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Materials and Methods
Immunocytochemistry
Cells were fixed for 15 minutes in 4% PFA/5% Sucrose/PBS, permeabilized for 5 minutes in 0.1% Triton-X-100/PBS and blocked for 30 minutes in 2% BSA/PBS. Primary antibodies were incubated overnight at 4°C as follows: mouse α-ß-tubulin (1∶5000; Sigma), mouse α-class III ß-tubulin (1∶1000; Covance), rabbit anti-ERK (1∶300; Cell Signaling), rabbit anti-P-ERK (1∶300; Cell Signaling). First antibodies were detected with Alexa 488 or 546-conjugated secondary antibodies (1∶1000; Molecular Probes). Cells were stained for F-actin with Texas Red-X Phalloidin (1∶100; Molecular Probes).
For visualization of P-ERK and Eph receptors, cultures were incubated with 1 µg/ml pre-clustered ephrin-A5 for 20 minutes, followed by fixation with 4%PFA/sucrose for 15 minutes. Ephrin-A5 bound to Eph receptors was visualized with anti-Fc Cy3 conjugated antibodies (1∶300 in block). Subsequently, P-ERK staining was performed as indicated above.

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