[實驗技術] 免疫螢光染色

玻片處理

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

[生活態度] 低頭謙遜 埋頭苦幹

認真充實智能   努力訓練技能

不要浪費所有的時間

努力的往前看   不要放棄自己

要持續的努力   努力   不要鬆懈

態度決定一切！！！

改變   改變   改變   全力尋求改變

磷酸根溶液 不是pbs

http://www.egr.msu.edu/biofuelcell/tools/phosphate/phosphate.html

八股式的科學論文

We sought to determine  whether XXX affects XXX.
XXX could be used as functional food-derived compound for .....
Thus, it is interesting and  valuable to determine the combined effect of XXX on XXX.
has been found to correlate to ~~
These results give rise to the possibility of  XXX not only in, but also XXX.
were enzymatically determined by ....

動物劑量與人體可用劑量交換

人體等酵劑量（mg/kg）= 動物劑量（mg/kg）×動物Km÷人體Km
The FASEB Journal • Life Sciences Forum
Dose translation from animal to human studies revisited
Shannon Reagan-Shaw,* Minakshi Nihal,*,† and Nihal Ahmad*,†,‡,1
*Department of Dermatology, †Paul P. Carbone Comprehensive Cancer Center; ‡Molecular and
Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin, USA