Chen-Ming Fan Lab,Carnegie Institute of Washington
Day 1:
Pretreatment and hybridization of embryos (or explants)
Wear gloves, use only DEPC treated solutions (RNase free!), have a heat block @ 70°
1. Take embryos from -20°C Methanol (100%) and rehydrate through PBT series:
75% MeOH / 25% PBT 5MIN or 10MIN*
50% MeOH / 50% PBT 5MIN or 10MIN*
25% MeOH / 75% PBT 5MIN or 10MIN*
100% PBT (2X) 5MIN or 10MIN each*
This and all subsequent washes (Days 1-3) done at room temperature gently rocking
unless otherwise stated. Adjust volumes and wash times appropriately:
*E9.5 and below ~4ml in small snap cap 5MIN each wash (this includes explants)*
*E10.0 and above ~10ml in large snap cap 10MIN each wash (to fully equilibrate)*
2. Bleach with 6% Hydrogen Peroxide in PBT 1HR at RT
Stock H2O2 is 30% stored @ 4° (1ml 30% 4ml PBT)
3. Wash 3X with PBT for 5MIN each at RT (Increase wash time for larger/older embryos)
4. Treat with (10μg/ml) Proteinase K in PBT for 15MIN at RT
Stock is 10mg/ml stored @ -20° (5μl 10mg/ml 5ml PBT)
Do not increase time for older embryos, this step is an exception to the rule
Change to a new pipet dropper after this step (avoid Proteinase K contamination)
5. Wash with freshly made (2mg/ml) Glycine in PBT for 5MIN at RT (10MIN for >E9.5 embryos)
6. Wash 2X with PBT 5MIN each at RT (This & all subsequent washes should be adjusted properly)
7. Refix with .2% Glutaraldehyde/4% Paraformaldehyde in PBT for 20MIN at RT
Stock Glutaraldehyde is 8% stored at -20° (125μl 8% Glu 4.875ml 4% Para)
8. Wash 2X with PBT for 5MIN each at RT
9. Add Prehybridization Solution (~0.25-0.5ml), mix briefly, remove, add final volume
(~1-2ml) of Prehybridization solution. Put at 70° for 1HR. Adjust volume for embryos
From this point on, maintain 70° to avoid nonspecific hybridization of probe
10. Add 70° Hybridization Solution(~0.25-0.5ml), mix briefly, remove, add final volume of
Hybridization solution (~0.5-1ml in small snap cap tube, 1-2ml for large). Hybridize in smallest
volume possible with digoxygenin probe @ [1μg/ml]. Place mineral oil over surface of solution
(prevents evaporation), then place at 70° overnight in heat blocks. Again, in appropriate volumes
Solutions:
PBT : 1X PBS (DEPC H2O) with .1% Tween-20
50 ml of 4% P a raformaldehyde (PFA ) : Dissolve 2g of PFA in ~25ml DEPC H2O with 6μl 10N NaOH,
place at 65° for ~1HR, chill to @ least RT, then add DEPC H2O to 45ml and add 5ml of 10x PBS
-Aliquot to ~10ml volumes and store @ -20° (good for ~a month)
Prehyb and Hybridization Solutions : 50% Formamide, 5X SSC pH 4.5 (Use citric acid to pH), 50μg/ml yeast
RNA,
1% SDS, 50μg/ml Heparin
-Aliquot to ~10ml and store @ -20° (good for a long time)
Day 2:
performed on rotator unless otherwise stated, increase volume & wash time for >E10 (see Day 1)
1. Transfer embryos with as little volume possible and no oil to a fresh snap cap tube
2. Wash 2X with prewarmed 70° Solution I using ~4ml for 30MIN each at 70°
3. Wash 1X with 1:1 mix of Solution I:Solution II, prewarmed to 70° for 10MIN @ 70°
4. Wash 3X with Solution II for 5MIN each at RT (as y'day: adjust wash time for full equilibration)
5. Wash 2X with 100μl/ml RNase A in Solution II for 30MIN each at 37° H2O bath
Put on rocker at RT for 5MIN initially in first wash to help RNase equilibrate
more rapidly into embryo. Stock RNase A is 10mg/ml (40μl 3.96ml Solution II)
6. Wash 1X with Solution II for 5MIN at RT
7. Wash 1X with Solution III for 10MIN at RT
8. Wash 2X with Solution III for 30MIN each at 65°
9. Wash 3X with TBST with 2mM Levamisole for 5MIN each at RT
Levamisole inhibits mammalian alkaline phosphatase (AP) activity (24mg/50ml)
10. Preblock embryos with 10% sheep serum in TBST (with Levamisole) for 2.5HR @ RT in
eppendorf tube. Block endogenous AP in sheep serum: heat @ 70° for 30MIN, store @ -20° or 4°)
11. While blocking embryos, weigh out 3mg of Embryo Powder (stored @ 4°) add 0.5ml
of TBST (with Levamisole) in eppendorf tube & place at 70° for 30MIN, vortex intermittently/
often throughout & at least 1MIN at the end. This is to "expose" as many sites as possible on
embryonic mouse proteins & block nonspecific Ab binding. (This volume is per 1 embryo)
12. Cool on ice, add sheep serum to 1% and goat or sheep anti-dig-AP conjugated Ab @ 1μl/0.5ml
Ab can be inactivated/denatured at high temperatures, be sure to cool the solution
13. Rotate the Ab-blocking solution and Ab for 1HR at 4°
14. Spin in microfuge for 10MIN at 4°
15. Dilute supernatant to 2ml, add ~1.5ml of 1% sheep serum in TBST (with Levamisole)
16. Remove Blocking Serum, add ~0.5ml of dig-Ab Solution, remove, and add final
hybridization volume of ~1.5ml per .5ml of blocking solution (~5X volume of embryos) at 4°
O/N on rocker in eppendorf. Use small snap caps if necessary (>E10 or multiple "older" embryos)
Solutions:
20ml Solution I : 10ml Formamide, 4ml 20X SSC pH 4.5, 1ml 20% SDS, 5ml DEPC H2O
40ml Solution II : 4ml 5M NaCl, 4ml 1M Tris pH 7.5, 40μl Tween-20, 32ml DEPC H2O
20ml Solution III : 10ml Formamide, 2ml 20X SSC pH 4.5, 8ml dH2O
50ml TBST : 1.4ml 5M NaCl, 135μl 1M KCl, 1.25ml 1M Tris pH 7.5, 50μl Tween-20, 47.2ml dH2O
Day 3:
Post Ab binding, AP substrate addition
All washes done on rotator at RT unless otherwise stated, make sure ALL solutions are
fresh and with 2mM Levamisole. NBT and BCIP Solutions are photosensitive, make sure all
solutions are protected from light. As always, adjust volumes appropriately
1. Transfer (if Ab binding performed in an eppendorf tube) to a fresh small snap cap tube, or large
snap cap for older embryos (>E9.5) or high volume of embryos (e.g., multiple #s of E9.5 embryos)
2. Wash 3X with TBST with Levamisole for 10MIN each at RT
3. Wash 5X with TBST with Levamisole for 1HR each at RT
4. Wash 3X with NTMT with Levamisole for 10MIN each at RT
5. Transfer with as little volume as possible to eppendorf tube, or use a small snap cap if this
volume is insufficient
6.. Add 0.5ml of freshly made NBT/BCIP Solution* to embryos, remove, add final substrate
volume ~1-1.5ml (larger appropriate volume if not in eppendorf tube) in light protected tube,
place on rocker at RT for 20MIN
*NBT added at 4.4μl/ml, BCIP added at 3.3μl/ml
7. Remove from rocker and place stationary until sufficient signal is obtained
Continue to keep embryos and solution in the dark
If not removed from rocker substrate will precipitate out of solution
Signal can appear anywhere from 30MIN to overnight
8. When reaction is complete:
A) Wash 2X in NTMT for 10MIN at RT while in dark
B) Wash in PBT pH 5.5 for 10MIN at RT
Both the acidity and phosphates inhibit AP activity
9. Alternately, to completely clear embryos, dehydrate in MeOH/PBT series 25%, 50%, 75%,
100% for 20MIN each at RT on rocker
10. Put in 1:2 Benzyl Alcohol:Benzyl Benzoate Solution for maximum of 2HR then back
through MeOH/PBT series for the following steps:
11. Post-fix embryos in 4% Paraformaldehyde in PBT 0.1% Glutaraldehyde for 1HR
Stock Glutaraldehyde is 8% stored at -20° (125μl 8% Glu 4.875ml 4% Para)
12. Wash 2X with PBT for 10MIN each
12. Store in PBT at 4°
Solutions:
50 ml TBST : 1.4ml 5M NaCl, 135μl 1M KCl, 1.25ml 1M Tris pH 7.5, 50μl Tween-20, 47.2ml dH2O
50ml NTMT : 1ml 5M NaCl, 2.5ml 2M Tris pH 9.5, 250μl 1M MgCl2, 50μl Tween-20, 46.2ml dH2O
NBT : .0075g into 100μl 70% Dimethyl Formamide
BCIP : .0050g into 100μl 100 Dimethyl Formamide
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