DAY ONE
Reagents:
Terminal Deoxynucleotidyl Transferase (TdT)
- Invitrogen Cat #: 10533-0651X TdT Buffer
- For 500 µl, add 400 µl PBS to 100 µl 5X Buffer
Digoxygenin-dUTP
- Roche Cat #: 11-093-088-910
PBS
1X SSC
Bleach (For 5ml)
- 0.125 ml 20X SSC
- 0.2 ml hydrogen peroxide
- 4.5 ml distilled water
- 0.25 formamide
Rehydration:
5 minute washes
o Methanol
o 75% Methanol
o 50% Methanol
o 25% Methanol
o 100% 1xSSC
o 100% 1xSSC
Bleach: o 1-2 hours in bleach under direct light
PBS Washes:
o 15 min in PBS
o 15 min in PBS
TdT Buffer Incubation:
o 1 hour in 500 µl 1X TdT Buffer at RT
Label Terminal Ends:
o Add 500 µl 1X TdT Buffer
o Add 1 µl of 15U/µl TdT enzyme per 100 µl buffer to make 150U/ml TdT
o Add 0.1 µl of DdUTP per 100 µl buffer (DdUTP stock is 1000X)
Leave Overnight at Room Temperature
DAY TWO
Reagents:
1 mM EDTA/PBS
Dilute .5 M EDTA with PBS
MAB
Anti-digoxygenin AP antibody
(Roche Cat #:11-093-274-910)
2% BMB Blocking Reagent in MAB
1 mM EDTA/PBS Washes at 65ºC:
o 1 hour
o 1 hour
PBS Washes at RT:
o 1 hour
o 1 hour
o 1 hour
o 1 hour
MAB Washes at RT:
o 5 minutes
o 5 minutes
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1ml Eppendorf tube is recommended to save on reagents.
MAKE SURE it is digoxygenin-dUTP (DdUTP). DNA gets labeled not RNA!
The buffer supplied by the kit is very limited. To make 5x Buffer: - Berger (1987) Meth. Enz. 152, 339.
1. Titrate 1 M Cacodylic acid to pH 7.2 with KOH.
2. Equilibrate K+ Cacodylate with Chelex 100 (ion exchanger from BioRad) at RT for 5 min. Chelex removes metals that can stimulate endonucleases.
3. Filter.
4. Add in order: H2O, DTT, CoCl2 to a final conc. of 0.5 M cacodylate, 1 mM DTT, 10 mM CoCl2***If CoCl2 is added before DTT, a ppt will form. Final buffer is tinted pink.
TdT is an enzyme that catalyzes the binding of digoxygenin conjugated dUTP to the 3¢ end of DNA strands.
EDTA is a calcium chelator that stops the transferase reaction.
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Blocking:
o 1 hour in 2% BMB blocking solution at RT
Antibody Incubation:
o Add 1/3000 dilution of anti-digoxygenin AP antibody in 2% BMB Blocking Reagent at 4˚C O/N
DAY THREE
Reagents:
Alkaline Phosphatase buffer (AP Buffer)
Contains 5 mM levamisol and .1% Tween
MAB
Bouin's Fixative
- 14 ml saturated Picric Acid
- 1ml glacial acetic acid
- 5 ml formaldehyde
NBT 75 mg/ml 70% DMF
BCIP 50 mg/mlin 100% DMF
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BMB is a blocking reagent to prevent non-specific antibody binding.
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5 MAB Washes at RT:
o 1 hour
o 1 hour
o 1 hour
o 1 hour
o 1 hour
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These washes are necessary to wash off excess antibody. The washes are flexible. One can do a quick wash then wash overnight at 4˚C.
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AP Buffer Washes at RT:
o 5 minutes
o 5 minutes |
AP buffer inhibits endogenous phosphatases.
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Staining:
o Transfer into NBT/BCIP 4.5 µl NBT + 3.5 µl BCIP per ml of AP buffer 4oC – may only take a few minutes
Stop Color Reaction:o Stop chromogenic reaction in quick MAB wash |
We use NBT/BCIP instead of BM purple. The reaction comes up quickly and BM Purple often turns different colors - pale blue to purple and is quite superficial. NBT/BCIP behaves better and stays purple and quite nuclear rather than diffuse.
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Fix Stain:
o Bouin's Fix O/N at RT
DAY FOUR
Reagents:
70% buffered Ethanol
Methanol
o Multiple 10 minute washes in 70% buffered EtOH at RT
o Multiple 5 minute washes in Methanol
Take pictures of uncleared tissue, or embryos can be dehydrated and cleared in BB:BA clearing agent (2 parts benzyl benzoate/1 part benzyl alcohol).
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The change in pH stops the color reaction and the formaldehyde fixes the stain.
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