TSA-based multiplex immunohistochemistry (mIHC) relies on sequential rounds of antibody staining, signal amplification via horseradish peroxidase (HRP)-catalyzed tyramide deposition, and antibody stripping between cycles. Proper antibody selection is critical to ensure signal specificity, minimize cross-reactivity, and maintain compatibility with TSA workflows. Below are key considerations for antibody selection in TSA-based mIHC:

1. Host Species of the primary antibodies

The TSA dye kit provided by our company contains two types of secondary antibodies: anti rabbit and anti mouse rabbit universal secondary antibodies. Other species of primary antibodies require separate preparation of HRP secondary antibodies

2. Antibody Validation for TSA Workflow

Paraffin section immunohistochemistry/immunofluorescence: Select IHC-P/mIHC;

Frozen section immunohistochemistry/immunofluorescence: Select IHC fr;

Cell crawling immunohistochemistry/immunofluorescence: Select cells containing ICC/IF.

*If the antibody instructions do not state whether IHC is P or Fr, we generally assume that both are suitable (please refer to the instructions for details).

**In principle, the host species of antibodies should not be consistent with the species of customer samples.

**The customer sample is a mouse, so try to avoid using mouse derived primary antibodies as much as possible, because if mouse derived primary antibodies are used, mouse derived secondary antibodies must also be used, which may bind to the IgG of the mouse body and produce non-specific results. If it is necessary to use primary antibodies of the same origin as the sample, it is recommended that the customer create a negative control (add secondary antibodies without primary antibodies to test for non-specific production).

3. Signal Optimization

High-Affinity Antibodies:

Use antibodies with low nonspecific binding to reduce background amplified by TSA.

Pre-titrate antibodies to determine the minimum effective concentration (TSA amplifies signals, so lower antibody dilutions often suffice).

Titration with TSA Dyes:

Optimize primary/secondary antibody concentrations alongside TSA dye dilutions (e.g., Nova 520, 570, 690) to balance signal intensity and spectral overlap.

4. Avoiding Cross-Reactivity

Sequential Staining Order:

Stain low-abundance targets first, as TSA generates strong signals that may mask weaker subsequent signals.

Example: Prioritize cytokines (low expression) before structural markers (high expression).

Cross-Adsorption Validation:

Test antibody pairs in singleplex and multiplex modes to rule out inter-target interference.

Use isotype controls (e.g., nonimmune IgG) to identify nonspecific TSA deposition.

5.Verified antibodies:

Priority should be given to antibodies that have been validated in IHC or mIHC, especially those that perform well in FFPE (formalin fixed paraffin embedded) samples.

TSA is commonly used to enhance the signal of low abundance antigens, therefore the specificity of the antibody itself is crucial.

6. Vendor-Specific TSA Kits

Kit Recommendations:

Use TSA kits compatible with your workflow

Follow kit protocols for optimal HRP activation and tyramide deposition.

Pre-Validated Panels:

7. Critical Controls

No-Primary Controls: Omit primary antibodies to confirm TSA signal specificity.

Singleplex-to-Multiplex Comparison: Compare multiplex results to singleplex stains to validate signal fidelity.

Epitope Stability Test: Verify retained antigenicity after multiple stripping cycles.

Key Tips

Batch Consistency: Use the same antibody batch across experiments to reduce variability.

Documentation: Record TSA dye lots, antibody dilutions, and stripping conditions for reproducibility.