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Troubleshooting

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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 antiobody 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 antibodies
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

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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

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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
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

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Other Resources

 

Vector - Troubleshooting Guide for Immunohistochemistry

Vector - Troubleshooting Problems Encountered Using Mouse Antibodies on Mouse Tissue

R&D Systems - Troubleshooting Guide: Immunohistochemistry

Troubleshooting Guide for Western Blot and Immunohistochemistry

Richard Allan Scientific - Tips for Troubleshooting Immunohistochemistry

CN Biosciences - Technical Tips - Troubleshooting Guide for Immunohistochemistry

Chemicon - Introduction to Antibodies - Troubleshooting

Upstate - Tips for Immunohistochemistry with Fixed Paraffin Embedded Tissues

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If you still can not find answers to your questions, please go to IHC World Forum to read, post, share and discuss your questions with other professional members.

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