F-actin stain for fixed cells
ActinGreen 488 ReadyProbes Reagent is a selective, high-affinity F-actin probe conjugated to our bright, photostable green-fluorescent Alexa Fluor 488 dye. Like other ReadyProbes reagents, the ActinGreen 488 reagent is formulated as a room temperature–stable solution and comes in a convenient dropper bottle.
This protocol can be used for:
- F-actin staining in fluorescence microscopy
This protocol should not be used for:
- Flow cytometry
You will need the following for this protocol:
- Cells growing in culture
- ActinGreen 488 ReadyProbes Reagent (Cat. No. R37110)
- Fluorescence microscope
Protocol
Labeling fixed cells
First, fix and permeabilize cultured cells with a protocol appropriate for your sample. Counterstain as desired. DAPI may be added directly to fixative.
 1. Wash the cells 1–3 times in PBS as needed. |
 2. Add two drops of ActinGreen 488 ReadyProbes Reagent per milliliter of medium. |
 3. Incubate for 30 minutes, protected from light. |
 4. Remove the stain solution. |
| 5. Wash the cells 2–3 times in PBS. |
 6. Image the cells. |
| ActinGreen 488 | |
|---|---|
| Excitation/Emission (nm) | 495/518 |
| Standard filter set | FITC |
| EVOS Light Cube | GFP |
| Storage conditions | Room temperature |
Protocol tips
- In some cases, more or fewer drops may be needed to achieve optimal staining intensity.
- Image quality may be improved by replacing the culture medium with Live Cell Imaging Solution (Cat. No. A14291DJ).
Multicolor staining of HeLa cells. HeLa cells were grown on coverslips and were fixed and permeabilized using the Image-iT Fixation/Permeabilization Kit. Cells were then stained to detect actin (green fluorescence from ActinGreen 488 ReadyProbes Reagent, B-catenin (red-orange fluorescence), and nuclei (blue fluorescence).
