Great for Fixation and Permeabilization for Intracellular Protein Staining

August 09, 2018

Johns Hopkins School of Medicine
Oncology
Graduate Student

Overall

Quality of Results

Ease-of-Optimization

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Company:

ebioscience/Thermo Fisher

Product Name:

eBioscience Foxp3 / Transcription Factor Staining Buffer Set

Catalog Number:

00-5523-00

Our lab studies the mTOR signaling pathway in T cell differentiation with an emphasis on both CD4 and CD8 T cells. We routinely conduct flow cytometry experiments to analyze the differentiation process into subsets based on transcription factor (TF) analysis. In order to look at Tregs or effector T cells, we analyze T cells for the expression of Foxp3 or Tbet along with other TFs. This kit is a quick and easy way to perform both critical steps in one step before proceeding to intracellular staining.

Experimental Design and Results Summary

Application

Flow cytometry

Starting Material

Single cells from tissue

Protocol Overview

Once single cells have been obtained, proceed to surface stain your cells of interest for 30 minutes at 4 deg. Wash away the stain, and then mix 1 part of the concentrate to 3 part of the diluent (Fix/Perm) based on the number of samples. I typically use 100ul of Fix/Perm for roughly 4-6e6 total cells. Either fix/perm overnight at 4 deg or 30 minutes at room temperature. Wash away the Fix/Perm with 1x wash buffer before proceeding to intracellular protein staining.

Tips

Determine conditions and duration for long fix/perm for certain proteins of interest.

Results Summary

We are able to routinely able to get great intracellular staining of proteins within cells for flow cytometry such as FoxP3 for Tregs.

DOI or PMID #

25849206

Additional Notes

N/A

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Summary

The Good

Very reliable, consistent, and easy to use!

The Bad

Cell morphology does shrink when using this kit but not a huge issue.

The Bottom Line

One of the best fix/perm kits on the market to really look at intracellular proteins but also nuclear proteins. This kit allows for analysis of transcription factors such as Foxp3.

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