One-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
Alcohol fixation and permeabilization:
Protocol Rationale: Fixation with alcohols is rapid and results in fixation and permeabilization at the same time. Thus, there is no need to include a permeabilization agent.
Cautions:
The alcohol should be kept in the freezer and added dropwise to the cells while it is still very cold. Because of low surface tension, the alcohol will start to drip out of a p1000 tip after you take it up. It is best to pipette up the alcohol immediately adjacent to the cell suspension and let it semi-passively drip (with a little encouragement or you will be there all day) into the cell suspension tube.
The mixing should take place on a vortex to ensure rapid mixing so that the cells don’t become fixed to one another and clump. If the alcohol is diluted by the addition of cells too much, fixation will become sub-optimal as a result.
It is important to wash off the ethanol after fixation so as to not affect the staining deleteriously. It is also important to wash after staining to remove unbound antibody.
Protocol:
· Place 100L of cells to be stained (106/mL) in a 1.5mL eppendorf tube (prestained for surface antigens and washed if this is applicable).
· While vortexing add 1mL of 80% methanol in a dropwise manner. I have found that fixation in 95-100% alcohol can be too harsh and remove antigen or destroy the epitope compared to 80% v/v.
· Fix on ice for 15 mins.
· Pellet the cells and wash in PBS 2x before staining for intracellular antigens and washing. The pelleting speed will depend on the cell type to an extent. Since they have been fixed they will be “lighter” so I would generally recommend 500xg 5 mins with a brake of 70%.
· Acquire samples on the flow cytometer or store at 4oC in the dark for up to a week depending on the cell type/stain.
Other fixatives commercially available include BD FACS Lysing solution (which lyses RBCs, fixes and permeabilizes cells), cytofix/cytoperm, etc.