Guide to Multiplex IHC with Aluora Spatial Amplification Assays

An Aluora dye is a fluorescent reagent that can be deposited onto a tissue sample through an enzyme-mediated reaction. The fluorescent Aluora dyes are resistant to downstream processing of antibody stripping, due to their covalent linkage to the samples, and allow subsequent detection of additional targets without affecting the signal strength or quality of existing labels. 

Iterative labeling of individual targets unlocks use of multiple primary antibodies from the same host species which is a major limitation in traditional IHC workflows. In addition, the Aluora workflow can be combined with directly conjugated primary antibodies that are introduced in a final step following the completion of the Aluora dye labeling.

Before beginning the Aluora spatial amplification assay, prepare the reagents and controls.  

1. Pair Aluora dyes to markers of interest
To begin designing a multiplex panel, start by pairing primary antibodies against the markers of interest to the Aluora dyes. When selecting dyes and antibody pairings, please consider:

• Co-expression: be sure to choose fluorophores with minimal spectral overlap when analyzing co-expression of markers
• Abundance: brighter fluorophores should be paired with lower expressing markers and dimmer fluorophores should be paired with more abundant markers. See Table 3 below for relative brightness ratings. 
  
  

2. Determine staining order
The staining sequence for the experiment should be based on the biology of the individual targets, extent of antigen retrieval required and co-localization of targets. Please consider the following:

• Epitopes: Some rare epitopes may be degraded by multiple exposures to heat and be placed earlier on in the staining order. To test the heat-sensitivity of epitopes, perform single-color controls with each primary antibody using the same number of HIER steps as in the multiplex. This will help determine if adjustments are needed to the staining order.
• HIER: multiple rounds of HIER can result in some epitopes being more exposed. This can lead to more signal when detected later in the protocol.
• Co-localization: assign the staining order so that consecutive antibody-fluor combinations will not co-localize in the same structure. 

3. Optimize primary antibodies

It can be challenging to optimize primary antibodies in terms of both dilution factor and antigen retrieval. Typically, once users have successfully completed their first multiplex panel development, other panels are easier to optimize. 

3.1. Determine primary antibody dilutions 

For each primary antibody, optimize and stain positive control slides, as you normally would during your IHC protocol development. The optimized primary antibody dilution should yield:

• The appropriate staining pattern
• Ideal specificity
• Appropriate intensity
• Low background 

We recommend referring to the dilutions suggested by the primary antibody vendor or as defined through previous IHC validation. For Aluora spatial amplification we recommend diluting the primary antibody at least 5-10–fold more than used for standard IHC. Many primary antibodies can be diluted in the region of 1:1,000 and sometimes even lower concentrations for robust targets. It is recommended to start with a dilution of 1:1,000 and select 2-3 additional dilutions to determine the optimal working concentration. See Table 4 for a working example.

3.2. Ensure antigen survival after multiple rounds of heat-induced epitope retrieval (HIER)

In this assay, HIER is used before labeling with each primary antibody to quench endogenous peroxidase activity, retrieve antigens, and to remove bound antibodies from a target. The Aluora fluorophores are covalently attached to the tissue so consecutive rounds of HIER will not remove the fluorescent label. However, there may be some bleaching of the fluorescent signal. If this becomes significant in some channels, the Aluora dye order may need to be optimized, placing the dimmer channel later in the multiplex order, thus reducing the number of stripping cycles it will undergo.

It is critical to ensure that the antigen can survive the rounds of HIER prior to the application of the corresponding primary antibody to the tissue. For antigens that may be lost with repeated HIER, these should be probed earlier on in the staining sequence.

To control for this, we advise that you perform control slides with the corresponding number of rounds of HIER prior to staining with the primary antibody.

4. Establish controls

Experimental controls are critical in verifying assay results. For the Aluora Spatial Amplification Assay, an unstained control is required. This control should be treated identically to the multiplexed sample without the addition of fluorescent reagents. In addition to the unstained control, each Aluora dye used should have a single-color control. 

4.1 Develop single-color control (SCC) slides

Single-color control slides are critical in defining the proper staining parameters for each of the individual antibody and Aluora dye pairings to enable spectral unmixing. These single-color controls should include one Aluora dye per sample, but otherwise treated identically to the multiplexed sample including the same staining order, same stripping, etc. This helps ensure that the SCC have the most representative spectral match to the multiplexed sample. Matching these spectral patterns is key to spectral unmixing and getting the best data from your samples.

It is also critical that all SCC samples are from the same lot of tissue. Tissue handling can significantly alter not only the autofluorescence profile of the tissue slices also relative antigen integrity and accessibility, which in turn, greatly effects the spectral unmixing.

After completing the necessary preparations, proceed to the assay protocol. The Aluora spatial amplification assay steps can be performed manually or with an automated slide stainer.

The fundamental principles of Aluora spatial amplification staining and developing a multiplex assay remain unchanged when performed on an automated platform. It is essential to perform the same optimization for multiplex development before beginning the assay. In addition, optimization of samples with the automation platform should be performed. Differences in experimental handling, such as HIER/stripping protocols, can significantly change the staining. Other adjustments may also be required depending on the automated staining platform. Refer to the user guides for your specific platform.

Find more details on performing automated staining in the Automated Multiplex IHC Staining with Aluora Spatial Amplification Reagents and Leica BOND protocol.

Aluora Spatial Amplification Kits are compatible with spectral imagers such as the EVOS S1000 Spatial Imaging System. To acquire images, follow these basic steps:

1. Setup the experiment protocol on the EVOS S1000 by selecting the fluorescent reagents you used in your samples. 
2. Set the initial channel exposures with your multiplex sample. 
3. Using the unmixing setup wizard, input your single-color control samples in order to setup the unmixing matrix. When the setup is complete, check the unmixing report to assess the quality of your unmixing matrix. 
4. Review the unmixing and ensure the results are satisfactory. If so, continue to image your multiplex sample.