Protocol Citation: 张 雪 2020. TUNEL. protocols.io https://protocols.io/view/tunel-bmv7k69n
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 30, 2020
Last Modified: September 30, 2020
Protocol Integer ID: 42655
Before start
1. 收集和固定： 收胚胎于1.5ml EP管中，移去egg water，加入4% PFA，4°C过夜
2. PBT 5min×3，剥皮
3. 脱水：100% MetOH 5min×3，-20°C过夜
Day1
Day1
复水：75%MetOH+25%PBT 1ml shake for 5min00:05:00
50%MetOH+50%PBT 5min00:05:00
25%MetOH+75%PBT 5min00:05:00
100%PBT 5min 1/400:05:00
Note
融化PK
100%PBT 5min 2/400:05:00
100%PBT 5min 3/400:05:00
100%PBT 5min 4/400:05:00
透化：Proteinase K: PBT = 1 : 2000 (终浓度5µg/ml），5min00:05:00
100%PBT 5min 1/200:05:00
100%PBT 5min 2/200:05:00
渗透：丙酮:乙醇（1:2）, -20°C，30min-20 °C 00:30:00
再固定：4%PFA 20min
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
00:20:00
1×PBT 5min 1/300:05:00
1×PBT 5min 2/300:05:00
1×PBT 5min 3/300:05:00
预杂交：+45µL底物（equilibration buffer，Rhoche，红盖），37°C，2～3h，此后避光37 °C 02:00:00
Note
此后所有操作避光
杂交：+5µL酶（Rhoche，蓝盖），37°C，1～1.5h37 °C 01:30:00
2×SSCT，37°C，20min 1/237 °C 00:20:00
2×SSCT，37°C，20min 2/237 °C 00:20:00
2×SSCT，室温摇床，20minRoom temperature 00:20:00
1×PBT 5min 1/300:05:00
1×PBT 5min 2/300:05:00
1×PBT 5min 3/300:05:00
+blocking PBTN 500µl，4°C摇床，2h4 °C 02:00:00 500 µL
+一抗（抗体:PBTN=1:1000，100µl，4°C摇床过夜） Dendra2：Rabbit-anti-Dendra2／ Kaeda：Rabbit-anti-kaeda ／ Mcherry：Mouse-anti-mcherry ／ CFP/GFP：Ab6658（goat），A1122（rabbit），Sc9996（mouse ）／ DsRed：Mouse-anti-DsRed 若实验对象带有两种及以上荧光，注意两种抗体应选择不同的来源100 µL 4 °C
Day2
Day2
回收一抗，保存在4°C冰箱
PT 20min 1/800:20:00
PT 20min 2/800:20:00
PT 20min 3/800:20:00
PT 20min 4/800:20:00
PT 20min 5/800:20:00
PT 20min 6/800:20:00
PT 20min 7/800:20:00
PT 20min 8/8（此后直接孵育二抗，不用加PBTN blocking）00:20:00
＋二抗（in PBTN，1:2000，200µl，4°C过夜）： 例donkey−goat 633，donkey-rabbit 488，donkey-rabbit 633 二抗与对应的所有一抗的来源都不能相同200 µL 4 °C
Day3
Day3
吸去二抗，不回收
PT 20min 1/800:20:00
PT 20min 2/800:20:00
PT 20min 3/800:20:00
PT 20min 4/800:20:00
PT 20min 5/800:20:00
PT 20min 6/800:20:00
PT 20min 7/800:20:00
PT 20min 8/800:20:00
直接拍照或1×PT在4°C摇床洗过夜，后者可以去除很多杂背景