由 natsumi 在 四, 10/09/2014 – 16:14 發表 Cholangiocarcinoma FGFR3 IHC
#1由 natsumi 在 四, 10/09/2014 – 16:30 發表。
PROTOCOL
Immunohistochemistry Staining (for paraffin-embedded sections)
1. Deparaffinize
¯ immerse slide in xylene for 7 min ´ 3 times (reused ~5 times).
¯ immerse slide in 100% ethanol for 3 min ´ 2 times (new).
¯ immerse slide in 95% ethanol for 2 min.
¯ immerse slide in 85% ethanol for 2 min.
¯ immerse slide in 75% ethanol for 2 min.
¯ immerse slide in 65% ethanol for 2 min.
¯ immerse slide in d.d.H2O for 3 min ´ 2 times.
¯ immerse slide in PBS for 5 min.
2. Antigen retrieval
¯TEG bfr. (10mMTris + 0.5mM EGTA pH9)
¯Pressure Cooker (預先加300~500 ml d.d.H2O), 121°C, 10 min (壓力上去下來約20 min)
¯recover in RT
¯immerse slide in PBS for 5 min ´ 2 times.
¯劃圈圈
3. 1% H2O2 in d.d.H2O, RT 15 min (避光進行).
¯ immerse slide in PBS for 5 min ´ 2 times.
4. Blocking, 0.1% BSA incubate for 0.5-1 hr at RT
5. Primary antibody in PBS, 4°C, O/N (Anti FGFR3 Ab: sc13121= 1:50 )
¯ immerse slide in PBST for 5 min ´ 4 times.
6. HRP-conjugated secondary antibody (DAKO), RT 1 h.
¯ immerse slide in PBST for 5 min ´ 4 times.
7. Final developmental DAB (DAKO)
¯利用顯微鏡觀察呈色程度 可再補加
¯immerse slide in d.d.H2O for 5 min ´ 2 times.
8. Hematoxylin staining for 30sec. 利用顯微鏡觀察 再補加時間
¯immerse slide in d.d.H2O for 5 min ´ 2 times.
¯流水, 10-15 min
9. Dehydration of array slide.
¯ immerse slide in 85% ethanol for 5 min.
¯ immerse slide in 95% ethanol for 5 min..
¯ immerse slide in 100% ethanol for 5 min ´ 2 times (new).
¯ immerse slide in xylene for 10 min ´ 3 times (new).
18. Mount slides.