Alexa488 conjugated donkey anti chicken

Immunofluorescence stainings of 4% paraformaldehyde (PFA)-fixed cultured cells were performed in 24 well plates. Immunohistochemical stainings of brain tissues were performed using free-floating sections. Briefly, anesthetized mice were intracardially perfused with 4% PFA in PBS. The brains were removed, fixed overnight in 4% PFA and cut in a Leica VT 1000 S vibratome in 50 μm slices. The slices were permeabilized and blocked using 5% BSA, 0.1% Triton PBS solution for 2 h and were next incubated with the primary antibody using 3% BSA, 0.1% Triton solution overnight. The next day the sections were washed with PBS 3 times and were incubated with the secondary antibody for 4 h in 3% BSA, 0.1% Triton solution. Sections were next washed 3 times with PBS and incubated with DAPI solution for 10 min, washed once again and mounted. Antibodies used were: mouse anti-BrdU (BD Biosciences, cat# 347580), rabbit anti-ACBP (Santa Cruz cat# sc30190), chicken anti-EGFP (Abcam, cat# ab13970), rabbit anti-DsRed (Clontech Living Colors, cat# 632496), mouse anti-ITGB1 (Abcam, cat# ab24693), Alexa 647 conjugated donkey anti-mouse and anti-rabbit (Thermo Fisher, cat# A21447, cat# A31573), Alexa 488 conjugated donkey anti-chicken (Jackson ImmunoResearch Laboratories cat# 703-545-155) and Cy3 conjugated donkey anti-rabbit (Jackson ImmunoResearch Laboratories cat# 711-165-152).

Dechorionated, formaldehyde-fixed, methanol devitellinized embryos were fluorescently stained as previously described [51 ]. The following primary antibodies were used: mouse anti-1D4/FasII [Developmental Studies Hybridoma Bank (DSHB); 1:100], mouse anti-Beta gal [DSHB; 1:150], mouse anti-Myc [DSHB (9E10); 1:500] rabbit anti-GFP [Invitrogen (#A11122); 1:500], Mouse anti-HA [Covance (16B12) 1:250], Chicken anti-GFP [Aves Labs (GFP-1020) 1:1000]. The following secondary antibodies were used: Alexa647- conjugated goat anti-HRP [Jackson Immunoresearch (#123-605-021); 1:500]. Cyanine 3-conjugated goat anti-rabbit [Jackson; 1:1000], Alexa488-conjugated goat anti-mouse [Molecular Probes; 1:500] and Alexa488-conjugated donkey anti-chicken [Jackson Immunoresearch; 1:500]. Embryos were mounted in 70% glycerol/PBS.