Table 1.

Double-labeling procedures for co-localization of intracellular sites known to compartmentalize eicosanoid synthesis

Sites of eicosanoid synthesis	Materials	Double-labeling procedures
Nucleus	DAPI (4,6-diamidino-2-phenylindole dihydrochloride)	For nuclear staining, after EDAC and Ab incubation steps, EicosaCell slide preparations should be extensively washed in HBSS−/− and then incubated with DAPI (DAPI working solution 100 ng/ml or 300 nM, see Note 4) for 5 min before aqueous mounting medium application
	TO-PRO-3 (642/661) 1 mM solution in dimethylsulfoxide (DMSO)	To better visualize perinuclear eicosanoid synthesis by EicosaCell, a double labeling with TO-PRO-3 is recommended. TO-PRO-3 must be added together with the secondary Ab for 1 h to label nuclei
Lipid Body	BODIPY® 493/503 (4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene)	To employ BODIPY 493/503 for lipid body labeling, incubate EicosaCell preparations with 1 μm BODIPY 493/503 for 45–60 min at 37 °C simultaneously with secondary Ab incubation. Then, to remove non-incorporated BODIPY 493/503, EicosaCell preparations should be washed at least twice in HBSS−/−
	mAb anti-ADRP Mouse monoclonal antibody (mAb) to adipose differentiation-related protein (ADRP)	For ADRP immuno-labeling, slides are incubated for 1 h with guinea pig anti-ADRP Ab together with the primary anti-eicosanoid Ab to localize lipid bodies within leukocytes. The cells are washed with HBSS for 10 min (3×) and incubated with Cy3-labeled anti-guinea pig secondary antibodies for 1 h
Phagolysosome	Abs anti-LAMP-1	For phagolysosome double-labeling, after incubation at 37 °C with EDAC, cells should be washed with HBSS−/− and incubated with mouse anti-eicosanoid Ab and rat mAb against LAMP-1 (2.5 μg/ml) in blocking buffer (5 % normal donkey serum) for 2 h at room temperature