Protocol Citation: 张 雪 2020. FISH and antibody staining. protocols.io https://protocols.io/view/fish-and-antibody-staining-bmurk6v6
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: 42609
Abstract
荧光原位杂交方法与一般原位杂交方法类似，只是为了不影响后续抗体显色，第一天所用的杂交温度较低，可选择55-65°C，还有第二天加入的是anti-dig-POD（4°C冰箱自取）而不是anti-dig-AP（领抗体），最后显色方法也有不同。
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 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
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×PT 20min 1/300:20:00
1×PT 20min 2/300:20:00
1×PT 20min 3/300:20:00
1×PBT 5min 1/400:05:00
1×PBT 5min 2/400:05:00
1×PBT 5min 3/400:05:00
1×PBT 5min 4/400:05:00
预杂交：500µl Hybe buffer（需提前预热），65°C水浴锅中预热2−5h，其中温度较为灵活(63-68.5°C), 温度低有利于探针的结合，温度高可使背景更干净03:00:00
杂交：6µl probe+200µl Hybe buffer，65°Covernight65 °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）750 µL 65 °C
Note
提前配制以下液体，并放在65°C水浴锅中预热，29）之前的操作均在水浴锅中进行
Hybe buffer 10min00:10:00
75% Hybe buffer+25% 2×SSCT 10min00:10:00
50%Hybe buffer+50% 2×SSCT 10min00:10:00
25%Hybe buffer+75% 2×SSCT 10min00:10:00
2×SSCT 10min00:10:00
0.2×SSCT 15min 1/400:15:00
0.2×SSCT 15min 2/400:15:00
0.2×SSCT 15min 3/400:15:00
0.2×SSCT 15min 4/400:15:00
以下步骤在室温水平摇床上进行 75% 0.2× SSCT+25% MABt 5min00:05:00 Room temperature 1 mL
50% 0.2× SSCT+50%MABt 5min00:05:00
25%0.2× SSCT+75%MABt 5min00:05:00
MABt 5min00:05:00
1×blocking 500µl（每管配700µl）2h，RT02:00:00
Room temperature
Anti-Dig-POD 1:1000，200µl，4°C摇床缓慢摇动过夜4 °C
Note
注意与in situ不同，为Anti-Dig-POD
Day3
Day3
吸去抗体不回收
1×MABt 15min 1/800:15:00
1×MABt 15min 2/800:15:00
1×MABt 15min 3/800:15:00
1×MABt 15min 4/800:15:00
1×MABt 15min 5/800:15:00
1×MABt 15min 6/800:15:00
1×MABt 15min 7/800:15:00
1×MABt 15min 8/800:15:00
PT 20min 1/300:20:00
PT 20min 2/300:20:00
PT 20min 3/300:20:00
PBS 5min 1/200:05:00
PBS 5min 2/200:05:00
加入〔1µl Cys TSA+49µl magnification buffer〕Green/Red，注意避光，室温摇床避光过夜（此后所有操作均应避光！！！）Room temperature 50 µL
Note
避光
Day4
Day4
吸去染液
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
+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
Day5
Day5
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
Day6
Day6
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