Table 1.
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) |