Phalloidin conjugated with a fluorophore will stain membrane actin and for nuclear staining as to avoid 405 nm laser we use far-red fluorophore
dye for staining of nucleus instead of DAPI. Phalloidin is not live cell compatible, but SYTO Deep Red is.
The following reagents are needed.
| Item | Item article number | Distributor | Vendor |
|---|---|---|---|
| UltraPure™ DNase/RNase-Free Distilled Water | 93901 | Thermofisher | Invitrogen |
| DEPC-Treated Water |
4387937 | Thermofisher | Invitrogen |
| 1xPBS pH 7.2 | |||
| Triton X100 | |||
| Phalloidin-Fluorescein conjugate | 23101 | AAT Bioquest | |
| SYTO Deep Red Fluorescent Nucleic Acid Stain | S34900 | Thermofisher | Invitrogen |
If you are starting from reagents directly from vendor make first the following two stocks.
First make sure you have a 10% Triton stock. Then make a permeabilization buffer:
| Reagent | Volume | Final concentration |
|---|---|---|
| 10x PBS | 5 ml | 1x PBS |
| 10% Triton | 1.5 ml | 0.3% |
| DEPC-water | 43.5 ml | |
| TOTAL: | 50 ml |
Make a stock solution of the 🔴SYTO Deep red. Add 50 µL of DMSO to a vial of 🔴SYTO Deep Red Nucleic Acid Stain to make 2000X SYTO™
Deep Red Stock Solution (2 mM).
| Reagent | Volume | Final concentration |
|---|---|---|
| 🔴 SYTO Deep Red Nucleic Acid Stain | 2 mM | |
| DMSO | 50 ul | |
| TOTAL: | 50 ul |
The ����Phalloidin should be aliquoted into 1 ul aliquots and used only once to avoid freeze thaw cycles.
| Reagent | Volume | Final concentration |
|---|---|---|
| 🟢 1 ul aliquot Phalloidin | 1 ul | |
| 🔴 2000x SYTO Deep Red Nucleic Acid Stain stock (2 mM) | 0.5 ul | 1 uM |
| 0.3% Triton DEPC-PBS permeabilization buffer | 998.5 ul | |
| TOTAL: | 1 ml |
⏰ Incubate for 20 min at room temperature.
Wash three times in 5 min each in PBST.