The following image provides an example of IHC staining.
Immunohistochemistry of formalin-fixed paraffin-embedded (FFPE) cancer tissue. Analysis was performed to compare Connexin 43 membrane staining in FFPE sections of human lung adenocarcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, heat-induced epitope retrieval (HIER) was performed using 10 mM sodium citrate (pH 6.0), followed by heating in a microwave for 8 to 15 minutes. After HIER, tissues were blocked in 3% H2O2 in methanol for 15 minutes at room temperature, washed with distilled H2O and PBS, and then probed overnight at 4°C in a humid environment with an Invitrogen Connexin 43 monoclonal antibody (Cat. # 13-8300), diluted 1:20 in PBS/3% (w/v) BSA. Tissues were washed extensively in PBS buffer containing 0.05% (v/v) Tween-20 (PBST). Detection was performed using an HRP-conjugated secondary antibody followed by chromogenic detection using DAB as the substrate. The sections were counterstained with hematoxylin and dehydrated with ethanol and xylene prior to mounting.
Immunohistochemistry of formalin-fixed paraffin-embedded (FFPE) tonsil tissue. Analysis was performed to compare SuperBoost (A) EverRed & (B) EverBlue staining in FFPE sections of human tonsil tissue compared (C) DAB staining. To expose target proteins, heat-induced epitope retrieval (HIER) was performed using 10 mM sodium citrate (pH 6.0), followed by heating in a pressure cooker for 20 minutes. After HIER, tissues were incubated in 3% H2O2 for 10 minutes at room temperature, blocked with blocking reagent, and then probed overnight at 4°C in a humid environment with an Invitrogen Ki-67 monoclonal antibody (Cat. No. MA5-14520), diluted 1:20 in PBS/3% (w/v) BSA. Tissues were washed extensively in PBS buffer containing 0.05% (v/v) Tween-20 (PBST). Detection was performed with the SuperBoost (A) EverRed Goat anti-Rabbit IgG (Cat. No. E40967), (B) EverBlue Goat anti-Rabbit IgG (Cat. No. E40968), or (C) DAB using SuperBoost Goat anti-Rabbit Poly HRP IgG (Cat. No. B40962). The sections were dehydrated with ethanol and xylene prior to mounting. Images were taken using an EVOS M7000 Imaging System (Cat. No. AMF7000) with 4x objective.
The following points are provided to help identify the cause of high background staining, which results in a poor signal-to-noise ratio. See also the additional notes sections at the bottom of this page for more information.
Incubate a test tissue sample with the detection substrate alone for a length of time equal to that of the antibody incubation. A strong background signal suggests interference from endogenous peroxidases or phosphatases.
Endogenous phosphatases can be inhibited with the endogenous alkaline phosphatase inhibitor, levamisole.
High background can occur when endogenous biotin is not blocked prior to adding the avidin–biotin–enzyme complex.
If the ABC complex is made with avidin, the highly-glycosylated protein can bind to lectins in the tissue sample.
The secondary antibody may show a strong or moderate affinity for identical or similar epitopes on non-target antigens.
Thermo Fisher Scientific is committed to adopting higher validation standards for the Invitrogen antibody portfolio. We have implemented additional specificity tests to help ensure the highest confidence levels in our products. You can identify the products that have already undergone this testing with the Advanced Verification badge, shown above. This badge can be found in antibody search results and at the top of product webpages. The data supporting the Advanced Verification status can be found in the product specific data galleries. To learn more about our testing standards, please visit Invitrogen Antibody Validation.
Egg white, which contains avidin, was often used to coat slides, dilute antibodies or block tissue samples because it is a readily available and inexpensive source of carrier proteins. It is used very rarely nowadays.
Nonspecific interactions between the primary antibody and non-target epitopes in the tissue sample occur regularly during incubation but at a level that does not influence background staining. A high primary antibody concentration will increase these interactions and thus increase nonspecific binding and background staining.
The primary antibody may also show a strong or moderate affinity for identical or similar epitopes on non-target antigens.
The primary antibody diluent may contain little or no NaCl, which helps to reduce ionic interactions.
See also the additional notes sections at the bottom of this page for more information.
Even when the tissue sample is properly prepared and labeled, the enzyme–substrate reaction must occur for the chromogenic precipitate to form. Deionized water can sometimes contain peroxidase inhibitors that can significantly impair enzyme activity. Additionally, buffers containing sodium azide should not be used in the presence of HRP. Finally the pH of the substrate buffer must be appropriate for that specific substrate.
A simple test to verify that the enzyme and substrate are reacting properly is to place a drop of the enzyme onto a piece of nitrocellulose and then immediately dip it into the prepared substrate. If the enzyme and substrate are reacting properly, a colored spot should form on the nitrocellulose.
Primary antibodies generally lose affinity for the target antigen over time, either due to protein degradation or denaturation caused by long-term storage, microbial contamination, changes in pH or harsh treatments (e.g., freeze/thaw cycles).
Test the primary antibody for potency by staining tissue samples known to contain the target antigen with various concentrations of the primary antibody; do the test concurrently with the test sample. If the positive control is not positive for the target antigen at all, then this suggests that the primary antibody has lost potency. In fact, it is good laboratory practice to always run a positive control sample through your staining protocol along with the experimental samples.
Solution: Ensure that the antibody diluent pH is within the specified range for optimum antibody binding (7.0 to 8.2) and that the antibody is stored according to the manufacturer's instructions. To prevent contamination of your antibody solutions, wear gloves when dispensing antibodies, and use sterile pipette tips, if appropriate. Even if you store your antibodies in a refrigerator, always divide them into separate small aliquots. This prevents contamination or loss of the whole vial of antibody if a problem arises.
While high concentrations of the secondary antibody can increase background staining, extremely high concentrations can have the opposite effect and reduce antigen detection.
To test if the secondary antibody concentration is inhibitory, stain positive control samples using decreasing concentrations of the secondary antibody. An increase in signal as the concentration decreases suggests that antibody concentration is too high.
Solution: Reduce the concentration of the secondary antibody.
If the diluent and/or blocking solution contains antigen-neutralizing antibodies, such as those found in serum, then the antibodies will block secondary antibody binding.
Solution: Remove the neutralizing antibodies or change to a different diluent and/or blocking solution.
If a fluorescent marker is being used, check to make sure that there is no autofluorescence in the unprocessed, fixed tissue. In particular, FFPE sections often show strong autofluorescence that may be difficult to inhibit. Many of the options listed above can then be tested to identify the cause of autofluorescence.
If there is autofluorescence in the test sample, then this suggests that either the tissue sample shows inherent autofluorescence (which is common) or that the fixation method is causing the sample to autofluoresce. To determine if the fixation step is the cause of the autofluorescence, test different fixatives (i.e., if aldehyde fixation is used, try a non-aldehyde fixative) to determine if autofluorescence can be reduced without sacrificing antigen detection. If aldehyde fixation is used and no other fixative can be used, then fixative-induced autofluorescence may be reduced by treating the sample with ice cold sodium borohydride (1 mg/mL) in PBS or TBS.
Another approach to reducing autofluorescence is to treat the tissue sample with dyes that quench fluorescence. These dyes include:
Paraffin-embedded samples are often more autofluorescent, even though the sample has been thoroughly de-waxed. Under the circumstances, switching to frozen sections may reduce autofluorescence.
If these approaches are not sufficient to reduce autofluorescence while maintaining tissue sample detection, then the only other alternative is to just choose a fluorescent marker that will not compete with the autofluorescence. For example, fluorescence from dyes that emit at near-infrared wavelengths, such as Invitrogen Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 750 and Alexa Fluor 790, are not affected by most tissue autofluorescence.
For Research Use Only. Not for use in diagnostic procedures.