GFP-Vimentin and Actin/Antibody Stain

Dominik Haudenschild May 28, 2008 TSRI

· Cytoskeletal Buffer (CB)

1. PIPES                             60mM                   (18.14g/L free-base)

2. HEPES                           27mM                   (6.50g/L)

3. **EGTA                        10mM                   (3.80g/L) **SKIP if using Alginate-embedded Cells

4. MgSO₄                          4mM                     (0.99g/L heptahydrate)
        pH to neutral (7.0 to 7.3) with NaOH

· PBS with Ca-Mg (PBS)

1. PBS                                1x          

2. CaCl₂                              0.9mM                  (0.132g/L dehydrate)

3. MgCl₂                            0.5mM                  (0.10g/L hexahydrate)
        Divalent cations help intact chromatin stay in nucleus during procedure

· PBS with Ca-Mg and Triton X-100 (PBStx)

1. PBS                                1x          

2. Triton X-100                0.05%                    (from 20% stock in CB)

· Mixed Aldehyde and Detergent Fixative (MADF)

1. Base is Cytoskeletal Buffer (CB)

2. Add                3% EM grade paraformaldehyde

3. Add                0.3% Triton X-100 (from 20% stock in CB)

4. Add                0.05% glutaraldehyde
        make fresh daily

· Procedure for Alginate-embedded Chondrocytes

1. Place alginate gels (6mm diameter, 2.5mm height) in wash buffer quickly

2. Fix gels for 30-40 minutes with gentile agitation at room temperature

3. Wash gels 1x 10 minutes with CB

4. Thinly slice gels with razor blade and place on slide within pap-pen circle

5. Wash 2x 10minutes with PBStx

6. Block 60 minutes with PBStx containing 10% serum from same species as secondary antibody

7. Add primary antibody at 10ug/ml in PBStx overnight at 4 degrees

8. Wash 3x 10 minutes with PBStx

9. Add secondary antibody (1:500 highly adsorbed AlexaFluor546) for 90 minutes

10. Wash 2x 10 minutes with PBStx

11. Counterstain nuclei with ToPro3 dye in PBStx for 5 minutes in last wash

(Alternatively, to see Actin structures, at step 6 use 1:40 AlexaFluor546-phalloidin for 30 minutes, then wash for 15 minutes PBStx and image)