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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。
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Y% m7 O: v9 ^0 x5 A( i免 疫 荧 光 步 骤
* O( r# N& R: g1. Add a coverslip into a 12-well plate and grow cells in culture media until they reach 50% confluence.
7 @7 F4 D' {$ h2. Aspirate media from plates and wash twice with PBS.- G1 F$ g( {+ k+ v# Q
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.
5 v$ J0 \2 d3 C6 \; H9 K4. 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.
, s. v* H9 ~* f6 h5. Wash twice for 5 min with 2 ml of 1X PBS.
1 `0 F0 E; V- x. `# y; C3 I6. 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.. D5 A; g/ f ?! S8 `
7. Wash 5 times for 5 min with 1X PBS-0.2% BSA（bovine serium albumin）. R- K7 i" s, o! [: j4 L
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.
- x( \, l2 k5 G) A9. Wash 5 times for 5 min with 2 ml of PBS.( ]2 O6 |9 Z/ l( s7 T" S3 O) z
10. Mount slide with anti-fading agent. |
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发表于 2010-2-23 10:57
发表于 2010-3-9 17:09
