本文介绍了斑马鱼原位杂交的一半步骤

In Situ Hybridization

1day

Fixation

1、dechorionate: in 35mm dish add 20mg/ml pronase 30ul to 1ml system water, room temperature 10min, wash twice with system water and transfer to 1.5ml EP.

2、fix embryos in 4% paraformaldehyde(PFA in PBS) overnight at 4℃(<14h).

3、wash PBST 2×10min(RT).

4、dehydrate with: methanol:PBST=1:2 10min

methanol:PBST=2:1 10min

100% methanol 2×10min.

5、stock at -20℃.

2day

Hybridization

1、rehydrate the embryos in: methanol:PBST=2:1 5min

methanol:PBST=1:2 5min

PBST 3×5min

2、fix for 20min in 4%PFA.

3、wash PBST 2×5min(RT).

4、treat with 10ug/ml proteinase K in PBST(1:1000 use), 精确 3min（5体节到24h）；24h 10min；48h 15 min。

5、用PBST轻洗，即加入后立刻吸出。

6、PBST中静置5min。

7、fix in 4%PFA 20min(RT).

8、wash PBST 2×5min(RT).

65℃(preheated)

9、add 400ul/tube HYB-,5min.

10、add 400ul/tube HYB+, 4-6h.

11、add preheated probe 2ug/ml, 300ul/tube, overnight.

3day

Post hybridization washs:现配现用，全部配好后放在65℃预热。

1、wash 5min in 1ml 50% formamide, 5×SSC:2×SSC=1:1, 0.1%Tween 20.

2、wash 10min in 1ml 50% formamide, 2×SSC, 0.1%Tween 20.

3、wash 30min in 1ml 50% formamide, 2×SSC, 0.1%Tween 20.

4、wash 15min in 1ml 2×SSC, 0.1%Tween 20.

5、wash 2×30min in 1ml 0.2×SSC, 0.1%Tween 20.

室温

6、wash 2×5min in MABT.

Immunolocalization of probes

1、add blocking solution 4℃ overnight.

4day