Immunofluorescence / Confocal Microscopy Protocol
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实验步骤
1. Wash cells 1x cold RPMI (no wash for cryostat sections).
2. Fix 20 min 4% paraformaldehyde in 0.1M phosphate buffer pH 7.4, 0.03M sucrose on ice. **
3. Wash 2x PBS/1%BSA. From now on everything can be at room temp, or on ice.
4. Permeabilize 5 min RT in 0.2% saponin, PBS, 0.03M sucrose, 1% BSA.
6. Block 15 min 5% normal goat serum (NGS) in PBS/BSA.
8. Diluted in PBS/BSA 60 min RT; 100 l per tube or section.
10. Block 15 min 5% NGS in PBS/BSA.
12. Diluted in PBS/BSA 30 min RT; 100 ul per tube or section.
13. Wash 3x PBS/BSA; (wash 1x in PBS/BSA then 2 x 10 min in Molecular Probes SlowFade Light buffer if using Slow Fade Light S-7461 to coversip); pellet cells and put up in two drops of Molecular Probes Slowfade Light antifade medium. Pipet about 15 ul on slide and coverslip. For chambered coverglass, just put two-three drops in each chamber after wash.注意事项
