Alexafluor 555 anti rabbit igg

Cells or tissues were stained per the manufacturer’s protocol using rabbit polyclonal (ab19857, Abcam, Cambridge, MA; 1:200 dilution) or murine anti-OCT-4 (ab91194, Abcam, Cambridge, MA; 1:200 dilution), and rat anti-Vimentin (ab115189, Abcam, Cambridge, MA; 1:200 dilution). Secondary antibodies were Allophycocyanin anti-mouse IgG (A-865, Life Technologies, Grand Island, NY; 1:1000), AlexaFluor 555 anti-rabbit IgG, and anti-Rat IgG AlexaFluor 647 (Abcam, Cambridge, MA; 1:1000 dilution). Cells were visualized with an Olympus FV1000 Confocal Microscope using 488 nm, 543 nm, or 633 nm for excitation. Tissue was visualized with an Olympus BX51 upright epifluorescence microscope (Olympus America, Center Valley, PA) using excitation/emission filters of 480 nm/535 nm, and 620 nm/700 nm.

In short, the cells were plated in 12-well culture plates with cover glass pre-coated with 10% polylysine. Cells were cultured for 3 days then fixed with 4% paraformaldehyde (PFA) in PBS, permeabilized with 0.1% Triton-X100 and blocked with 5% normal goat serum in PBS. Then, they were incubated overnight at 4 °C with: Rabbit anti-Ki-67, 1:200 (Abcam). After washing with PBS, cells were incubated with Alexa Fluor^®555 anti-Rabbit IgG, 1:4000 (Abcam) for 1 h at room temperature. Cells were then counter-stained with DAPI (Southern Biotechs). The images were captured using FLUOVIEW FV 10i (OLIMPUS).