Anti mouse igg alexa 488 conjugate antibody

Cells and blastocysts were plated on 0.1% gelatin-coated chamber slides (Thermo Fisher Scientific), treated as indicated, and then fixed with 3.7% formaldehyde diluted in PBS for 30 minutes. The slides were permeabilized with 0.1% Triton X-100 diluted in PBS, blocked with 10% BSA diluted in PBS, and incubated with an Oct3/4 antibody (Santa Cruz Biotechnology) and a Nanog antibody (Abcam) diluted in 5% BSA in PBS at a 1:200 dilution, for 2 hours. The slides were washed with PBS, and then were incubated with an anti-mouse IgG-Alexa 488 conjugate antibody (Thermo Fisher Scientific) and an anti-rabbit IgG-Alexa 548 conjugate antibody (Thermo Fisher Scientific) diluted in 5% BSA in PBS at a 1:400 dilution, for 1 hour. The slides were again washed with PBS and visualized by brightfield and fluorescent microscopy. All images were captured using IPLABS software (BD Biosciences) and processed using ImageJ software.

Cells grown on glass coverslips were treated as indicated, fixed and permeabilized with methanol. The slides were then blocked with 10% bovine serum albumin diluted in PBS. For each antibody used, the cells were incubated for 90 min using the following dilutions: CD63 (1:200, Abcam), LAMP1 (1:100, Cell Signaling Technologies), Cathepsin B (1:800, Cell Signaling Technologies), GFP (1:100, Cell Signaling Technologies). Anti-Mouse IgG-Alexa 488 Conjugate antibody (1:400, Thermo Fisher) and anti-Rabbit IgG-Alexa 568 Conjugate antibody (1:400, Thermo Fisher). DAPI (Sigma) was used to label nuclei, and conjugated Phalloidin (1:2000) was used to label actin. The cells were visualized with Super Resolution Structured Illumination Microscopy using a Zeiss Elyra Super Resolution Microscope with a 63x oil objective lens (Cornell University, Biotechnology Resource Center). Image processing and quantification was performed with ImageJ software.