Immunofluorescence Staining (2-plex): Dual Marker Detection with Nuclear Counterstain

Overview

2-plex immunofluorescence staining enables the simultaneous visualization of two protein targets in tissue sections, along with DAPI nuclear counterstaining. This technique is ideal for detecting co-localization, cell-type interactions, and tissue organization in research and preclinical studies.

At iHisto, our 2-plex immunofluorescence staining combines expert antibody pairing, TSA amplification, and Leica BOND RX automation to deliver clear, reproducible results with minimal variability.

How 2-Plex Immunofluorescence Staining Works

Tissue sections are incubated with two primary antibodies, followed by fluorophore-conjugated secondary antibodies selected for minimal spectral overlap. A nuclear counterstain (DAPI) is added to visualize cell structure.

Note: When two antibodies are from the same host species, iHisto uses a TSA-based (Tyramide Signal Amplification) method to prevent cross-reactivity and allow sequential detection with strong, non-overlapping signals.

iHisto Fluorophore Options:

• FITC

• CYAN

• CY3

• Texas-Red

• CY5

• CY7

• DAPI (standard nuclear counterstain)

Typical Visualization:

• Target 1: Green (e.g., FITC or CY3)

• Target 2: Red or far-red (e.g., Texas-Red or CY5)

• Nuclei: Blue (DAPI)

Step-by-Step Staining Process

1. Sectioning and Retrieval
   Tissue is sectioned (FFPE or frozen), with appropriate antigen retrieval applied.

2. Blocking
   Non-specific sites are blocked to minimize background and autofluorescence.

3. Primary Antibody Incubation
   Two validated primary antibodies are applied.

4. Secondary Antibody Detection
   Fluorophore-labeled secondary antibodies are added for each target.

5. Nuclear Counterstain
   DAPI is applied to label nuclei.

6. Mounting
   Slides are mounted in antifade media and sealed.

7. Imaging
   Captured via multichannel fluorescence or whole-slide scanning.

Optimized Workflow at iHisto

We ensure clean, well-separated 2-plex staining with:

• ✅ Automated staining available using Leica BOND RX platform

• ✅ TSA amplification for same-species antibody detection

• ✅ Balanced exposure and spectral separation using validated fluorophores

• ✅ Compatible with FFPE and frozen tissue

• ✅ Whole-slide scanning with multichannel output

• ✅ Custom antibody panel design and validation included

Applications of 2-Plex IF Staining

This method is ideal when evaluating two biomarkers in relation to tissue architecture, particularly when co-expression or interaction is relevant.

Common use cases:

• CD8 + PD-L1 (tumor microenvironment)

• GFAP + Iba1 (CNS glia interaction)

• CD3 + FOXP3 (T cell regulation)

• Ki-67 + cytokeratin (proliferation in tumor epithelium)

• CD68 + CD163 (macrophage phenotyping)

Advantages of iHisto’s 2-Plex IF Staining

• ✅ Clear, dual target detection with minimal overlap

• ✅ High specificity using validated antibody combinations

• ✅ Full support for FFPE and frozen tissue

• ✅ DAPI counterstaining included as standard

• ✅ Digital imaging and quantification available

We offer expert support from stain planning to data capture — helping you generate strong visual data from limited or archival tissue.

FAQs

• What is 2-plex IF staining?
  It’s a dual-target immunofluorescence method that detects two protein markers plus DAPI, ideal for co-expression analysis or comparative marker evaluation.

• Can I choose my fluorophores?
  Yes. You can select from our supported fluorophores (e.g., FITC, CY3, Texas-Red, CY5), or request help choosing based on your targets and microscope setup.

• What kind of tissues do you accept?
  We work with FFPE and frozen tissues from human, mouse, rat, and xenograft models.

• Do you offer scanned image files?
  Yes. High-resolution whole-slide imaging is available for fluorescence visualization and analysis.

Ready to Run Dual Marker Detection with Precision?

At iHisto, we provide 2-plex immunofluorescence staining with DAPI nuclear labeling and optimized dual-target detection. Whether you're mapping immune markers or characterizing cell types, we help you visualize interactions clearly — with results ready for review or publication.

📩 Contact us to request a quote or consultation.