Alexa fluor 680 donkey anti sheep igg

HUVEC were cultured on collagen-coated glass-bottom dishes (MatTek), fixed with 4% paraformaldehyde in PBS, permeabilized with 0.15% Triton X-100 for 20 minutes, and blocked with 5% donkey serum for 1 hour at room temperature. Cells were immunostained with primary antibodies at 1:330–1:1000 dilution range overnight at 4°C, washed, and incubated for 1 h at room temperature with DAPI and secondary fluorescent labeling antibodies (all at 1:2000 dilution), including Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 546 goat anti-mouse IgG, Alexa Fluor 488 goat anti-rabbit IgG, Alexa Fluor 594 goat anti-rabbit IgG, Alexa Fluor 680 donkey anti-sheep IgG (Molecular Probes), Cy3-AffiniPure Bovine Anti-Goat IgG (H+L) (Jackson ImmunoResearch Laboratories). Cells were imaged on an IX81 inverted confocal microscope with an FV1000 camera (Olympus) using sequential line scanning. FV10-ASW 3.0 (Olympus) software was used for image capture and analysis.

Squashes of w¹¹¹⁸ third instar larval salivary glands and immunostainings were performed as described previously [36 (link)] using guinea pig anti-Cyclin G (1:40), sheep anti-ASX N-ter (1:20) (described in [39 (link)] and [81 (link)], respectively), rabbit anti-H3K27me3 (1:40; pAb-069-050, Diagenode, Denville, NJ, USA), or rabbit anti-RNA polymerase II CTD phosphorylated on serine 2 (1:200; ab5095, Abcam, Cambridge, UK) antibodies. Secondary antibodies (Alexa Fluor® 488 goat anti-guinea pig, Alexa Fluor® 594 goat anti-rabbit IgG and Alexa Fluor® 680 donkey anti-sheep IgG, Molecular Probes, Eugene, OR, USA) were used at a 1:1,000 dilution.