免 疫 荧 光 步 骤

yuheng

免疫荧光技术在细胞学研究中愈来愈重要了，这里有个经典步骤，跟大家分享。/ S! ^$ F+ ?) H  c( @& Z, Q
  b* j6 `  O( W8 Z! K9 v' f$ j" ]1 k
免 疫 荧 光 步 骤6 m$ F0 I5 L3 t( M- B
1. Add a coverslip into a 12-well plate and grow cells in culture media until they reach 50% confluence.4 n: h# |4 H2 p8 a5 M; J, h
2. Aspirate media from plates and wash twice with PBS.' C% D" @6 _3 G4 q$ s
3. Fix cells with 4% paraformaldehyde solubilized in PBS-0.1% Triton-X100 for 20 min at room temperature (RT). There are multiple cell fixation procedures described in the literature. We recommend testing them until reaching the expected staining.
6 s& j6 f0 M/ ^2 p  w. \, D9 n* @4. Block for 1 hr with 2 ml of 1X PBS-1% BSA-4% goat serum. Note: always spin down any sera, antibodies, or antisera for 5 min at 10,000g before use, to remove small aggregates.
6 h+ R; d& Z& ^1 S5. Wash twice for 5 min with 2 ml of 1X PBS.
2 y# |2 C! b8 o/ T* t2 V( }6. Stain with primary antibody for 45 min at RT in 40 ml of 1X PBS-1% BSA by forming a drop on the coverslip. We recommend using at least two dilutions (1:200 and 1:1000) to start optimizing the staining.$ T! ^8 S6 Z+ H3 g6 D7 a( {9 J: m
7. Wash 5 times for 5 min with 1X PBS-0.2% BSA（bovine serium albumin）.# L3 K1 ~; D* `% Y. i& J/ W
8. Stain with conjugated secondary antibody for 30 min at RT in 40 ml of PBS-1% BSA. We recommend using 1:200 and 1:1000 dilutions.
/ S0 P7 ~7 X1 c+ j2 o" w9. Wash 5 times for 5 min with 2 ml of PBS.
; }4 ?$ Z# b: ?- ?$ d9 A10. Mount slide with anti-fading agent.