Re: [求救] 免疫螢光染色
先感謝板上回應我文章的各位 (agh0913、luhs、humwang)
我本身用的是frozen tissue sections 所以不需要前面幾個步驟
反而需要用到aceton 先固定sample
改善之後 果然染色變的比之前漂亮很多
至於protocol我是參照Immunohistochemistry / edited by S. Renshaw
和IHC world所提供的buffer配方
以下是我modified後的IF protocol：
1. Fix sections in pre-cooled acetone for 10 min
2. Pour off the fixative and allow acetone to evaporate from the tissue
sections for > 20 min at room temperature
3. Immerse the slides for 1min in PBS
4. Immerse the slides for 20 min in PBS containing 0.1% (v/v) Triton X-100
5. Immerse the slides three times for 10 min each in PBS
6. Incubate in PBS containing 0.3M glycine for 20 min
7. Immerse the slides three times for 10 min each in PBS
8. Incubate the slides in PBS containing 10% (v/v) normal serum + 0.5% (w/v)
BSA for 30 min
9. Immerse the slides for 1 min in PBS
10. For the primary antibody co-incubation, incubate the slides with both
primary antibodies optimally diluted in PBS containing
1% normal serum + 0.5% (w/v) BSA for 1 h to overnight
11. Immerse the slides three times for 10 min each in PBS
12. For the secondary fluorochrome-conjugated antibody co-incubation,
incubate the slides with both secondary antibodies optimally
diluted in PBS containing 1% normal serum + 0.5% (w/v) BSA
for 1 h to overnight in the dark
13. Immerse the slides three times for 10 min each in PBS in the dark
14. Mount in antifadant containing mounting media, coverslip, and seal with
nail varnish
15. View using fluorescence or confocal microscopy
附上我這次染色的結果 http://0rz.tw/Mg0QM
也請各位有想法也一起提出來討論吧 ^ ^
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