ImmunostainingofF-actinandvinculin.docVIP

Immunofluorescence staining of actin cytoskeleton and focal adhesion plaques Buffers: Stock PBS 10X: 2g KCl + 2g KH2PO4 + 80g NaCl + 28.86g Na2HPO4·12H2O in 1 L. TBS buffer: 50 mM Tris-HCl + 0.15 M NaCl + 0.1% NaN3, pH 7.4 Preparations: 1 PBS; 2 TBS; 3 3.7% Formaldehyde; 4 0.1% Triton X-100; 5 0.1% BSA; 6 primary antibody; 7 second antibody; 8 F-actin staining reagent; 9 Nuclear staining reagent; 10 Parafilm; 11 anti-fade reagent. Fixing: 1. Discard media. 2. 2 rinsing cells with DPBS. 3. Fix the cells with 3.7% Formaldehyde final concentration in DPBS at room temperature RT for 20 min. 4. 2 rinsing cells with DPBS. 5. Keep the samples at 4 oC for staining. Immunostaining 1. Fix the cells. 2. Permeabilize the cells: 0.1% Triton X-100 in TBS for 5 min. 3. Rinsing the cells 3 times with TBS. 4. Blocking the slide with 0.1% BSA in TBS for ~60 min RT. 5. Rinsing the cells 3 times with TBS. 6. Transfer the slides onto the Parafilm, make sure the cells are upturned. 7. Primary antibody incubation: 60 μL/片，30 min RT Monoclonal Anti-Vinculin antibody produced in mouse 用0.1% BSATBS溶液1:200稀释 8. Rinsing the cells 3 times with TBS, 5 min each. For 14 mm slides, 200 μL solution is OK 9. Second antibody incubation and F-actin staining: 60 μL/片，Alexa Fluor? 488 goat anti-mouse IgG antibody + Phalloidin–TRITC 均用0.1% BSATBS溶液1:00稀释储存液 10. Rinsing the cells 3 times with TBS, 5 min each. 11. Nucleus staining: 2 μg/mL in TBS for 30 min RT. DAPI 原液湘圣用无水乙醇稀释的，使用前0.1% BSA 的TBS 稀释至1μg/mL 12. Rinsing the cells 3 times with TBS, 5 min each. 13. 用移液枪吸取Milli-Q水清洗1次，除去无机盐 不必等候 ，不要吹N2。 14. Fix the slide onto the glass slides with anti-fade reagent overnight, make sure the cells downward. anti-fade reagent提前取出至室温下放至一会儿；轻轻压一下盖玻片，赶走气泡，但勿过于用力，以免破坏细胞膜、改变细胞形态 16. Wait for overnight, 用Milli-Q水清洗2次，N2吹干。 17. Take images. 鬼笔环肽的分子量小，很容易通过细胞，但Phanoidin–TRITC为毒覃肽的TRITC荧光性交联物，不能穿透大多数活细胞膜，需提前用Triton X-100处理细胞。 鬼笔环肽可选择性标染F–actin。激发波长为540–545 nm，发射波长为570–573 nm。 药品价格昂贵，0.1mg