I want to visualize EOMES, a transcription factor, in CD8 T cells. I am planning to perform an immunofluorescence test and I was wondering what kind of stain or chemicals I can use to enable the visualization of internal markers of a cell.
Targeting intracellular epitopes with immunoassays (fluorescent or histochemical) requires the cell membranes to be permeabilized. This is typically done by briefly incubating slides in PBS that contains Triton X-100 (typically 0.1 - 0.25% v/v).
Abcam provides a nice overview of these protocols here:
https://www.abcam.com/protocols/immunocytochemistry-immunofluorescence-protocol
You can also use Western blotting? flow cytometry or other assays for specific markers (for example caspases using kits for spectrophotometric determination).
Article Western Blot: Technique, Theory and Trouble Shooting
John Hardy Lockhart Thank you for the answer. Triton X-100 is not suggested for analysis in cells with an intact membrane if I am correct. I forgot to mention that I do want to keep the cells alive and their membranes intact, because I also would like to analyze some molecules that are expressed on the cell membrane. Do you have any suggestion which chemical I can use for this?
Fa Ro , that does make this quite a bit more complicated.
Fluorescently-conjugated small peptides (i.e. phalloidin) can be used to visualize some intracellular targets in live cells (actin filaments in the case of phalloidin), but this does not apply to most proteins. Unfortunately the size of normal antibodies prevents their entry into live cells, but a few different approaches for labeling intracellular targets in live cells (e.g. quantum dots and nanobodies) have been published. Unfortunately, none of these systems are in widespread use.
The most common approach for live cell visualization of a specific protein is constructing a fluorescent fusion protein. This can be done with transient transfection or through generation of stable cell lines. However, there is no guarantee that the fusion proteins will behave in the same manner as the wild-type due to steric hindrance.
One group has already reported a GFP-Eomes fusion protein in mice: Article Eomes and Brachyury control pluripotency exit and germ-layer...
. They used TALENs to insert the GFP into the first exon of Eomes, but you should be able to reconstruct this in a plasmid.
If you are going to do a flow cytometry experiment, try to use and Fix and Perm or any other commercially available solution. Some labs use Saponin I suggest to use a "ready to use" permeabilization solution which will allow you to detect surface markers simultaneously.
Best wishes,
You need to carry out membrane permeabilization step by using 50% chilled methanol for 30 min. before going for non-specific sites blocking step.
Hi, Triton-X100 can be supplied by Tween20 or chilled methanol
Compare both conditions with and without permeabilization.
FLow cytometry experiment to corroborate de membrane proteins.
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