Table 1.

Troubleshooting eicosanoid detection

Problems	Common causes	Solutions
Lack of eicosanoid detection (when few or no eicosanoid specific immunostaining is observed, but expected)	Improper fixation (when the eicosanoid is not cross-linked by EDAC, but washed-out from cells turning detection impossible) Inefficient cell stimulation	Slight increase in EDAC (by adjusting both EDAC concentration and/or incubation time with cells) Positive control (mandatory inclusion of a well known agonist)
Eicosanoid detection within nonstimulated cells (eicosanoids are nonstorable in the cell and newly formed upon stimulation, therefore nonstimulated cells should not show any immunostaining for the targeted eicosanoid)	Improper stimulation (cell activation during the procedures including cell incubation at 37°C or cell fixation/permeabilization with EDAC can lead to spontaneous, stimulus-independent eicosanoid synthesis) Nonspecific detection	Negative control (nonstimulated cells should always be included as an important negative control. Care is needed to ensure that cells are not mechanically, chemically, or immunologically stimulated) (Discussed below)
Poor preservation of cell morphology	EDAC cell toxicity (during EDAC incubation step, cell appearance may change from unimportant to severe modification of typical cell morphology)	Slight decrease in EDAC (by adjusting both EDAC concentration and incubation time with cells)
Non-specific detection (antibodies may non-specifically bind to other cellular structures or to other lipids found within cells)	Nonspecific binding to cell (since cross-linking properties of EDAC may favor the tendency for cells to be sticky) Nonspecific binding to other eicosanoids	Irrelevant antibody control (a proper control using host/isotype-matched antibodies in stimulated cells should always be included) Eicosanoid synthesis inhibitor control (stimulated cells treated with a synthesis inhibitor of the targeted eicosanoid is a mandatory control condition that should show no immune-labeling confirming specific detection of targeted eicosanoid)
High non-specific binding (when non-specific staining is too high with >10 % positive cells)	Improper detecting antibody conditions	Change detecting antibody dilution and/or host Incubation of detecting antibody with an adsorbing reagent Incubation of EDAC-treated cells with a normal serum (to effectively block out nonspecific sites) Centrifugation of the detecting antibody (to eliminate aggregates)
Losing cell adherence with EDAC (the ability of cells to stay adhered to coverslips or other substrates can be affected by EDAC incubation)	EDAC cell toxicity	Slight decrease in EDAC (previous careful setting of EDAC incubation step is obligatory and should be adjusted for each cell type)