Immunohistochemistry (IHC) is an antibody-based technique for detecting specific analytes in tissue sections. Because the tissue architecture is preserved, it is possible to study the distribution and relative abundance of targets of interest, both in the context of different cell types and/or subcellular compartments and under conditions of health and disease. Fluorescently-labeled antibodies are popular for IHC as they allow for multiplexing. However, choosing the right fluorophores is important for accurate interpretation of results. Here, we provide an overview of the fluorescent IHC workflow and suggest ways of addressing common challenges.


The Fluorescent IHC Workflow

A typical IHC workflow begins with collecting and preserving tissue samples. Usually, preservation is achieved by formalin fixation and paraffin-embedding (FFPE), although an alternative approach involves snap freezing the tissues in liquid nitrogen. The samples are then sliced into thin sections and applied to glass slides.

When working with FFPE tissues, the sections must be deparaffinized and rehydrated, and may also require epitope retrieval to disrupt protein cross-links, before immunostaining can proceed. Frozen sections should instead be fixed with alcohol or acetone prior to immunostaining.

If the aim of the IHC experiment is to detect intracellular antigens, FFPE tissue sections must be permeabilized for antibodies to access their targets. Frozen sections do not tend to require permeabilization as acetone and methanol serve this purpose. In either scenario, blocking is essential to prevent non-specific antibody binding.

Once all of these protocol steps have been completed, the samples can be incubated with fluorescently-labeled primary antibodies (direct detection), or with unlabeled primary antibodies and fluorescently-labeled secondary antibodies, incorporating a wash step in between (indirect detection). The workflow concludes with nuclear counterstaining, mounting, and microscopy-based analysis.


Common Challenges with Fluorescent IHC

Challenges with fluorescent IHC can be broadly divided into two groups: weak staining and high background. We’ve summarized the top ten causes for each here, along with suggested solutions.

Weak Staining

Cause Suggested solution
Low analyte expression Refer to online resources such as UniProt and The Human Protein Atlas, as well as to the antibody manufacturer’s datasheet, to confirm that the sample is expected to express the target; use FluoroFinder’s Spectra Viewer to identify a bright fluorophore for detecting analytes with low expression; consider incorporating a signal amplification step into your workflow
Epitopes have been masked by formalin fixation Decrease the fixation time, optimize the epitope retrieval step
Antibodies are unable to access intracellular targets Optimize the permeabilization step; consider replacing full-length antibodies with smaller antibody fragments such as Fab or VHH that offer better tissue penetration
Primary antibody does not recognize the native antigen Use FluoroFinder’s Antibody Search function to find antibodies that are validated for IHC
Primary antibody is too dilute Use the antibody manufacturer’s recommended antibody dilution as a starting point from which to optimize for your particular model system
Primary antibody incubation time is too short Adhere to the antibody manufacturer’s recommended incubation time/temperature
Incorrect secondary antibody Ensure that the secondary antibody matches the host species of the primary antibody (e.g., use a goat anti-rabbit secondary antibody to detect a primary antibody from a rabbit host)
Wrong excitation wavelength Use FluoroFinder’s interactive Spectra Viewer to find the right fluorophore for your microscope’s laser and filter configurations
Fluorophores have been exposed to light Store fluorescent reagents and samples in the dark, including during incubation steps
Samples have dried out Ensure that samples remain fully submerged in liquid throughout the IHC experiment


High Background

Cause Suggested solution
Autofluorescence Replace old formalin fixative solutions with fresh stocks; optimize the deparaffinization step to ensure paraffin is properly removed; use unstained samples to determine sample autofluorescence levels; consider replacing green-emitting fluorophores with red-emitting fluorophores (most autofluorescence occurs in the green range); try introducing an autofluorescence quencher (e.g., Sudan Black B) into your workflow
Insufficient blocking Optimize the blocking step (a 1 hour incubation with normal serum from the same host species as the secondary antibody is usually recommended)
Antibody concentrations are too high Optimize primary and secondary antibody concentrations
Primary antibody lacks specificity

Use appropriate positive and negative controls to confirm antibody specificity; consider switching to a different primary antibody


Sample and primary antibody share the same host species, resulting in secondary antibody binding to the sample  Ensure that the sample and primary antibody are from different host species; consider switching to direct detection with a labeled primary antibody; if species-on-species detection is unavoidable, try blocking endogenous IgGs with unconjugated anti-species Fab fragments
Secondary antibody is cross-reacting with the sample Use a secondary antibody that has been cross-adsorbed against the sample host species; consider switching to direct detection with a labeled primary antibody
Inadequate washing Increase the number and/or length of wash steps
Signal amplification is too high Reduce the amount of signal amplification (e.g., decrease the concentration of the streptavidin-fluorophore conjugate)
Spectral overlap Use FluoroFinder’s Spectra Viewer to check that fluorophores do not overlap into the same spectral range
Samples have dried out Ensure that samples remain fully submerged in liquid throughout the IHC experiment


Supporting Your Research

FluoroFinder has developed a suite of tools that can help to streamline fluorescent IHC. Use our Antibody Search function to find antibodies that are validated for IHC applications and our Spectra Viewer to identify the right fluorophores for your microscope’s laser and filter configurations.