Filipin Staining 非律平细胞染色
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Filipin Staining Protocol
1) Rinse cells 3x with PBS /PBS洗涤细胞三次
2) Fix with 3% paraformaldehyde for 30 minutes at RT
室温下用3%多聚甲醛固定30min
3) Rinse cells 3x with PBS 用PBS洗涤细胞三次
4) Incubate with 1 mL of 1.5 mg/mL Glycine for 10 minutes at RT
室温下，使用1ml 1.5mg/ml 甘氨酸培养10min
5) Rinse cells 3x with PBS 使用PBS洗涤细胞三次
6) Stain cells with 1 mL filipin working solution for 2 Hr at RT室温
下用1ml filipin 染液给细胞染色2h
a. Working solution (10 mL): 1 mL FBS, 9 mL PBS, 20 µl
Filipin Stock
染液(10ml):1ml FBS,9 mL PBS,20μl fili[in贮存液
贮存液50mg/ml储存在DMSO内，避光保存。
7) Remove chambers, wash slides 3x with PBS
转移 pbs洗涤三次
8) Mount and view cells in PBS
在PBS中观察细胞
Filipin Fluorescence Staining of Free Cholesterol
Materials: Filipin Complex: Sigma (F-9765) Stock: 50 mg/ml in DMSO final concentration 0.05 mg/ml in PBS Protect filipin solutions from light
Procedure: 1. Wash cells 3X with PBS. 2. Fix with
4% paraformaldehyde (freshly prepared) for 20min at RT. 3. Wash cells 3X with PBS. 4. wash with 20mM Amonium Chloride in PBS for 10 min at RT (quench the paraformaldehyde).
5. Stain cells with 1 ml of filipin for 1hr at RT.
6. Wash cells 3X with PBS.
7. View cells in using a UV filter set. Filipin fluorescence photobleaches rapidly One can avoid it using a density filter that
lets through only 5-10% of light.Could be used for cells that are fixated onto slides