Dylight 594 goat anti rabbit igg

The primary antibodies used were as follows: anti-HIFα rabbit polyclonal (1:200 for preimplantation embryos and 1:500 for the others, cat# NB100-479, Novus Biologicals); anti-Oct3/4 mouse monoclonal (1:200, cat# sc-5279, Santa Cruz Biotechnology); and anti-GFP mouse monoclonal (1:500, cat# G6539, Sigma-Aldrich). The following secondary antibodies were used at a dilution of 1:1000: DyLight 594 goat anti-rabbit IgG (cat# DI-1594, Vector Laboratories); DyLight 488 horse anti-mouse IgG (cat# DI-2488, Vector Laboratories); FITC-conjugated goat anti-mouse IgG2b (cat# ab98702, Abcam); and biotinylated donkey anti-rabbit IgG (cat# 711-065-152, Jackson ImmunoResearch).

Tissues were embedded in O.C.T.™ (Sakura), and 5-μm cryosections were generated and fixed in acetone before processing. Epidermal ear sheets were generated by separating epidermis from dermis with 3.8% ammoniumthiocyanate (VWR) and fixed in 4% PFA (Roth). Samples were blocked for 30 min at room temperature in 1% bovine serum albumin (Sigma-Aldrich) in PBS containing 5% goat serum (PAA) and 0.1% Triton (Sigma-Aldrich) and were incubated with the indicated antibodies overnight at 4°C in the same buffer. Apoptosis of epidermal LCs was assessed by co-staining with antibodies against Langerin and active caspase-3 followed by a secondary staining with the DyLight 594 goat anti-rabbit IgG (Vector Laboratories).