4-plex Immunofluorescence Staining: High-Resolution Spatial Detection of Four Targets

Overview

4-plex immunofluorescence staining allows for the simultaneous detection of four protein targets plus a nuclear counterstain (DAPI) within a single FFPE or frozen tissue section. This sophisticated multiplex IF service is ideal for analyzing complex cellular crosstalk, tumor microenvironments (TME), and immune profiling where single-marker stains are insufficient.

At iHisto, our 4-plex immunofluorescence service integrates validated antibody strategies, TSA (Tyramide Signal Amplification), and Leica BOND RX automation. We ensure precise, reproducible multiplex imaging with minimal cross-reactivity, providing the spatial context necessary for high-end discovery research.

How 4-Plex Multiplex IF Staining Works

Our IF stain service utilizes a strategic application of four primary antibodies, either sequentially or in groups, followed by TSA-amplified detection.

Technical Advantage: When panel markers require antibodies from the same host species, iHisto employs a TSA strategy to isolate signals. This advanced multiplex IF stain service ensures clean signal amplification without cross-reactivity.

High-Plex 5-Channel Visualization (DAPI + 4 Targets):

• 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)

Our Optimized 4-Plex Workflow

iHisto provides a robust laboratory framework for your immunofluorescence service needs:

• Fully Automated Protocols: Standardized runs via the Leica BOND RX platform for superior consistency.

• TSA-Based Precision: Sequential detection capabilities for complex antibody pairings.

• Spectral Integrity: Rigorous panel validation to eliminate bleed-through and spectral overlap.

• Digital Pathology: Multichannel whole-slide scanning with high-resolution fluorescent output.

• Broad Compatibility: Expertise in processing human, rodent (mouse/rat), NHP, and xenograft models.

Applications of 4-Plex Multiplex IF Services

This IF stain service is the gold standard for studying complex microenvironments in limited tissue samples.

• Immune Cell Mapping: CD3, CD8, FOXP3, and PD-L1 panels.

• Neuroinflammation Panels: Simultaneous detection of NeuN, GFAP, Iba1, and CD68.

• Tumor–Immune Interaction: Analyzing CK, Ki-67, CD45, and PD-1 spatial relationships.

• Stem Cell Differentiation: Comprehensive profiling with OCT4, SOX2, Nestin, and GFAP.

Why Choose iHisto for 4-Plex IF Staining?

• Superior Resolution: Clear four-marker separation with distinct nuclear context.

• Reduced Background: Advanced blocking and TSA technology for high signal-to-noise ratios.

• AI-Ready Imaging: Expert pathologist review combined with high-resolution digital capture.

• Flexible Delivery: Optional digital quantification and secure cloud delivery of your multiplex IF stain service data.

Frequently Asked Questions (FAQs)

Q: Why choose 4-plex over lower-plex immunofluorescence services? 

A: A 4-plex IF service allows you to characterize complex cell phenotypes (e.g., proliferating regulatory T-cells) and their proximity to other structures in a way that 2-plex or 3-plex cannot.

Q: Can your immunofluorescence service handle antibodies from the same host? 

A: Yes. We use TSA amplification and sequential stripping to distinguish targets cleanly, even when all primary antibodies are from the same species (e.g., all Rabbit).

Q: Is digital quantification available for 4-plex staining? 

A: Yes. We offer optional AI-driven quantification to measure marker intensity and spatial distance between cells across the entire slide.

Ready to Visualize Complex Biology with 4 Targets?

At iHisto, we deliver high-quality 4-plex immunofluorescence staining backed by TSA amplification and Leica BOND RX automation. Whether you’re analyzing immune panels or tumor architecture, our multiplex IF service helps you see the complete biological picture on one slide.

📩 Contact us today to request a quote or consultation for your 4-plex IF stain service.