A major advantage of immunohistochemistry (IHC) over other immunoassay techniques is that it preserves the architecture of harvested tissue material. This allows researchers to study antigen distribution and relative abundance, which often differ between conditions of health and disease. However, because IHC involves a greater number of protocol steps than many other immunoassay formats, there are more places where things can potentially go wrong. Here, we’ve compiled some common IHC issues and suggested ways of resolving them.
While every immunoassay workflow requires researchers to make certain choices, IHC takes things that much further. As well as deciding between direct or indirect detection, choosing whether to use enzyme-labeled or fluorophore-labeled antibodies, and determining if additional signal amplification is required, researchers performing IHC must also consider whether they will work with frozen or formalin-fixed paraffin-embedded (FFPE) tissue and if epitope retrieval is necessary for antibody reagents to access their targets. Moreover, where FFPE samples are used, the IHC workflow will include steps such as deparaffinization and rehydration—yet more steps requiring careful optimization.
“Each step of the IHC workflow contributes to the final result,” reports Erika Leonard, Director of Quality Control and R&D at Vector Laboratories. “People tend to focus on the primary antibody and the detection reagent, but it is important to make a conscious decision at every point. This includes making sure that every reagent—from the blocking buffer to the mounting media—is compatible with each other as well as the specimen, and optimizing conditions based on the antigen content of your tissue and how your tissue was fixed. For example, some blocking solutions can interfere with primary antibody binding, while the choice of substrate for chromogenic detection can have a significant impact on assay sensitivity. Proper controls are critical throughout to avoid inconsistent or misleading results.”
In recent years, multiplexed IHC has become increasingly popular. According to David Schwartz, Ph.D., CEO and CSO at Cell IDx, this is due to the fact that singleplex staining confirms only that the target of interest is present. “With multiplexed staining, researchers can uncover contextual information of value,” he says. “For example, using multiplexed IHC, it is possible to address questions such as is PD-L1 expressed on tumor cells or immune cells, which specific immune subsets are infiltrating a tumor, and does this information correlate with response to particular therapies?” Historically, multiplexed IHC was limited to just two biomarkers. However, by using Cell IDx’ UltraPlex technology, which is based on cocktails of tag-conjugated primary antibodies that are detected with anti-tag rabbit monoclonals, this is no longer the case.
Although frozen and FFPE tissue samples involve different IHC staining protocols, both present similar issues. The following table lists some common IHC complaints and potential solutions, with application-specific problems shown in parentheses.
Prepare fresh slides
Store slides at 4°C
Decrease the fixation time (formalin fixation should not exceed 24 hours)
Select primary antibodies that have been validated for FFPE IHC
Compare different epitope retrieval methods (heat-induced epitope retrieval versus protease-induced epitope retrieval) for unmasking
Increase the deparaffinization time
Prepare fresh dimethylbenzene (xylene) stocks
Confirm that an appropriate sample type was used by referring to sites such as UniProt, PAXdb, or proteinatlas.org, and to antibody manufacturers’ datasheets, for information about protein expression
Consider including a signal amplification step
Check that primary antibodies have been validated for the chosen sample type (paraffin sections versus frozen samples) and species
Run a positive control sample, such as a tissue known to endogenously express the target protein or a paraffin-embedded cell pellet, to verify antibody performance
Perform western blots using native and denatured forms of the target protein to confirm the antibody recognizes native protein
Switch to using different primary antibodies
Titrate primary and secondary antibodies to determine optimal concentrations
Use fresh antibodies for each experiment (do not re-use antibodies)
Increase the duration of the primary antibody incubation step
Check the expiry dates of antibody reagents
Confirm antibodies have been stored correctly
Avoid freeze-thaw cycles
Optimize the permeabilization step and include the permeabilizing agent in the blocking buffer and antibody diluent
Confirm that the correct secondary antibody was used (e.g., if the primary antibody was raised in rabbit, an anti-rabbit secondary is required for detection)
Ensure tissues are covered in liquid throughout the course of the experiment
Avoid using sodium azide with horseradish peroxidase (HRP) based detection / phosphate buffer with alkaline phosphatase (AP) based detection
Switch to a different detection system
Consider performing signal amplification (e.g., using VECTASTAIN® ABC)
Increase the exposure time
Quench endogenous peroxidase activity using 3% H2O2 in methanol / endogenous phosphatase activity using 1mM Levamisole prior to incubation with primary antibodies
Optimize the blocking conditions (1 hour blocking with 10% serum from the same host species as the secondary antibody is usually recommended)
Include the blocking agent in antibody diluents
Consider using a glycoprotein-free blocking buffer such as Carbo-Free Blocking Solution
Reduce the duration of the primary antibody incubation step
Consider direct detection (with enzyme- or fluorophore-labeled primary antibodies) or try using a biotinylated primary antibody with a conjugated streptavidin detection reagent
Include a mouse-on-mouse blocking step (e.g., using a M.O.M.™ Immunodetection Kit)
Use secondary antibodies that have been cross-adsorbed against the host species of the sample
Replace anti-species secondary antibodies with tagged primary antibodies and anti-tag secondaries (e.g., UltraPlex technology from Cell IDx)
Run secondary antibody-only controls
Try pre-incubating secondary antibodies with a blocking solution containing 1-2% serum from the same species as the sample
Block endogenous avidin/biotin with 0.05% biotin/avidin in PBS, or using a commercial kit (e.g., the Avidin/Biotin Blocking Kit) prior to incubation with primary antibodies
Block endogenous lectins using 0.2 M alpha-methyl mannoside in antibody diluent prior to incubation with primary antibodies
Avoid introducing potential sources of biotin, such as non-fat dry milk or non-IHC grade BSA
Increase the number, volume, and/or duration of wash steps
Dilute the substrate
Rinse the substrate off the slides sooner
Decrease the exposure time
Ensure fluorophore-labeled antibodies do not share the same spectral range
Switch to using infrared fluorophores for detection to minimize overlap
Consider quenching autofluorescence with a product such as the TrueVIEW® Autofluorescence Quenching Kit
Optimize the deparaffinization step
Try using frozen tissue
Compare different fixation methods
Consider using charged/coated slides (e.g., poly-L-lysine, gelatin, or aminoalkylsilane coated glass) or coat the slides in-house with a reagent such as VECTABOND™
Try using a slide warmer after floating sections onto slides
Optimize the fixation protocol
Optimize the epitope retrieval method
Bake slides prior to epitope retrieval
Use only gentle agitation during incubation and wash steps