paraformaldehyde.  Stir to dissolve.  Fix keeps one week in the fridge.

The quality of the frozen sections seems to be somewhat better with this fix.

Note:  Fixed tissue may be stored in 100% methanol at -20˚C. Dehydrate in methanol series to PBS before sectioning.


Embedding medium

1.0 g low-melt agarose

0.9 g agar (use bacteriological grade)

5.0 g sucrose

100 mL distilled H2O

Place ingredients in a flask and set flask in a beaker of water.  Stir on  magnetic stirrer and then heat until dissolved.

Solution should be clear.  Store in 20 ml scintillation vials at 4˚C.


Hybridization Buffer           

                                                                 50 ml                Final Conc.

10X Salt                                                      5 ml                 1X

formamide                                                 25 ml                  50%           

dextran sulfate                                             5 g                   10%

rRNA (50 mg/ml)                                          1 ml                  1 mg/ml

50 X Denhardt's                                           1 ml                  1X

ddH2O                                                        18 ml           


Dextran sulfate sodium salt

(Sigma: Cat# D8906-100G) Does not go into solution easily.  Shake vigorously and warm to 70o C.  Alternatively, a 50% stock solution can be used, replacing an equivalent volume of water.


Yeast RNA

( Sigma: Cat#R-6750-1G) (Ribonucleic acid from baker’s yeast)

Dissolve in DEPC treated or RNAase free H2O 10 mg/ml final concentration.


50X Denhardt's  (100 ml)

1 gm Bovine serum albuminute                       1% w/v

1 gm FicollTM                                                       1% w/v

1 gm PVP  (Mol. Wt. 360,000)                         1% w/v

Make up to 100 ml with ddH2O.  Freeze in polypropylene tubes.


10X Salt

NaCl                                          114 g 

Tris HCl    pH 7.5                      14.04 g   

Tris Base                                   1.34 g

NaH2PO4                                      7.8 g

Na2HPO4                                   7.1 g

0.5 M EDTA                               100 ml

Bring to 1 L with ddH2O