Alexa fluor 555 conjugated wga

As the CellMask plasma membrane stain does not survive permeabilization, dissected pupal wings were immediately stained with a 1:300 dilution of CellMask Deep Red (Invitrogen C10046) for 10 min. After removing the staining solution, the wings were fixed in 4% PEM-PFA at room temperature for 15 min. Following washes in PBS, pupal wings were stained with a 1:200 dilution of Alexa Fluor 555-conjugated WGA (Invitrogen W32464) for an hour at room temperature. In order to prevent permeabilization, the buffers for CellMask stains did not contain any detergents (e.g., Triton).

Wheat germ agglutinin (WGA) was previously used to visualize butterfly scale cell growth and is thought to initially stain plasma membrane before switching to chitin at later developmental stages (Dinwiddie et al., 2014 (link)). Phalloidin is a standard method to visualize F-actin. For AF-555 WGA and AF-647 phalloidin double-staining, pupal wings were incubated in 1:200 dilution of Alexa Fluor 555-conjugated WGA (Invitrogen W32464) and 1:40 dilution of Alexa Fluor 647-conjugated phalloidin (Invitrogen A22287) for an hour at room temperature. For FITC WGA and TRITC phalloidin double staining, P. palinurus pupal wings were incubated in 1:100 dilution of FITC WGA (EY Labs F-2101-5) and 1:100 dilution of TRITC phalloidin (Sigma P1951) for an hour at room temperature. Arp2 is the ATP-binding component of the actin Arp2/3 complex, which functions as an actin nucleator in branched actin networks (Goley and Welch, 2006 (link); Smith et al., 2013 (link)). After blocking, pupal wings were incubated with a 1:500 dilution of rabbit anti-Arp2 (Abcam ab47654; pblast search revealed UniProt #P61160, Human Arp2 has 82% sequence similarity to XP_013178655.1, P. xuthus Arp2) at 4℃ overnight. After washing, the wings were incubated in buffer with a 1:300 dilution of Alexa Fluor 594 goat anti-rabbit secondary antibody (Abcam ab150088) for an hour at room temperature.