Goat anti chicken alexa fluor 488 secondary

Embryos were fixed in 4% paraformaldehyde solution in PBS at 4°C overnight, stored at −20°C in methanol and stained according to a previously described fluorescence immunohistochemistry protocol (Turner et al., 2014 (link)). The following antibodies were all used at a dilution of 1/500: mouse anti-mCherry (Clontech) with goat anti-mouse Alexa-Fluor^®-568 secondary (Invitrogen); chicken anti-EGFP (Abcam) with goat anti-chicken Alexa-Fluor^®-488 secondary (Invitrogen) (Du et al., 2017 (link); Hall et al., 2014 (link)).

Embryos were fixed with a standard 18.5% formaldehyde/heptane fix for 20 min followed by methanol devitellinization. mCherry embryos were visualized directly after fixation, rehydration, and mounting. srp-moe::GFP embryos were rehydrated and underwent antibody staining, using standard protocols and overnight incubation with a 1:500 dilution of GFP antibody (Aves Labs, Tigard, OR), followed (after washing) by incubation for 2 hr with a 1:500 dilution of Goat anti-Chicken Alexa Fluor 488 secondary (Invitrogen, Carlsbad, CA). Embryos to be stained with Lz Ab (DSHB, Iowa City, IA) were heat-fixed using standard protocols and incubated overnight with a 1:20 dilution of the antibody, followed after washing by incubation for 2 hr with a 1:500 dilution of Goat anti-Mouse Alexa Fluor 488 secondary antibody (Invitrogen). After washing, they were mounted in Vectashield Mounting Medium (Vector Labs, Burlingame) on 76 × 26 mm slides from Glasfabrik Karl Hecht (Sondheim, Germany) with 22 × 40 mm coverslips, No. 1 thickness (VWR International, Radnor, PA).