Rhodamine cytoskeleton

Staining the Actin Cytoskeleton 1. Intracellular buffer (2x). 280 mM KCl, 2 mM MgCl, 4 mM EGTA, 40 mM Hepes of pH 7.5, 0.4% low endotoxin albumin from human serum (Sigma) see Note 5). 2. Fixation buffer (2x). 640 mM sucrose, 7.4% formaldehyde (Sigma) in 2x intracellular buffer store at 4°C (tee Note 6). 3. Stain buffer. 0.2% triton, 2 pl/ml rhodamine phalloidin (Invitrogen) in lx intracellular buffer (rccNote 7). 

Fig. 3 Actin cytoskeleton and focal adhesion. F-actins were visualized using rhodamine phalloidin (red), and vinculins found in focal adhesions were stained using fluorescently conjugated antibodies. (A) Immunofluorescent images of a thin filamentous actin meshwork and vinculins showed that osteoblasts contain fewer and smaller focal adhesions. (B) In contrast, hMSCs showed thick actin stress fibers, and multiple and large adhesion contacts.

Cellular interactions on the developed nanoparticles-enriched coatings were also examined by inverted fluorescence microscopy. After 24 and 72 hours of incubation, the replicate surfaces were harvested and washed thrice with PBS. The cells that attached to the surfaces were fixed with paraformaldehyde (4%) for 10 minutes and permeabilized with Triton X-100 (0.1%) for 5 minutes. The actin filaments of the cytoskeleton were labeled with rhodamine phalloidin for 2 hours at room temperature. The surfaces were then mounted using Vectashield with DAPI and examined by an inverted fluorescence microscope with a magnification of 20X. 

Rhodamine tubulin (10- j,l aliquot at -20°C Molecular Probes or Cytoskeleton) (Optional) Fluorescein or rhodamine actin (Cytoskeleton)... 

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