Alexa fluor 488 conjugated phalloidin

Actin filaments were visualized by fluorescence staining of F-actin, and their alignment was determined. Cultured cells were rinsed twice with pre-warmed PBS, fixed with 3.7% (v/v) formaldehyde for 10 min, and permeabilized with 0.1% Triton^® X-100 in PBS for 5 min. Fixed cells were pre-incubated with 1% BSA in PBS for 20 min, to avoid non-specific protein binding and to increase fluorescence intensity. Filamentous actin was stained with Alexa Fluor^® 488-conjugated phalloidin (1:100; Lonza) according to the manufacturer’s protocol. Subsequently, the nuclei were stained with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI, 1:1000 in PBS; Nissui Pharmaceutical, Tokyo, Japan). Images of stained sections were acquired using a confocal microscope (Nikon ECLIPSE TE2000-U, Nikon Corporation, Kanagawa, Japan).

Overnight cultures of the strains were diluted to an OD₆₀₀ of 0.2 in 10 ml YPD. After 3 h of incubation at 30°C, formaldehyde was added to a final concentration of 4.4% and the cultures were incubated for 45 min at room temperature. The cells were washed three times with PBS and stained with Alexa Fluor 488-conjugated Phalloidin (Lonza) overnight at 4°C. Images were captured with a Leica DMI6000 fluorescence microscope.