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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 minute. 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 wash solutionTNT, 10 minutes each at room temperature.
18. Add 2% 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.
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24. Transfer slides to Coplin jars containing 40ml 40 ml of TNT.
25. Wash in TNT 4 X for 10-15 minutes at room temperature.
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29. Replace amplification diluent with 100-200µl 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.
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31. Quench development reaction by washing in 2%H2% H2O2/ in TNT for 15 minutes.
32. Wash 4 X 5 minutes in TNT.
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36. Wash slides 4X in TNT at room temperature over 1hour 1 hour total.
37. Wash five minutes in 100-200 µl Amplification Diluent.
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53. Quench development reaction by washing for 15 minutes in 2%H2% H2O2/ in TNT.
54. Wash 4 times in TNT.
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Slides are ready for imaging.
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Appendix
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DNP Probes: DIG probes: Fluor Probes:
1-2µg 2 µg linear plasmid plasmid 1-2µg 2 µg linear plasmid plasmid 1-2µg 2 µg linear plasmid
1µl 1 µl 20X NTP 2µl 2 µl DIG NTP mix 2µl 2 µl Fluor NTP mix
1µl 1 µl 20X DNP-11-UTP
2µl 2 µl 10X TXN buffer 2µl 2 µl 10X TXN buffer 2µl 2 µl 10X TXN buffer
1µl 1 µl RNAse inhibitor 1µl 1 µl RNAse inhibitor 1µl 1 µl RNAse inhibitor
2µl 2 µl T7/SP6/T3 2µl 2 µl T7/SP6/T3 2µl 2 µl T7/SP6/T3
Nuclease free H2O to 20 µl Nuclease Nuclease free H2O to 20 µl Nuclease free H2O to 20 µl
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3. Mix in 2 µl DNAse I
4. Incubate 15 minutep 30 minutes at 37˚C
5. Add 0.8µl 8 µl 0.5M 5 M EDTA pH 8.0
6. Add 2µl 2 µl 5M LiCl
7. Add 75µl 75 µl prechilled 100% ethanol
8. Place @ -80˚C for 45 minutes to overnight
9. Centrifuge 10 minutes at 12,000 rpm @ 4˚C
10. Remove liquid
11. Rinse with 200 µl prechilled 80% ethanol
12. Centrifuge 5 minutes @ at 12,000 rpm@ 4˚C
13. Remove as much liquid as possible
14. Air dry 5-10 minutes at room temp
15. Resuspend in 60µl 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 60µl water 60 µl H2O (using with 65˚C water H2O could help to elute more RNA, optional).
17. Check the probe by heating 1 or 2 µl probe in 50% formamide 3 mins minutes at 68˚ C and running this heated mixture on an 1.5% agarose, 1x TBE or TAE gel @ 130V
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Mix: 10 µl each of ATP, GTP, CTP with 6.5µl 5 µl UTP in 13.5µl 5 µl nuclease free H2O
Resulting in: 20 mM each ATP, GTP, CTP, 13mM UTP stock.
20X DNP-11-UTP stock: Obtain a 250 nmol/25 µl stock of DNP-11-UTP from Perkin-Elmer (cat# NEL555001EA). Add 10.7µl 7 µl nuclease free H2O for a 7mM 7 mM stock
TSA Cyanine 3 and Fluorescein system: Perkin Elmer Cat# NEL753001KT.
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2. Wash twice in PBST (1X PBS, 0.25% tween-20).
3. Dechorionate embryos using a pair of watchmakers 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 minute minutes in each solution.
5. Store the embryos in 100% methanol at --20˚C up to several months one year before sectioning.
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Antibodies:
anti-Fluorescein peroxidase (1:2000) in TBSTB
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100 ml 10X PBS, 12.5 ml 20% tween-20, Sterile H2O to 1 L.
store Store at room temp
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TNT: (0.1 M Tris-HCl HCl pH 7.5; 0.15 M NaCl; 0.5% Tween20)
100 ml Tris pH 7.5, 30 ml 5M 5 M NaCl, 25 ml 20% tween-20, Sterile H2O to 1 L.
store Store at room temp
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TBSTB: (TNT with 0.5% Perkin-Elmer blocking powder)
0.25 g Perkin Elmer Blocking Powder in 50ml 50 ml TNT,
Mix well, and heat @ 68˚ C for one hour to get powder into solution.
Store Aliqoute and store at -20˚C, and once thawed, never refreeze.
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20X SSC:
NaCl 140 g , NaCitrate NaCitrate 88 g, EDTA-Di Di 7.4 g, H2O less 1 L , adjust pH with 4% NaOH to pH 7.4 and add H2O to 1 L.
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50% formamide, 1X SSC, 0.25% tween-20)
10 ml 20X 20 X SSC, 100 ml formamide, 2.5 ml 20% tween-20, Sterile sterile H2O to 200 ml .
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4% PFA
Put 40 g paraformaldehyde in 1 L of 1x PBS. Heat to dissolve. Filter the solution and and aliquot in 40 ml tubes, and keep . 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.
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8 g sucrose 4% 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
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Solution should be clear. Store in 20 ml scintillation vials at 4˚C.
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Hybridization Buffer
50 ml Final Conc.
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ddH2O 18 ml
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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.
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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.
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50X Denhardt's (100 ml)
1 gm Bovine serum albuminute 1% w/v
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Make up to 100 ml with ddH2O. Freeze in polypropylene tubes.
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10X Salt
NaCl 114 g
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