Immunohistochemistry Troubleshooting

 

Immunohistochemistry Troubleshooting

 

 

 

Weak or No Staining

 

Sources

Solutions

Inadequate deparaffinization Deparaffinize sections longer or change fresh xylene
Inactive primary antibodies Replace with a new batch of antibodies
Antibodies do not work due to improper storage Aliquot 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 low Increase 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 time Increase antibody incubation time
Inadequate or improper tissue fixation Increase duration of postfixation or try different fixatives
Tissue overfixation Reduce the duration of postfixation. If the tissue has already been overfixed, perform an appropriate or recommended antigen retrieval procedure.
Incompatible secondary and primary antibodies Use secondary antibody that will interact with primary antibody. For example, if primary antibodies are raised from rabbits, use anti-rabbit secondary antibodies
Inactive secondary antibody Replace with a new batch of antibody
Inactive ABC reagents Replace with a new batch of reagents
Defective or incompatible enzyme substrate system Replace with a new batch of reagents
Inadequate substrate incubation time Increase the substrate incubation time
Incorrect mounting medium Choose a correct mounting medium
Reagents applied in wrong order or steps omitted Check notes or procedure used

 

Overstaining

 

Sources

Solutions

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

 

High Background

 

Sources

Solutions

Inadequate washing of sections Wash at least 3 times between steps
Tissue contains endogenous enzyme such as peroxidase or alkaline phosphatase Block 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 activity Block 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 high Non-specific binding  may be reduced by using higher dilution of primary antibodies
Non-specific binding of secondary antibodies to tissue Treat 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 fixation Increase duration of postfixation
Mouse antibodies used on mouse tissues Treat tissue with MouseOnMouse blocking reagent prior to the primary antibody incubation

Sections dried out

Avoid sections being dried out