F-actin stain for fixed cells
ActinRed 555 ReadyProbes Reagent is a selective, high-affinity F-actin probe conjugated to the red-orange fluorescent dye tetramethylrhodamine (TRITC). Like other ReadyProbes reagents, ActinRed 555 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
- ActinRed 555 ReadyProbes Reagent (Cat. No. R37112)
- 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 ActinRed 555 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. |
| ActinRed 555 | |
|---|---|
| Excitation/Emission (nm) | 540/565 |
| Standard filter set | TRITC |
| EVOS Light Cube | TRITC |
| 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 BPAE cells. BPAE cells were fixed and permeabilized using the Image-iT Fixation/Permeabilization Kit. Actin was stained using ActinRed 555 ReadyProbes Reagent, and nuclei were stained with NucBlue Fixed Cell ReadyProbes Reagent.
For Research Use Only. Not for use in diagnostic procedures.