Dear Amanda Rocha

You have to pay attention your fluorochromes and your proteins molecular weight.

So please see these links for more clarification, nomenclature and procedure:

2 links for details:

https://microscopy.duke.edu/guides/paraformaldehyde-formaldehyde-formalin

https://bitesizebio.com/22141/fixation-and-flow-cytometry/

Nomenclature:

http://www.cyto.purdue.edu/cdroms/cyto3/1/topics/prep/0011.htm

Prepration:

http://flowcytometry.utoronto.ca/wp-content/uploads/2016/02/FixingCells_PFA.pdf

All the best,

Abdolvahid

To carry out intra-cyto labeling on monocytes in culture, cold acetone containing 5% water is a very reliable method, and an antibody coupled to a fluorophore at a concentration of 1 μg / ml can be used. For the FACS, saponin (QuilA Bark) is used at 0.2%, and the antibody concentration can be lowered to 0.1 μg / ml.

Dear you should be able to see it from the manual provided by the manufacturer.

I have used commercially available fixatives from BD or ebiosciences it worked well. I have done extensive 11 color flow cytometry on clinical samples and i did not found much of a difference on the fluorochrome leaching except for methanol fixation

Hello Amanda,

I would try Biolegend's FOXP3 Fixation/Permeabilization Buffer, it has been VERY reliable for intracellular/nuclear labelling of all intracellular antigens we have evaluated. Home made solutions - fix => 2% methanol-free PFA(buffered pH7.2) and perm => 0.25% molecular grade saponin in buffer-saline supplemented with 0.1% BSA-V and 0.25% sodium azide. You may need to determine optimal incubation times - standard intracellular protocol 15/45 minutes fixation ->(wash) and permeabilization. For intranuclear, antigens, I extend the permeabilization(antibody labelling) up to 18 hours. ALWAYS use isotype controls, or FMOs, to confirm unbound antibodies are washed out before calculating specific binding. We use Biolegends buffer(quality controlled) for the staining but home made saponin solution for washing steps, so large volumes can be used with minimal cost.

Good luck