Nuclear and membrane co-staining of Paramecium

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.

🚛 Reagents needed 📦 :

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
Stock solutions

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.