Total protein was assessed with IN Cell analyser 2000 (GE Healthcare, Amersham, UK). Cells were fixed with 4 % (w/v) paraformaldehyde then stained with mouse MUC1 ND antibody (Santa Cruz Biotechnology, Heidelberg, Germany) prior to incubation with an anti-mouse Texas Red conjugated secondary antibody (emission 620 nm; Molecular Probes, Thermo Fisher UK). Cell nuclei were counter stained with 4’,6-diamidino-2-phenylindole, DAPI (emission 470 nm; Invitrogen, Thermo Fisher, UK), where PBS wash steps followed each staining process. Five low magnification images of approximately 1000 cells in total were achieved. An object segmentation protocol within the IN Cell Developer software masked the nuclei by segmenting on intensity in the DAPI channel. Information about the fluorescence output in the Texas Red channel (MUC1) within this ‘cell region’ was recorded and each cell assigned an output value. All data shown is based on a minimum of three biological repeats, analysed as parametric data using 2 tailed T test. Significance given as * p < 0.05, **p < 0.001.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.