Alexa 488 conjugated wheat germ agglutinin

Isolated myocytes were plated on poly-L-lysine–coated coverslips and fixed immediately (0 hour) or after 3 hours in either 6 or 30 mM K⁺ PSS at 37°C. Myocytes were labeled with Alexa 488–conjugated wheat germ agglutinin (Invitrogen), a plasma membrane stain, before permeabilization with 0.1% Triton X-100. Myocytes were blocked with 5% BSA in PBS and incubated with the same K_v1.5-specific antibody used for Western blotting experiments at 1:100 in 0.5% BSA in PBS overnight at 4°C. Myocytes were washed with PBS and incubated with Alexa 546–conjugated secondary antibody. After washing, coverslips were secured to slides using mounting media (1:1 PBS/glycerol), and images were acquired using a laser scanning confocal microscope (LSM Pascal, Carl Zeiss). Alexa 488 and 546 were excited at 488 and 543 nm with emission detected at 505 to 530 and ≥560 nm, respectively. Pixel colocalization was measured using weighted colocalization coefficient values with Zeiss Pascal system embedded software. The RG2B Colocalization plugin for ImageJ was used to isolate colocalized pixel data with automatic selection threshold values and express the data as the average of the corresponding red and green channels.