將玻片放在dish上，add 1N NaOH
↓
Shaker 10min
↓
Washing 3 times by MQ buffer
↓
用酒精浸泡一下
↓
照UV
以染PPARa/STAT1為例
CON, IF, FR(0.2 μM)+IF, GW(5 μM)+IF 四組
藥品配置
1. Stock:
IFN: Take 1 μL stock dil with 14 μL 106 U/mL (1000 U/μL);
Fluoxetine: 10 mM;
Fludarabine 10 mM;
5 μL(10 mM) dil with 245 μL ddH2O (5 μL x 10 mM/250 μL);
5 μL (200 μM) dil with 4995 μL culture medium (5 μL x 200 μM/5000 μL)
GW6471 10 mM
2. Final culture medium (1.5 mL)
IF
取1.5 uL IFN + 1.5 uL Flu dil 1500 uL
FR+IF
取 dil Fludarabine 1.5 uL for 30 min, 取1.5 uL IFN + 1.5 uL Flu dil 1500 uL
GW+IF
取 GW 0.75 uL for 30 min, 取1.5 uL IFN + 1.5 uL Flu dil 1500 uL
1. Cover slides wiped clean with 75% ethanol and UV for 30min 要注意棉絮殘留在玻片
2. 1 cover slide for 1 well
3. seeding 2x10^4/well in 24-well; 0.5 mL/well; 37℃, overnight
4. Change medium to 1.5mL/well; add FR/GW for 0.5 hr and then add IF for 6 hr
5. 1x PBS rinse x 1 times; 500 μl/well
6. 4% paraformaldehyde 500μl /well, 20min, RT
7. 1x PBS rinse x 3 times; 500μl /well,
8. Triton X100 0.2% in PBS, 10 minutes for nuclear protein staining;
9. 1x TBST, wash 3min x 3 time, 70rpm; 500 μl /well
10. Primary antibodies for 6hr treatment, STAT1 (nuclear protin; mouse) and PPARa (nuclear protein; rabbit) in TBST (1:˙50) 同時加入染; 4℃, overnight; 200 μl /well
(上染劑應要慢慢染，轉數可以調慢)
11. Primary antibody for 24hr treatment, Glut2 (membrane protein; rabbit) in TBST (1:100); 4℃, overnight; 250 ul/well 1x TBST, wash 5min x 6-12 times, 70rpm; 500 μl /well (手也可以先搖一下、轉一下，清洗應要略快，轉速可以調快，不可以溢出)
12. 2°Ab (mouse, green 488 and rabbit, red 568) (1:200) in 1x TBST, 1hr, RT, 70rpm, 避光: 200 μl /well
Mouse (488) - Green (1:200) dilute 1000 = 5 μl +995 μl TBST;
Rabbit (568) - Red 1:200 dilute1000 = 5 μl +995 μl TBST
13. DAPI in 1x TBST (1:100,000), 與 2°Ab 同時加入染 1hr, RT, 70rpm, 避光
14. 1x TBST wash 5min x 10 times, 70rpm, 500ml/well; 避光
15. 封片, 使用封片膠; 避光
16. store at 4℃/-20℃
根據11/06的結果 下列事項要進行修正
1. 紅光太強 – PPARa 一抗濃度改為 1:80
2. 綠光太弱 – STAT1 一抗濃度改為 1: 25
PPARa(1:˙80) 10 uL STAT (1 :25) 32 uL
以TBST 758 uL 混合成 800uL
加入細胞 反應overnight
二抗
同時混合二抗
Green (1:200): 4 uL
Red (1:200): 4 uL
以TBST 792 uL 混合成 800uL (1:200)
DAPI 分開染
DAPI(1:10,000)/ 2 min
3.玻片要前處理，背景會比較乾淨
4. 所有的溶液要先以0.22 uM過濾，降低雜質的干擾
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