Alexa fluor 488 and 555 conjugates

Third instar wing imaginal discs were dissected on ice in PBS and fixed in 4% paraformaldehyde in PBS for 20 minutes. The discs were then washed with 0.1% Triton X-100 in PBS followed by a 1 hour incubation in PBS with 0.5% Triton X-100 and 10% BSA. Wing discs were incubated with guinea pig anti-Senseless (1:2000) (Nolo et al., 2000 (link)), mouse anti-Wg (4D4, Developmental Studies Hybridoma bank [DSHB], 1:20), and mouse anti-Engrailed (4D9, DSHB, 1:20) at 4°C overnight in PBS with 0.1% Triton X-100. Discs were subsequently incubated with secondary antibodies (anti-guinea pig and anti-mouse Alexa Fluor 488 and 555 conjugates, ThermoFisher Scientific, 1:500) for 2 hours at room temperature. The discs were stained in 2 μg/ml DAPI and mounted in Prolong Gold (Invitrogen). All fluorescent images were obtained using a Nikon A1RSi confocal microscope.

Third instar larval wing imaginal discs were dissected in PBS and fixed in 4% paraformaldehyde in PBS for 20 minutes. After fixation, discs were washed with PBS with 0.1% Triton X-100 followed by incubation in PBS with 0.1% Triton X-100 and 10% BSA for 1 hr. at room temperature. Wing discs were incubated with guinea pig anti-Senseless (41 (link)) or rabbit anti-dsRed (Clontech/TaKaRa, Cat. No. 632496, 1:500, to detect the sens reporter mScar:T2A:sens), mouse anti-Wg (4D4, Developmental Studies Hybridoma bank [DSHB], 1:500), and mouse anti-Engrailed (En) (4D9, [DSHB], 1:20) at 4°C overnight in PBS with 0.5% Triton X-100, followed by incubation with secondary antibodies (anti-guinea pig and anti-mouse Alexa Fluor 488 and 555 conjugates; ThermoFisher Scientific, 1:500) for 2 hr. at room temperature. Specimens were stained with DAPI (2 ug/ml) and mounted in Prolong Gold (Invitrogen). Fluorescent images were obtained on a Nikon A1Rsi confocal microscope.