Donkey anti chicken igy alexa488

Embryos were isolated in PBS⁺ dissection medium [PBS containing Mg²⁺ and Ca²⁺]. Isolated embryos were fixed for 20 min at RT in 2% PFA in PBS⁺ followed by permeabilizing for 10-15 min in permeabilization solution [0.1 M glycine/0.1% Triton X-100]. Embryos were transferred into blocking solution [0.1% Tween-20; 10% FCS; 0.1% BSA; 3% Rabbit, Goat or Donkey serum]. Primary antibodies were added into the blocking solution and incubated o/n at 4 °C. The following antibodies were used: Foxa2 (Abcam, ab40874, 1:1000), Sox17 (Acris/Novus, GT15094,1:1000), GFP (Aves, GFP1020, 1:1000), AFP (R&D, AF5369, 1:1000) and IAP-GAG (Cullen lab, 1:1000). The next day, embryos were kept at RT for 2 hours. After 3 washes with PBST, embryos were incubated with secondary antibodies Donkey anti rabbit Alexa488 (Jackson Immuno Research, 711-545-152, 1:800), Donkey anti goat Alexa 555 (Invitrogen, A-21432, 1:1000), Donkey anti Chicken IgY Alexa488 (Jackson Immuno Research, 703-545-155, 1:800), Donkey anti mouse Cy3 (Jackson Immuno Research, 715-165-150, 1:800), Donkey anti rabbit Alexa647 (Jackson Immuno Research, 711-605-152, 1:500) for 3 hours at RT, followed by three washes. Embryos were then embedded in antifade medium (Invitrogen, P36930) for microscopy analysis.

Cells were carefully washed once with PBS. Fixation was carried out with 3.7% formaldehyde (Carl Roth) in PBS for 10 min at RT. Cells were then permeabilized with 3 mM sodium citrate tribasic dehydrate (Merck), 0.1% v/v Triton X-100. Permeabilized cells were washed twice with PBS and twice in washing solution [PBS, 0.1% v/v Tween 20, 0.2% w/v BSA] for 5 min. Cells were blocked with blocking solution [PBS, 0.1 % v/v Tween 20, 2.5% w/v BSA] for 30 min and incubated overnight at 4 °C with primary antibodies in blocking solution. The following antibodies were used: GFP (Aves, GFP1020, 1:1000), IAP-GAG (Cullen lab, 1:1000). Cells were washed three times in washing solution for 10 min before incubation with secondary antibodies Donkey anti Chicken IgY Alexa488 (Jackson Immuno Research, 703-545-155, 1:800) and Donkey anti rabbit Alexa 555 (Invitrogen, A-31572, 1:1000) in blocking solution containing 10% normal goat serum (Dianova-Jackson Immuno Research) at RT for 1 h. After washing three times in PBS, 0.1% Tween 20 for 10 min, cells were embedded with Vectashield/DAPI (Vector Laboratories) on standard microscope slides (Carl Roth). The immunofluorescence staining was examined with Axiovert 200 M inverted microscope for transmitted light and epifluorescence (Carl Zeiss Microscopy) with the help of the AxioVision Special Edition Software (Carl Zeiss Microscopy).