Fixation is a process that helps to lock proteins in place on cells you plan to analyze. There can be a variety of reasons to fix your cells, including:

  • Timing/Covenience (experiments that cannot be completed in one day)
  • Safety (biohazardous samples that must be fixed/killed prior to analysis)
  • Intracellular analysis of targets (also includes permeabilization)

Fixation is a common practice, but it creates some difficulties if samples are fixed prior to staining for surface antibodies. Fixation can alter the epitope, making it difficult for your antibody to recognize its target. The information provided below is based on our in-house testing of our clones on unfixed cells (no fixation) and cells fixed with 4% paraformaldehyde prior to staining (post-fixation). We have provided data where possible. Black histograms/dot plots represent unstained samples. Purple histograms/dot plots represent staining with the indicated test antibody.

 

Please note that this data is based on several variables, including the types of cells analyzed, the antibody's fluorophore, and relative abundance of the antigen. Costains are also recommended to help you identify your population of interest. If you are planning to use a live/dead stain (such as Zombie Dyes), stain with them prior to fixation.

 

While these results will not necessarily guarantee the success or failure of staining a fixed epitope, it will at least provide you with an idea of how your samples might look.

 

View Tips!

Chemokine receptors often display high false positives across lymphocytes, monocytes, and granulocytes when staining post-fixation.

 

Lymphocyte, monocyte, and granulocyte gates are based on FSC and SSC profiles.

 

FL-1 indicates the FITC Channel


Learn more about our fixation solutions:


True-Phos™ Perm Buffer is specially designed for the flow cytometric staining of phospho-specific targets like pSTAT3. 

Fixation Buffer – for use with Permeabilization Wash Buffer (10X) to detect intracellular targets such as cytokines, chemokines, or other cytoplasmic antigens.

FluoroFix™ Buffer – for fixation of immunofluorescence stained cells, optimized to stabilize tandem dyes.

True-Nuclear™ Transcription Factor Buffer Set - for optimal staining of intranuclear and cytoplasmic antigens. Cytokine staining may not be optimal with this buffer as loss from cells will be evident with this method of fixation and permeabilization.  Refer to our Fixation and Permeabilization buffers above for cytokine staining.

RBC Lysis/Fixation Solution (10X) - optimized for one-step lysing of red blood cells (RBCs) and fixation of the remaining blood cells following immunofluorescent staining with fluorochrome-conjugated antibodies, suitable for either lyse/wash or lyse/no wash staining procedures.

 

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