Intracellular Staining

Staining intracellular antigens (phospho-proteins, transcription factors, etc) involves fixing the cells so that the intracellular contents do not float away and permeabilization of the cell membrane so that the antibodies/fluorochromes can enter the cell and label their targets of interest. The two most common ways of doing this are either a one-step fix/perm protocol (ethanol/methanol/commercial fix/perm reagents) or a two-step fix/perm protocol (most often PFA/Saponin) both of which are detailed in the respective protocols available on this site. The general pros and cons are:

Methanol/ethanol fixation (see Facilities-Protocol1-Onestep-Fix-perm) is very quick, fixes and permeabilizes at the same time and is very inexpensive. However, alcohols can lead to lipid extraction and removal of some protein and generally does not preserve intracellular structures well which is one of many reasons it is used less in microscopy.

PFA/Saponin fixation and permeabilization (see Facilities-Protocol2-Two-step-Fix-perm) is milder in this regard and tends to preserve epitopes and more “loosely” membrane-bound proteins. This process requires that saponin is in all wash steps as saponin pores are transient and you need to wash unbound antibody out of the permeabilized cell. In either case you will need an appropriate negative control as the permeabilized cell has more surfaces for non-specific binding and can passively trap unbound antibody inside. In most cases the appropriate control is an isotype control which is the same species, antibody type (i.e. mouse IgG1k), and has the same fluorophore in the same fluorophore/antibody ratio (i.e. Alexa488 cannot be used in place of FITC and if your antibody has 10 FITC moieties attached to it, your isotype control cannot have 20 FITC moieties attached to it).

 

intracellular staining, fixation and permeabilization

As is the case with any fixation step, both of these protocols will result in changes to epitopes and antigens in different ways. If it is unknown what fixation method works best for your antibody/antigen pair then it is best to do a small pilot experiment to determine which fixative results in the best positive staining and low background. For many markers there are compatibility resources for fixatives which can help save you time (see the fixation compatibility links).

Other than the specifics of fixation and permeabilization the two main challenges of intracellular staining are 1) getting the antibody in to stain the antigen well and washing unbound background antibody out, and 2) sensitivity (increased background, autofluorescence, low signal due to epitope fixation). To this end many of the multicolour staining rules from the Immunophenotyping section are relevant. In particular:

  1. Stay away from short wavelength fluorophores when you can as they have worse autofluorescence (which is only exacerbated in fixed samples).
  2. Since signal intensity/background is generally more of a problem with intracellular stains, use your brightest fluorophores for these antigens where possible. For the same reasons, appropriate blocking (BSA etc) is crucial.
  3. If you are using PFA make sure there is no bleach in the waste container or you will form toxic chlorine gas.
  4. Remember that your fluorophores don’t create light from nothing; they have mass. Bigger fluorophores are harder to get into a cell and to wash back out if they are not bound to antigen. I recommend that you do NOT use PE or PE tandem conjugates as they are huge. While very bright, you can often find a smaller fluorophore that will give you just as good of a signal. This isn’t to say you can never use PE, just that there are other options that can be advantageous. If you are using a proprietary dye from a company ask how big it is so you know if it will affect your intracellular staining (i.e. Alexa 647 is small and will work well. Some violet fluorophores are actually tandem conjugates of varying sizes).
  5. An unstained control is insufficient. Because permeabilized cells are effectively little cages to trap unbound antibody you must use controls like isotype controls (which have to have the same fluorophore and F/P ratio to be relevant).
  6. You generally can’t titrate down intracellular antibodies like you can with extracellular antibodies.
  7. If you are combining intracellular and extracellular staining, stain surface things first. Then fix/perm. Then stain intracellular targets.
  8. If you want to perform dead cell exclusion you can’t use PI/7AAD/etc but there are several fixable viability dyes that are available (see the links section).

Useful links:

Fixable Viability Dyes:

Experiment Planning:

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