Zenon alexa fluor 647 rabbit igg labelling kit

URECs were fixed in ice-cold methanol for 10 min. After 30 min saturation with 5% BSA in PBS, cells were incubated for 1 h at room temperature with the following primary antibodies in blocking solution: rabbit anti-ARL13B (Proteintech, 17711–1-AP); mouse anti-acetylated α-tubulin (Sigma T6793); mouse anti-ARL13B (Proteintech, 66739-1-Ig); rabbit anti-IFT88 (Proteintech, 13967-1-AP). Following washes in PBS, cells were incubated at room temperature for 1 h with the following secondary antibodies: donkey anti-rabbit Alexa Fluor 488 (Thermo Fisher); donkey anti-mouse Alexa Fluor 594 (Thermo Fisher). Following further washes in PBS, cells were incubated overnight at 4 °C with primary rabbit anti-Pericentrin antibody (Abcam ab 4448) directly labelled using Zenon Alexa Fluor 647 rabbit IgG labelling kit (Thermo Fisher), washed with 0.1% PBS Tween and post-fixed with 4% PFA for 15 min. Following final washes in PBS, cells were mounted in Vectashield (Vector Laboratories Ltd., H-1200). Images and z-stacks were captured in a blinded fashion, using a Nikon (A1) confocal inverted microscope. Each experiment was performed in duplicate.