The following antibodies were used for expression analysis:, mouse anti-CadN (DN-Ex#8) at 1:20039 (link) Developmental Studies Hybridoma Bank), rabbit anti-RFX at 1:5000 (a gift from Anne Laurençon and Benedicte Durand)40 (link), rabbit anti-Wnd at 1:500 (a gift from Aaron DiAntonio)31 (link), rabbit anti-GFP at 1:250 (Invitrogen), mouse anti-DsRed at 1:250 (Clontech), rabbit anti-TagRFP at 1:500 (Evrogen), rabbit anti-Dendra2 at 1:5000 (Evrogen), rabbit anti-Killerred at 1:1000 (Evrogen), mouse anti-Flag at 1:250 (Sigma-Aldrich), mouse anti-StrepII at 1:200 (Thermo Scientific), mouse anti-S at 1:100 (Thermo Scientific), mouse anti-V5 at 1:2000 (Invitrogen), mouse anti-c-Myc at 1:250 (Abcam) and mouse anti-HA at 1:200 (Covance).
Drosophila embryos (0 to 24 hours) were collected on grape-agar plates and were subsequently fixed for 20 minutes in a 1:1 mixture of 0.38% formaldehyde in PBS and heptane. The fixative was then removed and methanol added. After vigorously shaking, the heptane-methanol mixture was replaced by methanol, whereupon methanol was replaced by ethanol. Upon rehydration in PBS/0.2% Triton, embryos were blocked for 1 hour in PBS, 10% normal goat serum and incubated overnight with primary antibodies. Fluorescently labeled or HRP-conjugated secondary antibodies were obtained from Jackson ImmunoResearch and were used at a 1:250 dilution.
Drosophila embryos (0 to 24 hours) were collected on grape-agar plates and were subsequently fixed for 20 minutes in a 1:1 mixture of 0.38% formaldehyde in PBS and heptane. The fixative was then removed and methanol added. After vigorously shaking, the heptane-methanol mixture was replaced by methanol, whereupon methanol was replaced by ethanol. Upon rehydration in PBS/0.2% Triton, embryos were blocked for 1 hour in PBS, 10% normal goat serum and incubated overnight with primary antibodies. Fluorescently labeled or HRP-conjugated secondary antibodies were obtained from Jackson ImmunoResearch and were used at a 1:250 dilution.
