Mulitplex Immunohistochemistry (IHC): Guidance, Tips, and Reagents

Introduction

Tissues and organs consist of different cell types, which work together to perform physiological processes. Therefore, it is essential to study these cells in-situ to understand the complex biological functions. Such methods for studying tissues or organs at a single cell resolution include transcriptomics, immunohistochemistry (IHC), immunofluorescence (IF), mass spectometry, and cytometry. For this article, we will focus on IHC.

Multiplexing IHC/IF

Multiplex IHC is a powerful technique whereby scientists can study multiple proteins, using antibodies, on the same tissue/organ slide. Through accessing a large range of reagents, scientists are now able to simultaneously analyze up to 100 proteins. Depending upon which set of reagents you select determines how many proteins you can analyze.

Indirect detection - multiplexing for <5 proteins:

Indirect detection is beneficial when you wish to analyze a low number of proteins (<5). In summary, indirect detection involves staining with the unconjugated primary antibodies followed by the addition of fluorescently labeled secondaries. Due to the amplification affect generated through using a secondary antibody, indirect detection is perfect for analyzing proteins which are expressed at low levels.

For further information, see The Fundamentals of Immunofluorescence - A Brief Guide

Generally, scientists are restricted to staining 5 proteins when using indirect detection due to cross species reactivity of the fluorescently labeled secondaries, plus the majority of primary antibodies are raised in either rabbits or mice, thus limiting choice. When staining 6+ proteins, preference is to adopt the direct detection method.

Direct detection - multiplexing for 6+ to 100 proteins:

When analyzing 6+ proteins, the preferred approach is to directly label the antibody with a fluorescent dye/protein (other labeling options may include metal tag, fluorophore DNA oligos and enzymes). Of the various antibody based imaging methods, fluorescence multiplexing is the most popular method and tends to be limited to visualizing 4-7 proteins within a single image cycle, due to spectral overlap of selected fluorophores. This limitation has been overcome through hyperspectral analysis (capturing and processing images at a large number of wavelengths) using 21 different channels and incorporating fluorophores with diverse excitation and emission spectra.

Even higher multiplex analysis can be achieved through a cyclic or multi-step process involving:

1. Staining the tissue slide with fluorescently labeled antibodies.
2. Capturing images using a microscope.
3. Fluorophore inactivation or removal of antibodies.
4. Repeat steps 1-3 until the desired number of proteins have been analyzed.

The table below details the various multiplexing techniques for fluorescently labeled antibodies:

Although these multiplex IHC methods are powerful, success still depends upon selecting the correct set of reagents, and performing the necessary controls to ensure your experimental results are accurate and reproducible.

Hints and Tips for reagent selection

Tissue selection - a user has the option of selecting either fresh frozen or formalin-fixed paraffin embedded (FFPE) tissues with each method having advantages and disadvantages:

Antibodies are obviously critical tools for multiplexing, and therefore reagent selection and validation are key considerations. Several factors should be considered:

• Confirming antibody specificity through single-plex IHC using positive and negative controls.
  • To demonstrate the antibody is protein specific, it is important to use positive and negative controls (cells or tissues) that express the target protein of interest. This is where a thorough review of the scientific literature is essential to identify tissue or cell types which fit your criteria. For tissues, in general, a range of sample types (often stained using a tissue micro-array approach) is used for antibody validation including clinical specimens with known genetic alteration that impact protein expression.
  • To enhance confidence in your selected reagents, one approach is to use knock-out cell lines or tissues where the target gene has been removed, thus eliminating expression of the protein.
  • One point to note - when analyzing any tissue or cell line for antibody testing, staining should be evaluated for subcellular localization/tissue specific staining.
    
    
• Signal to noise.
  • The importance of this factor should not be underestimated. If a protein is expressed at low levels, and staining with an antibody delivers a low signal to noise ratio, it will be difficult to analyze the results. Therefore, a rigorous design approach will help by implementing the following:
    • The antibody should be labeled with the brightest fluorescent protein or dye to increase the signal to noise ratio.
    • During the staining procedure, targets with low expression/low signal-to-noise ratios should be prioritized first, to prevent the immunogen from being compromised, especially through subsequent antibody removal steps when using the cyclic staining approach.
      
      
• Tissue and subcellular location.
  • Deciding on whether to use specific antibodies at the same time or spread across cycles really depends on the scientific questions you are attempting to answer. For instance, in multiplexing, protein-protein interaction is often critical, therefore under these types of circumstances it does make sense to use antibodies against the proteins within the same staining cycle.
  • On the other hand, having a three-laser microscope obviously limits the number of proteins analyzed per staining cycle. Therefore, if you intend to perform several rounds of cyclic staining, it is important to separate the antibodies which stain either the same tissue or subcellular location. For instance, avoid a cycle whereby all antibodies stain the nucleus.
    
    
• Antibody compatibility in a multi-plex panel.
  
  • Once the individual antibodies have been shown to be target specific, it is important to validate the full panel. For instance, when antigen retrieval is required for any of the antibodies, the same method of retrieval must work for all antibodies. If antibody staining in a single-plex vs multi-plex experiment is observed, further adjustments will be needed.
    
    
• Autofluorescence - native tissue and other types.
  • Autofluorescence varies widely from tissue types, disease states, sample preparation, and fixation methods. If a tissue does exhibit autofluorescence, it is important to attempt to quench the signal.
  • The table below outlines the most common reasons for autofluorescence with potential solutions to overcome the issue:

Key Reduction in autofluorescence: No (-), Minimal (+), Moderate (++), Significant (+++)

1. 1. 1. Avoid fixatives with aldehydes or use a fixative with a detergent.
      2. Quenching with sodium borohydride or pre-treatment with H₂0₂.
      3. Light irradiation.
      4. Capture image using microscopes with defined excitation sources, narrow filters and/or detector ranges.
      5. Select fluorescent dyes with high quantam yields.
      6. Amplify signal in autofluore channels with secondary antibodies.
      7. Incorporate computational methods to remove autofluorescence.

Modified table from - Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging.

Antibodies Inc Research Tools to Support Multiplex IHC

Designing a panel of fluorescent conjugates for multiplex IHC can be a complex and challenging process, often presenting numerous obstacles. To address these challenges, Antibodies Inc. offers a comprehensive range of reagents designed to enhance the likelihood of achieving successful experimental outcomes.

NeuroMab portfolio:

The NeuroMab collection of mouse monoclonal antibodies have been highly validated in brain (ICC, IHC, WB, and IP), and several have been shown to be protein specific through knock-out validation. Furthermore, NeuroMabs have been cited in >6,200 publications.

By offering our NeuroMab antibodies directly conjugated to ultra-bright fluorophores, scientists can now use these rigorously validated antibodies to design multicolor staining panels without limitations.

Conjugated Primary Antibodies:

Perfect for IHC and ICC, our conjugated primary antibodies help to reduce background and save you time. Available in 4 different wavelengths - 490 nm, 550 nm, 59 nm, and 650 nm - they work great in multiplexing applications, and are available in both single vial and bulk formats.

NeuroMab Carrier-free formulations (COMING SOON):

The range is also available in a carrier free format (conjugation ready), delivering the following benefits:

• Ideal for antibody labeling (conjugation) as the antibody PBS only formulation is free of BSA, sodium azide, and tris.
• The carrier free formulation is compatible with a variety of different labels such as fluorescent dyes/proteins, enzymes, biotin/streptavidin, oligonucleotides, and heavy metals
• Provided at the optimal concentration (1mg/ml) for conjugation.
• 400+ NeuroMabs available.
• Products ready to ship within 3 working days, NOT weeks.

Antibody Labeling Kits (COMING SOON):

We now offer a range of easy-to-use antibody labeling kits (aka antibody conjugation kits). Each of our antibody labeling kits are designed to label 100µg of antibody requiring only 30 seconds hands-on time, without having to be a technical expert in conjugation. Additional benefits include:

• Simple: To label an antibody involves only 3 simple steps. The labeled antibody is ready to use in less than 11 minutes.
• Convenient: Protocol is optimized for labeling 50-100µg of purified antibody.
• Choice of Flexibility: Range consists of fluorescent dyes, R-phycoerythrin (R-PE), biotin, and HRP.
• Comprehensive Kits: All components are included in each kit, except the purified antibody, microcentrifuge, and pipettes.

References & Resources

A framework for mutiplex imaging optimization and reproducible analysis

Spatial mapping of protein composition and tissue organization: a primer for multiplexed antibody-based imaging

High-plex immunofluorescence imaging and traditional histology of the same tissue section for discovering image-based biomarkers

The Fundamentals of Immunofluorescence Staining - A Brief Guide

The Power of Polyclonal Chicken Antibodies in Multiplex Immunofluorescence

Immunocytochemistry and Immunohistochemistry using Fluorscein-labeled Secondary Antibodies