You first make the single cell suspension of the cells. Now if you want o stain with surface marker, do it now: Incubate with surface antibodies for 15-30 minutes at 37 degree/dark. after that wash the cells with wash buffer/ stain buffer once. After centrifugation, add fixation buffer (most of those are methanol based). The you need to treat cells for permeabilization. If you are using saponin based perm buffer, then you need to stain cell during perm step, because saponin based perm is reversible. If you are using other perm buffers (use manufacturers instructions). Then stain your cells with intracellular antibody (Arginase 2). Also add Hoechest dye at this time. After 30-60 minutes of incuabtion, wash the cells twice with staining buffer and Go for Acquisition on flowcytometry.

My questions are;

1. Do you want to stain cells for FACS or microscopy (as the protocol i said is for FACS)

2. Do you have conjugated antibody for Arginase 2 or a primary secondary combination?

3. What buffers do you have with you for fixation ad permeablization?

You first make the single cell suspension of the cells. Now if you want o stain with surface marker, do it now: Incubate with surface antibodies for 15-30 minutes at 37 degree/dark. after that wash the cells with wash buffer/ stain buffer once. After centrifugation, add fixation buffer (most of those are methanol based). The you need to treat cells for permeabilization. If you are using saponin based perm buffer, then you need to stain cell during perm step, because saponin based perm is reversible. If you are using other perm buffers (use manufacturers instructions). Then stain your cells with intracellular antibody (Arginase 2). Also add Hoechest dye at this time. After 30-60 minutes of incuabtion, wash the cells twice with staining buffer and Go for Acquisition on flowcytometry.

My questions are;

1. Do you want to stain cells for FACS or microscopy (as the protocol i said is for FACS)

2. Do you have conjugated antibody for Arginase 2 or a primary secondary combination?

3. What buffers do you have with you for fixation ad permeablization?

Its not for flow cytometry. Its for direct immunofluorescence staining

After fixing/permeabilizing the cells, add the DNA dye when it's most convenient for you. It will stick pretty well. Optimize the staining procedure for the antibody.