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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, or use pre-adsorbed 2nd antibody.

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