Sep 29, 2020

Public workspacein situ hybridization

 Forked from FISH and antibody staining
This protocol is a draft, published without a DOI.
  • 1西南大学
  • Zhang Xue
张 雪
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Protocol Citation: 张 雪 2020. in situ hybridization. protocols.io https://protocols.io/view/in-situ-hybridization-bmvzk676
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 29, 2020
Last Modified: September 29, 2020
Protocol Integer ID: 42649
Before start
1. 收胚胎于1.5ml EP管中，移去egg water，加入4% PFA，固定至少一晚 2. PBT洗2遍，剥皮 3. 脱水：1ml MetOH，shake for 5min×3 4. -20°C储存至少一晚（胚胎可保存数月）
Day1
Day1
复水：75%MetOH+25%PBT 1ml shake for 5minDuration00:05:00

50%MetOH+50%PBT 5minDuration00:05:00

25%MetOH+75%PBT 5minDuration00:05:00

100%PBT 5min 1/4Duration00:05:00
Note
融化PK


100%PBT 5min 2/4Duration00:05:00

100%PBT 5min 3/4Duration00:05:00

100%PBT 5min 4/4Duration00:05:00

透化：Proteinase K: PBT = 1 : 1000 (终浓度10µg/ml），24h前：不消化；24h：5min；36h：10min；48h：15min；60h：20min；72h及以上：30minDuration00:00:00 Duration00:10:00 Duration00:15:00 Duration00:20:00 Duration00:30:00

再固定：4%PFA 30min
Note
加PFA时，加完一管应立即放在摇床上，否则容易在底部凝结成团
Duration00:30:00

1×PBT 5min 1/4Duration00:05:00

1×PBT 5min 2/4Duration00:05:00

1×PBT 5min 3/4Duration00:05:00

1×PBT 5min 4/4Duration00:05:00

预杂交：500µl Hybe buffer（需提前预热），68.5°C水浴锅中预热2−5hTemperature68.5 °C Duration03:00:00

杂交：6µl probe+200µl Hybe buffer，68.5°C overnightTemperature68.5 °C

Day2
Day2
回收探针，保存在-20°C，可重复使用（以下步骤仍在水浴锅中进行，且不用盖盖子，根据热胀冷缩原理，所吸液体量减至700-800µl）Amount750 µL Temperature68.5 °C
Note
提前配制以下液体，并放在68.5°C水浴锅中预热，29）之前的操作均在水浴锅中进行



Hybe buffer 10minDuration00:10:00

75% Hybe buffer+25% 2×SSCT 10minDuration00:10:00

50%Hybe buffer+50% 2×SSCT 10minDuration00:10:00

25%Hybe buffer+75% 2×SSCT 10minDuration00:10:00

2×SSCT 10minDuration00:10:00

0.2×SSCT 15min 1/4Duration00:15:00

0.2×SSCT 15min 2/4Duration00:15:00

0.2×SSCT 15min 3/4Duration00:15:00

0.2×SSCT 15min 4/4Duration00:15:00
Note
以下步骤在室温水平摇床上进行


75% 0.2× SSCT+25% MABt 5minDuration00:05:00 TemperatureRoom temperature Amount1 mL

50% 0.2× SSCT+50%MABt 5minDuration00:05:00

25%0.2× SSCT+75%MABt 5minDuration00:05:00

MABt 5minDuration00:05:00

1×blocking 500µl（每管配700µl）2h，RTDuration02:00:00
TemperatureRoom temperature Amount500 µL

Anti-Dig-AP 1:2000，200µl，4°C摇床缓慢摇动过夜Temperature4 °C Amount200 µL
Note
注意与FISH不同，此处为Anti-Dig-AP



Day3
Day3
回收抗体，保存在4°C冰箱
1×MABt 15min 1/8Duration00:15:00

1×MABt 15min 2/8Duration00:15:00

1×MABt 15min 3/8Duration00:15:00

1×MABt 15min 4/8Duration00:15:00

1×MABt 15min 5/8Duration00:15:00

1×MABt 15min 6/8Duration00:15:00

1×MABt 15min 7/8Duration00:15:00



1×MABt 15min 8/8（配制NTMT，现配现用，每管需要3.5ml）Duration00:15:00
20mlNTMT
2ml1M pH9.5 Tris-HCl
2ml1M NaCl
0.5ml10% Tween
15.5mlddH2O

Equilibrate with NTMT，5min 1/3Duration00:05:00


转移至24孔板，NTMT 5min 2/3Duration00:05:00

Note
配显色液：BCIP in NTMT（1:50，500µl每管）

NTMT 5min 3/3Duration00:05:00

显色：BCIP in NTMT（1:50，500µl），37°C避光，每隔15min观察一次（BF，磨砂玻璃）Amount500 µL Temperature37 °C Duration00:30:00
Note
避光


1ml stop solution 终止反应后，将胚胎置于甘油中拍照