Granato Laboratory
Last Modified January 2007
Fluorescent In Situ & Antibody Staining
COLLAGENASE PERMEABILIZATION
**This protocol is for embryos between 5 somites and 4 dpf, and will work with most,
but not all primary antibodies**
Fixation
1. Fix embryos in 4% PFA + 1% DMSO in 0.1M phosphate buffer (pH7.4)
Fixation times: 2-4 hours at room temperature OR O/N at 4 oC.
**Use fresh fix: < 2 weeks old.
2. Wash embryos 3x 10 minutes in 0.1M phosphate buffer (rotate on wheel). Be careful,
embryos are sticky at this point and will try to stick to sides of tube/pipette.
**Important: Do not put embryos in contact with any detergent prior to collagenase
treatment**
If you are not ready to go on to step 3, embryos can be stored at 4 oC in 0.1M phosphate
buffer for up to one week prior to staining
Alternatively: Embryos may be stored in 100% MeOH at –20 oC for up to several
months. However, the level of background may be higher. Rehydrate through a MeOH
series before proceeding on to step 3.
Collagenase digestion
3. Incubate in collagenase solution (Sigma C-9891, 0.1% w/v in 0.1M phosphate buffer).
The following are rough estimates of incubation times, as each batch of collagenase is
different. Determine the correct time for each batch you make! Collagenase solution can
be stored in Eppendorf tubes at –80 oC. Once the tube is thawed, do NOT reuse.
Up to 24 hpf: 6 minutes
24 hpf-36 hpf: 9 minutes
48 hpf: 45 minutes
3 days: 75 minutes
4 days: ??
5 days: don’t use collagenase, peel larvae using sharp forceps instead
4. Replace collagenase solution with PBST.
5. Wash 3x 15 minutes in PBST, rotating on wheel (Volume in tube: ~400 ul).
Acetic anhydride treatment (Blocks positively charged groups)
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6. Replace PBST with dH2O (as quantitatively as possible).
7. As quickly as possible, replace H2O with a fresh mixture of 2.5 µl acetic anhydride in
1ml of 0.1M triethanolamine (pH7.0)
8. Incubate for 10 min at RT.
9. Wash 2 times for 10 min each in PBST.
Pre-hybridization
10. Remove PBST and replace with 300µl of HYB¯. Incubate 5 min at 55 °C.
11. Replace HYB¯ with HYB+.
12. Pre-hybridize at 65°C for at least 3 hrs in HYB+.
Hybridization
13. Use 60µl of fresh HYB+ containing1 µl of RNA probe. Heat the probe in Hyb+ for 5
min at 65 °C before adding to the embryos (Note: different probes may require different
dilutions).
14. Remove as much of the pre-HYB+as possible without letting the embryos touch air
and add preheated probe.
15. Incubate overnight at 65°C.
Probe removal
16. Replace probe with preheated 50% formamide in 2x SSCT. (Keep probe and store at
–20 °C; it can re-used at least twice).
17. Incubate embryos 2x 30 minutes at 65°C in 50% formamide in 2xSSCT.
18. Wash 15 min at 65°C in 2xSSCT.
19. Wash 2 times for 30 min each at 65°C in 0.2x SSCT.
Block endogenous Peroxidase
20. Incubate embryos in 0.3 % H2O2 in 1x PBS for 30 minutes.
21. Wash 5x5 minutes in 1x PBST.
In situ probe detection
22. Block for 4 hrs with 2% BM block in 1x MAB (no FCS)
23. Incubate with anti-Dig-POD (Roche) diluted 1:500 in 2% BM block overnight at 4 oC
or 5 hrs at RT.
24. Wash 3x10 minutes in 1 x MABT.
25. Wash 3x10 minutes in TNT buffer.
26. During last wash, dilute fluorescent tyramide stock 1:50 in 1x Amplification Diluent
(provided in TSA FL system (NEN Cat.# NEL 701A for fluorescein and NEL 702 for
rhodamine).
27. Cover tubes with foil and incubate in diluted tyramide for 10 minutes. Tubes should
stay covered in foil from this point on. ** NOTE: Incubation time may vary; for new
probes try different times in the 10-15 minute range).
28. Wash 3x10 minutes with TNT buffer. Wash longer to reduce background if
necessary.
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Fluorescent Antibody staining
29. Wash 3x15 minutes in I.B.
30. Remove I.B. and replace with primary antibody diluted to appropriate concentration
in I.B. (Volume in tube: 50-200 ul).
31. Incubate O/N at 4 oC or 4 hours at RT.
32. Remove primary antibody and save. Most antibodies can be re-used up to 4 times.
Store saved antibody at 4 oC. (Add 0.02% sodium azide as preservative, if necessary).
33. Wash 3x 15 minutes in I.B., rotating on wheel.
34. Remove I.B. and replace with fluorescently conjugated secondary antibody diluted to
appropriate concentration in I.B. (Volume in tube: 50-200 ul).
35. Incubate O/N at 4 oC or 4 hours at RT.
36. Remove secondary antibody and save. Most antibodies can be re-used up to 4 times.
Store saved antibody at 4 oC. (Add 0.02% sodium azide as preservative, if necessary).
37. If doing triple labeling, go back to step 29 and repeat through step 36.
38. After final secondary antibody incubation, wash 3x15 min in I.B.
39. Remove I.B. and replace with 1 drop Vectashield. Let embryos settle to bottom of
tube overnight. Store covered embryos at 4 oC.
40. Mount embryos in Vectashield.
Notes:
Most goat anti-mouse IgG secondary antibodies are diluted 1:400.
If doing double labeling, do IgG2a staining before IgG1, as 2a tends to cross-react, while
1 does not.
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SOLUTIONS
0.1M Phosphate Buffer (pH7.4)
Make 1L stocks of 0.2M Na2HPO4 and 0.2M NaH2PO4
For 400 ml phosphate buffer, mix: 162 ml 0.2M Na2HPO4
28 ml 0.2M NaH2PO4
200 ml pico tab H20
Incubation buffer: 0.1M phosphate buffer + 0.2% BSA + 0.5% Triton-X
Aliquot in 50 ml tubes and store at –20 oC.
Working tube should be kept at 4 oC.
16% PFA stock:
Add 16g PFA pellets to 100 ml of 0.1M phosphate buffer (pH7.4). Stir on hotplate (IN
HOOD, COVERED) at 60-80 oC until PFA dissolves. Let cool.
Filter 16% PFA through 2 micron bottle top filter.
Store in 5ml aliquots at –80 oC.
For use, dilute to 4% in 0.1M phosphate buffer (pH7.4) and add 1% DMSO.
MABT (maleic acid buffer + Tween):
100 mM Maleic Acid
150 mM NaCl
pH 7.5
Adjust w/ NaOH
Autoclave
Add Tween-20 to 0.1%
For 500 ml MAB:
5.81g Maleic acid
4.39 g NaCl
TNT
0.1 M Tris-HCl
0.15 M NaCl
0.05% Tween-20
Hyb50% formamide
5x SSC
0.1% Tween-20
Hyb+
Hyb- with 5 mg/ml torula RNA
20x SSC (1L)
NaCl
175.3 g
NaCitrate
88.2 g
Adjust to pH 7.0 with NaOH or HCl
Bring to 1L with dH2O and autoclave
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