Prepare stock solutions：

Prepare a 200T/mL reserve solution with 0.5mL methanol. A unit (T) of labeled phalloidin is defined as the amount of dye used to stain a glass slide loaded into a cell. For labeled phalloidin, the recommended dilution ratio is 1:40-1:200, with one unit equivalent to 200uL total chromosomal product added to the 1-5uL 200T/mL reserve solution.

Note: The dilution ratio can be adjusted according to the actual dyeing effect.

Fixed cell staining：

1.1 Wash cells three with pre-warmed phosphate-buffered saline, pH 7.4 (PBS).

1.2 Fix the sample in 3.75% methanol-free formaldehyde solution in PBS for 15 minutes on ice.

Note: Methanol can disrupt actin during the fixation process. Therefore, it is best 

to avoid any methanol containing fixatives. The preferred fixative is methanol-free 

formaldehyde.

1.3 Wash three times with PBS.

1.4 Permeabilize the sample in 0.5％Triton X-100 in PBS for 10 minutes. 

1.5 Wash three times with PBS.

1.6 Dilute the Phalloidin reservoir with 200?uL PBS, add a cover glass or hole, incubate 20min at room temperature for dyeing.

Note: Chromosomal products can be adjusted according to the sample conditions. To avoid the spread of dye during incubation, the cover glass can be placed in an airtight container.

1.7 Wash three times with PBS.

1.8 Fluorescence microscope observation. The YF dye-labeled Phalloidin is very light-stabilized and the sample can be imaged in PBS, but for best results, it can also be observed with anti-fluorescent quenching agents.

Live cell staining：

Phalloidins are usually not cell-permeable and have therefore not been used extensively 

with living cells. However, living cells have been labeled.Pinocytosis appears to 

be the method of entry for some cells, although hepatocytes “avidly” take up the dye 

by an unknown mechanism.In general, a larger amount of the dye is needed for 

staining living cells. Rhodamine phalloidin has been microinjected into fibroblasts 

without noticeable changes in shape or ruffling.Injections of phalloidin into living 

cells appear to alter the actin distribution and cell motility.Consult the literature to 

find the procedures suitable for your experiments