Gfp antibody

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).

PTU-treated 5 dpf old Shp2^D61G embryos in the Tg(cd41:GFP, kdrl:mCherry-CAAX) transgenic background were fixed in 2% PFA overnight and stained as described in Choorapoikayil et al., 2012 (link). Primary pHis3 antibody (1:500 in blocking buffer, Abcam, Cambridge, UK, Ref: ab5176) and secondary GFP antibody (1:200 in blocking buffer, Aves Labs Inc, Tigard, OR, GFP-1010) were used. Embryos were mounted in 0.3% agarose, their CHT was imaged using the SP8 confocal microscope, and 3D images were subsequently reconstructed using Imaris.