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。 7 D5 _$ j, f0 |2 I- s |1 C! Z - b# ^! \8 T2 e, V$ T! ^免 疫 荧 光 步 骤 , @3 ?: l; B. T( h7 n* e: T& ]8 K1. Add a coverslip into a 12-well plate and grow cells in culture media until they reach 50% confluence.6 l% s Z( G2 q0 F
2. Aspirate media from plates and wash twice with PBS.0 }2 Y3 x3 D4 J# J
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.! R% N, c: e! ? g8 C& }3 _
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. / j; n: T2 V5 d. p- \2 r2 \# [5. Wash twice for 5 min with 2 ml of 1X PBS.) O9 j$ h% c- i1 X& P# b
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. 0 x b3 X# `% I1 w7. Wash 5 times for 5 min with 1X PBS-0.2% BSA（bovine serium albumin）. 2 @- D2 k7 K. D1 n( o- {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.$ p7 t. @! ?# q1 @0 X: _
9. Wash 5 times for 5 min with 2 ml of PBS. # k" P+ q9 m5 K I; c# X10. Mount slide with anti-fading agent.作者: janeyu 时间: 2010-3-9 17:09