Troubleshooting for IHC
Immunohistochemistry (IHC) is the detection of antigens in tissue sections through specific antibodies. The unique advantage of IHC over other protein detection methods is the ability to correlate the presence of an antigen with its location in a tissue or cell. This is very important for the study of cell function in normal and pathological tissues, but IHC requires procedures that have many steps that can go wrong, even with appropriate antibodies and in experienced. Here we review some of our methods for IHC troubleshooting investigative procedures. In our experience, the most common problem is the quality of the primary antibody used in IHC on FFPE sections. Any deviation from an optimal protocol can result in nonspecific background staining, less than optimal specific staining, or no staining at all. Careful evaluation of all components involved in each step of the IHC technique and correction of deficiencies will likely result in optimal staining. Creative Biolabs has built a strong technology platform to provide a full range of NAA services and products.
Fig.1 A schematic representation of various factors which may influence the standardization and reproducibility of the IHC process. (O'Hurley, 2014)
-
Excessive Background Staining
-
Prestaining Problems
- Inadequate fixation, necrosis, and autolysis.
- Tissue sections allowed to dry out. Reduce incubation time; incubate in a humidified chamber.
- Sections not completely deparaffinized. Use fresh dewaxing solutions.
- Slide adhesive inappropriate or too thick. Use adhesives specific for IHC or positive-charged slides.
- Tissue section too thick. Prepare thinner sections.
- Inappropriate antigen retrieval used. Reevaluate antigen retrieval conditions.
- Incubation temperature too high. Reduce temperature.
-
Blocking Problems
- Endogenous enzyme activity not suppressed. Increase concentration of blocking agent.
- Inadequate protein blocking. Use a different blocking agent.
- Inadequate blocking of endogenous avidin-binding activity. Use an avidin-biotin-blocking step or use a nonavidin-biotin detection method.
- Inadequate blocking of endogenous biotin. Use an avidin-biotin-blocking step or a nonavidin-biotin detection method.
- Blocking serum from improper species. Use blocking serum from same species as the link (secondary) antibody.
-
Primary Antibody Problems
- Primary antibody too concentrated. Retitrate the primary antibody.
- Primary antibody incubation time too long. Reduce incubation time.
- Primary antibody is from a similar or an identical species as the test tissue (mouse on mouse, rat on mouse, etc.). Use specific protocols or additional blocking steps.
- Inadequate buffer washes (inappropriate buffer ion concentration). Modify ionic strength of the buffer solution.
-
Secondary Antibody Problems
- Secondary antibody and label concentration too high.
- Secondary antibody and label incubation time too long.
- Buffer washes insufficient.
- Secondary antibody recognizes endogenous (tissue) immunoglobulins.
-
Chromogen and Counterstain Problems
- Chromogen concentration too high. Reduce concentration of chromogen.
- Chromogen allowed to react too long. Reduce incubation time with chromogen.
- Buffer washes insufficient. Prolong buffer washes.
- Counterstain obscures the IHC reaction. Use a different counterstain that does not interfere with immunohistochemical staining.
-
Prestaining Problems
-
Inadequate or No Staining of the Test Slide and Adequate Staining of the Positive Control Slide
- The antigen in question is not present in the test tissue.
- The antigen is present in the test tissue, but in a concentration below the method's detection limits. Consider using an amplification procedure, or increasing the primary antibody concentration, incubation time, or temperature, or a combination thereof.
- The test tissue is over- or under-fixed. Modify antigen retrieval protocol.
- The test tissue is from a different species than the control tissue and has different reactivity with the primary antibody. Validate the IHC test with same species control and test tissues.
-
Weak or No Staining of Positive Control and Weak or No Staining of Test Slides
- All slides from some of the primary antibodies used in the run are affected. Check for inadequacy of the primary antibody, method incompatibility, primary and link antibody incompatibility, or inadequate antigen retrieval.
- The whole run is affected. Check assay log and adequacy of reagent volumes and sequence of reagent delivery to the slide. Determine whether reagents were delivered to all slides.
- If it is hit and miss throughout the run, consider technical problems or problems with the tissues. Check for inadequate sequence of reagents, unbalanced autostainer, and inadequate drop zone.
-
No Staining of Positive Control Slide and Adequate Staining of the Test Slide
- Technical error in the staining or handling of the positive control slide: The log or check list should be reviewed; if everything is in order, the assay should be repeated. For control blocks for infectious diseases, the antigen to be tested may not be present through the entire block.
- Tissue section aging: This is probably the result of tissue photooxidation and dehydration during prolonged storage of tissue control sections. Test the tissue control section with a known positive case by IHC or test a freshly cut tissue control section.
Creative Biolabs offers well-established and innovative One-Stop-Shop NAA solutions. We will find a way to manage both the entire supply chain and the entire value chain to meet your needs. When partnering with us there is no need to change providers, which can be both time-consuming and expensive. If you are interested in our NAA services and products, please contact us for more detail.
Reference:
- O'Hurley, G.; et al. Garbage in, garbage out: a critical evaluation of strategies used for validation of immunohistochemical biomarkers. Molecular oncology. 2014, 8(4): 783-798.