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Very low signal: how long can I stain for?

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mollyryan

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Posts: 1

Joined: Wed Sep 13, 2017 3:40 pm

Post Tue Jan 30, 2018 5:21 pm

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!!
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RDevine

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Posts: 3

Joined: Tue Sep 13, 2016 3:38 pm

Post Mon Feb 12, 2018 2:53 pm

Re: Very low signal: how long can I stain for?

Hello,

I guess a couple questions would be are you staining extracellular and intracellular at the same time? Or do you do an extracellular followed by a permeabilization of some kind and then do intracellular? Fixation can also have an affect on the staining quality.

A good public resource on staining is from the Nolan lab; https://www.cytobank.org/nolanlab/experiment_protocols/

It could also just be a clone or antibody issue. If the signal is weak on Western blotting it wouldn't be unreasonable to think the antibody is the case. In the SDS-PAGE system your protein is totally (at least ideally) unfolded and all the peptides are accessible to antibody binding. If your antibody isn't binding well in that case to give a good signal it isn't unreasonable to think that in the cell 3D environment the antibody is having even more issues.

As for the time limit if your cells are fixed when staining then you could probably let it go quite a while in a staining medium. The last thing that popped into my head is have you titrated the antibody with different concentrations? There's nothing wrong with your proposed method of sussing it out but you may not have hit the ideal antibody concentration which could be sorted by a titration.

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