Hi,
I hypothesize that transcription factor X is activated in cell type A (in this case cells of the tumor microenvironment) but not in cell type B (cancer cells). Can I test this hypothesis with indirect immunofluorescence staining on tissue sections and judge activation of the TF by nuclear localization vs no signal or cytoplasmatic signal? (I will also perform co-staining to identify the specific cell types.)
Unfortunately, I don't have single cell suspensions to generate cell lysates..
I'd appreciate any help/ suggestions.
Best, Johann
Hi johann, you can also try to do a microdissection of your tissue and get the two cell population you want to interrogate (cancer cells) (a pool of microenvironment cells) , this way you will get only the cellular fraction that you want to interrogate, then extract protein from the tissue and perform WB with your TF antibody.
i also think that the aproach you are plannig is correct as you will include aditional markers to clearlly identify cancer vs other cells. it will depend of how ubicous is the expression of your TF.
best.
Dear Johann,
In theory your approach will work, but it will also depend on the quality of the antibody to your transcription factor. Even with a good primary antibody, you may not be able to get a good signal if you only rely on a labeled secondary. I have only had experience with an antibody to pSTAT3 in neurons (not cancer cells), and that needed amplification to be detected.
Good Luck!
Jill
That sounds good. Do not forget to stain DAPI or Hoechst 33342 for nuclear marker. By the way, nuclear localization is only part of the event that indicates the activation, but there is also possibility that the said factor gets transported back to cytoplasm. Best way to indicates activation of certain pathway by transcription factor is to use reporter assay, for example luciferase-based reporter assay.
To complement the assay, you can perform RNAscope (ISH for mRNA) of downstream target genes of transcription factor X to validate your findings (If X is active, it's downstream targets should be increased). This can be quantified by dots per cell. Fancy technique, I have used it in two recent publications.
Yes, your plan is correct.
In addition to this, if suitable, you can perform flow cytometry with "localisation of protein" protocol. In this way, you can immunophenotype your cells along with localisation (nuclear vs cytoplasmic) of your transcription factor. The best part of this approach is that you will know the exact % of your transcription factor inside the nucleus in each of your tissue.
You can follow the following protocol to prepare cell suspension from your tumor/non tumor tissues for flow cytometry analysis.
Chapter Preparing Viable Single Cells from Human Tissue and Tumors f...
Another possibility is to use an antibody targeted against the phosphorylated form of the TF – if available and if the activated TF is phosphorylated…. Good luck, Karsten :-)
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