先感谢板上回应我文章的各位 (agh0913、luhs、humwang) 我本身用的是frozen tissue sections 所以不需要前面几个步骤 反而需要用到aceton 先固定sample 改善之後 果然染色变的比之前漂亮很多 至於protocol我是参照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 附上我这次染色的结果 http://0rz.tw/Mg0QM 也请各位有想法也一起提出来讨论吧 ^ ^ --

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