Hello everyone,
I am trying to label two different antigens in two different cells of interest, and I want to also be able to label them when the cells are cultured together. I am trying to figure out how to label a cell surface receptor on one cell and a nuclear receptor on another cell. I plan on using Triton X-100 and I fear that if I use the triton X-100 I will dissolve my cell surface receptor. Does anyone have any comments on dual-staining procedures in general, or comments specific to my experiment? Thank you.
Hi Sebastian.
Assuming your membrane receptor antibody recognizes an extracellular epitope,
Try first fixing with Paraformaldehyde and do routine staining for the membrane protein (complete staining, meaning primary and secondary antibodies). Permeabilize the cells with Triton, repeat blocking step and go with routine staining for the nuclear protein. Watch out the compatibilities of the secondary antibodies used! If nuclear staining is not good perhaps you may need an antigen retrieval step to strength signal.
Good luck!
Hi Sebastian,
you can try to do an antigene retrieval, but you can also try to play a litle bit with the concentration of the primary ab. If this leads not to sufficient results try do to use a biotinylated secondary ab and streptavidin with fluochrome. This 3-step method is more sensitiv than the 2-step method. If Triton X-100 is to strong, use Tween 20 for permiabilisation.You can also play with the incubation time of the primary ab. That means: 3-4 hours increase to over night incubation or stay for 2-5 days in the primary ab at 4°C, This methods which I discribed here are not so strong like the antigene retrieval but they will help you to get good results as well. Good luck!
Hi Sebastian,
I think Ute's idea for using biotinylated secondary Ab and fluorochrome-conjugated streptavidin can do wonders here. Of course, you need to do the Ag retrieval with that.
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