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Immunohistochemistry Troubleshooting
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Immunohistochemistry Troubleshooting

Weak or No Staining

Inadequate deparaffinizationDeparaffinize sections longer or change fresh xylene
Inactive primary antibodiesReplace with a new batch of antibodies
Antibodies do not work due to improper storageAliquot antibodies into smaller volumes and store in freezer (-20 to -70 C) and avoid repeated freeze and thaw cycles. Or store antibodies according to manufacture's instructions.
Antibody concentration was too lowIncrease the concentration of primary and/or secondary antibodies. Or run a serial dilution test to determine the optimal dilution that gives the best signal to noise ratio
Inadequate antibody incubation timeIncrease antibody incubation time
Inadequate or improper tissue fixationIncrease duration of postfixation or try different fixatives
Tissue overfixationReduce the duration of postfixation. If the tissue has already been overfixed, perform an appropriate or recommended antigen retrieval procedure.
Incompatible secondary and primary antibodiesUse secondary antibody that will interact with primary antibody. For example, if primary antibodies are raised from rabbits, use anti-rabbit secondary antibodies
Inactive secondary antibodyReplace with a new batch of antibody
Inactive ABC reagentsReplace with a new batch of reagents
Defective or incompatible enzyme substrate systemReplace with a new batch of reagents
Inadequate substrate incubation timeIncrease the substrate incubation time
Incorrect mounting mediumChoose a correct mounting medium
Reagents applied in wrong order or steps omittedCheck notes or procedure used


The concentration of primary and/or secondary antibodies was too highReduce antibody concentration or perform a titration to determine the optimal dilution for primary and secondary antibodies
Incubation time was too longReduce incubation time
Incubation temperature was too highReduce incubation temperature
Substrate incubation time was too longReduce substrate incubation time
Sections dried outAvoid sections being dried out

High Background

Inadequate washing of sectionsWash at least 3 times between steps
Tissue contains endogenous enzyme such as peroxidase or alkaline phosphataseBlock endogenous enzyme activities using 3% hydrogen peroxide (block peroxidase) in methanol or levamisole solution (block AP) prior to incubation of primary antibodies.
Tissue contains endogenous biotin activityBlock endogenous biotin activity using the avidin/biotin blocking reagent prior to incubation of primary antibodies.
Non-specific binding of primary antibodies to tissue or antibody concentration was too highNon-specific binding  may be reduced by using higher dilution of primary antibodies
Non-specific binding of secondary antibodies to tissueTreat tissue with normal serum from the same species as secondary antibodies.
Secondary antibodies cross react with similar species of tissue, i.e. rabbit anti-rat IgG may cross react with mouse tissue.Use pre-adsorbed 2nd antibody, i.e. use rabbit anti-rat IgG, mouse adsorbed, on mouse tissue, or use rabbit anti-mouse IgG, rat adsorbed, on rat tissue.
Diffusion of tissue antigen due to inadequate fixationIncrease duration of postfixation
Mouse antibodies used on mouse tissuesTreat tissue with MouseOnMouse blocking reagent prior to the primary antibody incubation
Sections dried outAvoid sections being dried out

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