Problem 1: Lack of Staining
Possible Source	Test or Action
Lack of antigen.	Check protein expression by in situ hybridization    (in some rare cases translation may be blocked even though mRNA is detected).
Antibodies do not work due to improper storage.	Follow storage instructions on the datasheet. In    general, aliquot antibodies into smaller volumes sufficient to make a working    solution for a single experiment. Store aliquots in a manual defrost freezer    (-20 to -70℃) and    avoid repeated freeze-thaw cycles.
Inactive primary or secondary antibodies.	Test reporter system independently to assess reagent    viability.
Inadequate tissue fixation.	Try increasing the fixation time or try a different    fixative.
Tissue overfixation.	Reduce the duration of the immersion or post-fixation    steps. If immersion fixation cannot be avoided (for example, collection of postmortemtissues    or biopsies in pathology lab), antigens may be unmasked by treatment with    antigen retrieval reagents.
Incompatible secondary and primary antibodies.	Use a secondary antibody that will interact with the    primary antibody. For example, if the primary antibody was raised in rabbits,    use an anti-rabbit secondary antibody.
Antigen was destroyed before incubation with the primary    antibody.	If quenching of endogenous peroxidase was done prior to    the addition of primary antibodies, block peroxidase after incubation with    the primary antibody.
Epitope altered during fixation or embedding procedure.	Try restoring immunoreactivity through various antigen    retrieval techniques.            Embed tissue at 58℃ or    below.
Antigen retrieval was ineffective.	Increase the time of treatment or change the treatment    solution.
Reagents omitted or used in wrong order.	Repeat staining and confirm that correct reagents are    used and are added in the correct order.

 

Problem 2: High Background
Possible Source	Test or Action
High concentration of primary and or secondary    antibodies.	Titer antibody to determine optimal concentration    needed to promote a specific reaction of the primary and the secondary    antibodies.
Hydrophobic interactions of the antibody and proteins    in the tissue.	Lower the ionic strength of the antibody diluent    (particularly monoclonal antibodies respond well to reducing the salt    concentration).
Non-specific binding of primary and/or secondary    reagents to tissues.	Use blocking step just prior to primary antibody    incubation (we use 1% bovine serum albumin with 10% normal donkey serum).    Non-fat dry milk is another option.
Non-specific binding of secondary antibody.	Use an antibody that has cross-reactive IgG species    removed (absorbed against sample species).
Tissue dried out.	Avoid letting the tissue dry during the staining    procedure.
Reagents sticking to old or poorly prepared slides.	Start over with freshly prepared or purchased slides.
Background due to ionic interactions.	Increase the ionic strength of the diluent buffer.

 

Problem 3: Cell/Tissue Morphology is Destroyed
Possible Source	Test or Action
Antigen retrieval methods are too harsh.	Empirically determine the conditions that preserve    tissue morphology while restoring the immunoreactivity of the antigen.
Tissue sections falling off slide. (more common with    frozen sections)	Increase fixation time. Empirically determine an    additional or alternative fixative.            Use freshly prepared, adequately charged slides.
Tissue section appears torn or folded. Air bubbles    under section.	Re-cut sections using a sharp blade, or ignore damaged    areas when analyzing the results.
Poor resolution of tissue morphology	Cut thinner tissue sections. Ice crystals may have    destroyed morphology of frozen sections.
Underfixation has physically damaged the tissue or    cells during the staining process	Increase fixation time and/or add a post-fixation step.    Increase the fixative/tissue ratio.            Cut smaller pieces of tissue for more thorough immersion fixation.
Autolysis of tissue leading to staining of necrotic    debris.	Increase the fixation time, ratio. Consider using    cross-linking fixative.

 

Problem 4: Staining is Inappropriate
Possible Source	Test or Action
Fixation method is inappropriate for the antigen.	Try a different fixative or increase the fixation time.
Antigen retrieval may be inappropriate for this antigen    or tissue.	Try different antigen retrieval conditions.
Electrostatic charge of the antigen has been altered.	Try adjusting the pH or cation concentration of the    antibody diluent.
Delay in fixation caused diffusion of the antigen.	Fix tissue promptly. Try a cross-linking fixative    rather than organic (alcohol) fixative.

 

 

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