Immunofluorescence Staining (4-plex): High-Resolution Detection of Four Targets with Nuclear Counterstain

Overview

4-plex immunofluorescence staining allows simultaneous detection of four protein targets plus DAPI in a single FFPE or frozen tissue section. This approach is ideal for analyzing cellular crosstalk, tumor microenvironments, immune profiling, and spatial biology where single-marker stains aren’t enough.

At iHisto, our 4-plex staining protocols integrate validated antibody strategies, TSA amplification, and Leica BOND RX automation to ensure precise, reproducible multiplex imaging with minimal cross-reactivity.

How 4-Plex Immunofluorescence Staining Works

Four primary antibodies are applied (either sequentially or strategically grouped), followed by corresponding fluorescent secondary antibodies or TSA-amplified detection for markers from the same species. Nuclear counterstaining with DAPI provides cellular context.

Note: When two or more antibodies are from the same host species, we use a TSA (Tyramide Signal Amplification) strategy to isolate signals without cross-reactivity.

iHisto Fluorophores:

• FITC

• CYAN

• CY3

• Texas-Red

• CY5

• CY7

• DAPI (nuclear counterstain)

Typical Visualization:

• Marker 1: Green (e.g., FITC or CY3)

• Marker 2: Red (e.g., Texas-Red)

• Marker 3: Far-red (e.g., CY5)

• Marker 4: Infrared (e.g., CY7)

• Nuclei: Blue (DAPI)

Step-by-Step Staining Process

1. Sectioning & Antigen Retrieval
   FFPE or frozen sections prepared with optimized protocols for multiplex compatibility.

2. Blocking & Quenching
   Reduces autofluorescence and eliminates background signal.

3. Sequential or Combined Primary Incubation
   Antibodies are applied based on species or isotype compatibility.

4. TSA Amplification or Direct Fluorophore Detection
   TSA enables clean signal amplification for same-species antibodies.

5. Nuclear Counterstaining
   DAPI is applied to stain nuclei.

6. Mounting
   Slides are sealed with antifade media.

7. Imaging
   Performed using Leica BOND RX or high-resolution whole-slide fluorescent scanners.

Our Optimized Workflow

• ✅ Fully automated protocols via Leica BOND RX platform

• ✅ TSA-based sequential detection when needed

• ✅ Panel validation to avoid bleed-through or spectral overlap

• ✅ Custom antibody pairing consultation

• ✅ Whole-slide scanning with multichannel fluorescence output

• ✅ Human, rodent, and xenograft samples accepted

Applications of 4-Plex IF Staining

This method is ideal for studying multiple markers within limited tissue or understanding complex microenvironments.

ommon use cases:

• Immune cell mapping – CD3, CD8, FOXP3, PD-L1

• Neuroinflammation panels – NeuN, GFAP, Iba1, CD68

• Tumor–immune interaction studies – CK, Ki-67, CD45, PD-1

• Stem cell differentiation – OCT4, SOX2, Nestin, GFAP

Advantages of iHisto’s 4-Plex IF Staining

• ✅ Accurate four-marker resolution + nuclear context

• ✅ Reduced background via TSA and signal separation

• ✅ High-throughput automation via Leica BOND RX

• ✅ Expert pathologist review and AI-ready image capture

• ✅ Optional digital quantification and cloud delivery

FAQs

• What is 4-plex IF staining used for?
  It’s used to detect four proteins + DAPI on a single slide, revealing spatial relationships and co-expression in tissue.

• What if I have antibodies from the same host species?
  We apply TSA amplification to distinguish targets cleanly.

• Can I scan and share the slides digitally?
  Yes — we offer high-resolution whole-slide scanning with multichannel overlay support.

• What sample types do you support?
  We process FFPE and frozen sections from human, mouse, rat, NHP, and xenograft models.

Ready to Visualize Complex Biology with 4 Targets?

At iHisto, we deliver high-quality 4-plex immunofluorescence staining backed by TSA amplification, expert antibody pairing, and Leica BOND RX automation. Whether you’re analyzing immune panels or tumor architecture, we help you see it all on one slide.

📩 Contact us to request a quote or consultation.