Strategy to Run a Full Multiplex SeqIF

Using these three protocols sequentially helps ensure that your antibodies are not only compatible with the COMET platform but also optimized for multiplexing performance, minimal background, and reproducibility.

The Screening protocol is your first checkpoint. It helps determine whether a given antibody:

• Binds properly
• Produces a specific signal
• Is compatible with COMET

This is especially critical when using third-party or in-house antibodies.

• Use a single antibody (or a simple 2-antibody mix) in one staining cycle.
• Apply the antibody with standard conditions:
• e.g. 1:100 dilution, 8 min incubation
• Detection:
• Use Alexa Fluor™ Plus secondaries
• Optionally include DAPI
• No elution → one-pass test
• Image and assess performance

Verifies antibody performance on your specific tissue (FFPE or frozen)

Identifies:

• ❌ No signal: poor Ab or missing antigen
• ⚠️ Diffuse background: non-specific staining
• 🧭 Mislocalization: off-target effects

Filters out unsuitable antibodies before running full panels

To test epitope stability across cycles — essential when using markers prone to degradation during multiple staining/elution rounds (e.g. transcription factors, phospho-epitopes).

The same antibody is stained and imaged in multiple consecutive cycles (e.g., 5–6).

Each cycle includes:

• Staining
• Imaging
• Elution

After the run, compare signal intensity and morphology across cycles.

Helps determine optimal cycle placement:

• Fragile markers → early cycles
• Stable markers → later cycles
• Ensures consistency and avoids dropouts
• Prevents underestimation of rare targets

If FOXP3 shows strong signal in Cycle 1, but weak or no signal by Cycle 4, it must be placed in Cycle 1–2 of your panel.

To fine-tune:

• Antibody dilution
• Incubation time

This maximizes signal-to-background and ensures robust, reproducible staining.

Test a single antibody (or mix) at:

• Varying dilutions (e.g. 1:50, 1:100, 1:200)
• Varying incubation times (e.g. 4, 8, 12 min)
• Each test goes into a separate staining cycle
• Compare images across conditions for:
• Signal intensity
• Uniformity
• Background noise

• Prevents over-/under-staining
• Matches antibody strength to optimal fluorescence channel
• Essential for high-plex designs where channel crosstalk and background buildup must be avoided
• Promotes panel consistency across users and tissues

• Start with vendor-recommended dilution
• Shorter incubation times save time if signal is strong
• Use blocking buffer if background is high

If your objective is to move straight into a full multiplex SeqIF run without performing preliminary antibody optimization, it is possible — but only with careful experimental design, validated reagents, and an understanding of the limitations of spectral, tissue, and fluidic constraints. The COMET™ system allows this flexibility, but success hinges on informed decisions across several critical parameters.

Choose antibodies from:

• SPYRE™ panels 🧪
• Lunaphore COMET Marker Table 📄
• Well-cited IF/IHC studies with stock concentrations 📚
• Only use unconjugated primary antibodies.
• Avoid all direct-conjugates unless explicitly validated on COMET.

• Cycle 1–2: Sensitive/low-abundance targets (e.g., FOXP3, phospho-proteins)
• Cycle 3–6: Stable immune markers (e.g., CD3, CD8, CD20, CD68)
• Later cycles: Structural/stromal markers (e.g., αSMA, CK)

Tip: Duplicate a core marker (e.g., CD3) in early and late cycles to monitor epitope degradation 🔁

Use:

• Alexa Fluor™ Plus 555 for TRITC channel
• Alexa Fluor™ Plus 647 for Cy5 channel

Benefits:

• High quantum yield and low background
• Superior photostability under COMET’s LED excitation

Flow cytometry fluorophores (such as PE, PerCP, APC, FITC, and their tandem conjugates like PE-Cy7, APC-H7, etc.) are engineered for cytometric systems, not for imaging. Their use in COMET or any fluorescence microscopy workflow is highly discouraged for several scientific and practical reasons:

• Spectral incompatibility with microscope filters
  • Many flow dyes emit over broad, overlapping ranges or require excitation/emission filters not available in microscopes.
• Large conjugates → poor tissue penetration
• Photobleaching and unstable emission
  • Flow dyes (especially tandem conjugates) are extremely sensitive to light.
  • In flow cytometry, excitation is brief (~microseconds); in microscopy, exposure is sustained and repeated during acquisition.
  • Result: Rapid photobleaching and total signal loss during imaging.
• High non-specific background

COMET uses a gentle elution buffer to strip off antibodies between cycles. This is validated with indirect staining using unconjugated primaries and Alexa Fluor™ Plus secondaries. Flow-conjugated antibodies are not validated for COMET:

• No guarantee of elution efficiency
• Risk of residual signal between cycles (ghost staining)
• May compromise the entire run

In lung, liver, or pigmented tissues:

• Avoid FITC/TRITC channels

Assign low-abundance or critical markers to:

• Cy5 (647 nm)
• Cy7 (750 nm)

Reference: Zrazhevskiy et al., Nat Biomed Eng (2021)

The COMET chip supports 12.5 × 12.5 mm imaging area

Load:

• Different tissue types (tonsil, tumor, normal) on the same chip
• Replicate sections to test staining consistency

Benefits:

• Maximizes data per run 💰
• Reduces per-tissue cost 📉