| RNA Whole Mount In Situ Hybridization【Harvard University】 | 点击： 作者：51protocol收集 来源： 时间： 2007-03-28 本站论坛
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Preabsorb the antibody, if desired. (Note: A number of people do not preabsorb the antibody for chick embryos and still get good results, using a dilution of 1:5000 to 1:10,000).
For chick embryos, put roughly 3 mg of chick embryo powder into a microtube with 0.5 ml TBST. Heat at 70° C for 30 minutes, vortex for 10 minutes. Cool on ice, add ml sheep serum and 1 m l anti-dig AP antibody. Shake gently at 4° C for 1 hr. Spin in microfuge for 10 minutes at 4° C. Collect supernatant into a 15 ml tube. Dilute with 1% sheep serum/TBST to a volume of 2 to 8 mls per vial of embryos, depending on the desired antibody dilution.
For mouse embryos, follow the above procedure but use roughly 3 mg of mouse embryo powder and 0.5 ml TBST plus Boerhinger blocking reagent.
Remove blocking solution from embryos. Add antibody and incubate overnight at 4°C.
Day 3: Post-antibody washes
- Wash 3 times for 5 minutes each with TBST at room temperature.
- Wash 5 times for 1 to 1.5 hours in TBST at room temperature.
- Wash overnight in TBST at 4°C with gentle rocking.
Day 4: Color development
- Wash 3 times in NTMT for at least 10 minutes each at room temperature.
- Remove NTMT, add reaction mix (125 mg/ml BCIP and 250 m g/ml NBT in NTMT) and allow the reactions to develop at room temperature with gentle rocking. Throughout the development reaction, keep samples covered with aluminum foil to protect them from light and check them periodically to monitor the progress of the reaction.
- When the reaction is judged complete, wash with PBS or PBT and post-fix in 4% paraformaledehye/ 0.1% glutaraldehyde.
- Wash two to three times in PBS.
Solutions
All solutions should be made with high-quality reagent grade water that is RNase-free. (Note: We routinely use water directly from our purification system without DEPC treatment.) In addition, all solutions containing Tween-20 should be filtered and kept sterile.
10X PBS (1 liter):
80 g NaCl 2 g KCl 14.4 g Na2PO4 2.4 g KH2PO4 (adjust to pH 7.4 with HCl and bring to 1 liter with water)
PBT:
1X PBS plus 0.1% or 1%* Tween *NOTE: For mouse embryos, it is recommended that all solutions with Tween contain 1%, rather than 0.1%. Tween is routinely used at 0.1% in the chick protocol.
Hybridization solution:
50% formamide 5X SSC, pH 4.5 (use citric acid to pH) 1% SDS 50 mg/ml yeast tRNA 50 mg/ml heparin
Solution I:
50% formamide 5X SSC, pH 4.5 1% SDS
Solution III:
50% formamide 2X SSC, pH 4.5 0.1% Tween (NOTE: this is only included for young chick embryos, approx. st 4-st 9)
Sheep serum:
inactivate by heating to 70°C for 30 minutes, store in aliquots at -20°C.
10X TBS (for 1 liter):
80 g NaCl 2 g KCl 250 ml 1M Tris-HCl, pH 7.5 (bring to 1 liter with water)
TBST:
1X TBS plus 0.1/1% Tween 2 mM (0.5 mg/ml) Levamisole (NOTE: most people no longer add levamisole to TBST)
NTMT:
100 mM NaCl 100 mM Tris-HCl, pH 9.5 50 mM MgCl2 0.1/1% Tween-20 2 mM (0.5 mg/ml) Levamisole
200X NBT:
50 mg/ml NBT in 70% DMF
200X BCIP:
25 mg/ml BCIP in water
Embryo preparation
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