|
  
- 积分
- 757
- 威望
- 757
- 包包
- 5169
|
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。
h" w1 a! P9 j' w) [( B9 b3 D
: a- T; [( z/ ^$ n# ^0 Z) }免 疫 荧 光 步 骤
5 J; z! m- E0 b& N' ?+ r1 e1. Add a coverslip into a 12-well plate and grow cells in culture media until they reach 50% confluence.
3 A! }/ |: R! b O( Y5 A2. Aspirate media from plates and wash twice with PBS.
4 |3 s) Q- d& |' k X3. 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.
) y( k8 P4 ~# L1 g! q4. 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.
, _" f) Y7 P$ ^( e+ ]/ i, v5. Wash twice for 5 min with 2 ml of 1X PBS.
/ P' ~; D ]& |7 }' g; g5 ~1 z6. 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.
1 x. v4 s1 p( \7 l3 v8 X7. Wash 5 times for 5 min with 1X PBS-0.2% BSA（bovine serium albumin）.8 }4 ^4 n, c9 U m3 I. x% @1 @
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.; c& W: W# g$ C, A/ G
9. Wash 5 times for 5 min with 2 ml of PBS.- ?; z s( ~) Q/ d$ V: Y+ g
10. Mount slide with anti-fading agent. |
-
总评分: 威望 + 10
包包 + 10
查看全部评分
|
发表于 2010-2-23 10:57
发表于 2010-3-9 17:09
