IF protocol

Introduction

IF protocol (before DNA FISH)

Materials

• 0.15% TritonX-100
• 10% TritonX-100 as stock in PBS at RT
• 0.1% PBS–Tween 20
• 10% Tween 20 as stock in PBS at RT
• 5% BSA in PBS–Tween20
• store at 4°C

Procedure

Day 1

Prepare working buffer

Solution	Component	Volume / amount	Final
0.15% TritonX-100	10% Triton X-100	0.75 ml	0.15% TritonX-100
	PBS	49.25 ml
	Total	50 ml
0.1% PBS–Tween 20	10% PBS–Tween 20	0.5 ml	0.1% PBS–Tween 20
	PBS	49.5 ml
	Total	50 ml
5% BSA in 0.1% PBS–Tween20	BSA (W/V)	0.5 g	5% BSA
	0.1% PBS–Tween 20	10 ml

Steps

1. NCCIT cells should be cultured on cover slips for 2 days (0.3 M, 1.5 Day)
2. PBS twice
3. 4% PFA (diluted with PBS) 20 min at RT
4. PBS twice
5. 0.15% TritonX-100 (diluted with PBS) 15 min at RT
6. PBS twice
7. 5% BSA (diluted with 0.1% PBS–Tween20) 1 h at RT
8. PBST twice (0.1% PBS–Tween20)
9. Primary antibody solution (diluted with 0.1% PBS–Tween20) 4 h at RT / overnight at 4°C

Day 2

1. PBST four times
2. Secondary antibody solution (diluted with 0.1% PBS–Tween20) 2–4 h at RT
3. PBST five times
4. DAPI 5 min
5. PBS twice
6. Seal the coverslips

Notes

• 一抗/二抗/DAPI——180ul，二抗（1:500）
• Seal封片剂，加的时候不要等片子风干，湿漉漉的时候即可封片
• PBST 2min/times

后期拍摄的nd2格式图片（尼康显微镜产出的图片格式），可以用FIJI（一种imageJ的分支）来处理，这里有一个宏脚本很好用。不过要注意AutoScale等参数，必须理解含义。