Very low signal: how long can I stain for?
Hi, I'm relatively new to CyTOF and I've been working on optimizing my panel of antibodies. I've been staining both surface and intracellular antibodies at room temperature for 30 min, and I've noticed that for certain antibodies, no matter now much I incubate with, I never get good staining. The same antibodies I'm struggling with in CyTOF also give a weak signal when I do western blots (I have to expose the film for longer), so I'm thinking a solution for this could be to incubate my cells for longer with the antibodies during my CyTOF prep. I've seen on here that people range from 30 min to 1.5 hrs and at room temperature, 4C, or on ice.
Does anyone have any insights to how I might be able to stain my samples to obtain a better signal? I was going to set up a few different experiments in parallel and do a 1:50 dilution of the antibody and incubate for various amounts of time (1.5 hrs at room temp, 2 hrs on ice, etc). Is there an upper limit to how long I could stain them before I jeopardize the integrity of my sample?
Thank you!!
Does anyone have any insights to how I might be able to stain my samples to obtain a better signal? I was going to set up a few different experiments in parallel and do a 1:50 dilution of the antibody and incubate for various amounts of time (1.5 hrs at room temp, 2 hrs on ice, etc). Is there an upper limit to how long I could stain them before I jeopardize the integrity of my sample?
Thank you!!