Two-step fix/perm Protocol
**These protocols are meant to be modified with your experiment specifics in mind. This can be done in
conjuunction with the RCF staff if you require any assistance
PFA/Saponin fixation and permeabilization:
Protocol Rationale: PFA (4%) acts as a fixative and is used in conjunction with an agent that
permeabilizes the cell (saponin, digitonin, etc). In the absence of fixative, the permeabilizing
agent would allow access to the intracellular compartments but much of the protein would leak
out and be lost. Below is a sample protocol for the PFA/saponin combination.
Cautions:
When using PFA, fixation typically is done at 4% but prolonged fixation/storage can lead
to long, rigid backbones rich in double bonds and massively increasing autofluorescence.
After fixation it is recommended to remove the PFA or at least dilute it to below 1%.
PFA and bleach combine to make toxic gas. If you are using PFA fixed samples ensure that
the waste container does NOT have bleach in it.
Formalin contains some methanol so it will not work as “well” as PFA
Protocol:
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Prepare 106
any extra unbound antibody washed off. Following the last wash, resuspend the pellet
in 4% PFA (diluted in PBS) ensuring rapid mixing. If 4% PFA does not work, try fixing with
lower concentrations as this may help to preserve your epitope(s).
Fix for 15 minutes at room temperature.
Pellet and resuspend the cells in PBS/0.1% saponin/4%PFA for 15 mins. Some protocols
leave out the PFA at this stage but it will depend on your antigen. You have to remove
the PFA before adding antibody in any case.
Pellet and wash cells with 2x with PBS followed by resuspending in PBS/0.1% saponin
with 1-2% BSA or other serum protein for blocking purposes.
Add appropriate dilutions of intracellular staining antibody(ies) (preferably directly
conjugated to fluorophore in order to eliminate the extra wash steps associated with
indirect labeling).
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Incubate for the recommended time (usually 30 mins at room temperature since
intracellular staining is a bit “slower”).
Wash twice with PBS/0.1% saponin in order to maintain permeabilization during the
wash steps.
Acquire data via flow cytometry.
Other permeabilization agents include Digitonin, 0.1% Triton X-100 or NP-40 (nonionic
detergents) or as a last resort detergents like SDS or DOC which are almost never used.