Immunofluorescence Staining (3-plex): Three Marker Detection with Nuclear Counterstain

Overview

3-plex immunofluorescence staining allows simultaneous visualization of three protein targets and nuclear counterstaining (DAPI) within a single tissue section. This technique enables spatial mapping of complex tissue biology, making it ideal for immune profiling, tumor microenvironment studies, and co-expression analysis.

At iHisto, our 3-plex immunofluorescence staining combines expert antibody pairing, TSA amplification, and Leica BOND RX automation to deliver clear, reproducible results with minimal variability.

How 3-Plex Immunofluorescence Staining Works

Tissue sections are incubated with three 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 or more 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 Fluorophores:

• FITC

• CYAN

• CY3

• Texas-Red

• CY5

• CY7

• DAPI (nuclear counterstain)

Typical Visualization:

• Marker 1: Green (e.g., FITC or CYAN)

• Marker 2: Red (e.g., CY3 or Texas-Red)

• Marker 3: Far-red (e.g., CY5 or CY7)

• Nuclei: Blue (DAPI)

Step-by-Step Staining Process

1. Sectioning and Antigen Retrieval
   Tissue sections (FFPE or frozen) are prepared with optimized retrieval protocols.

2. Blocking
   Prevents non-specific binding and background autofluorescence.

3. Primary Antibody Incubation
   Three validated primary antibodies are applied sequentially or in combination, depending on species.

4. TSA or Fluorescent Secondary Detection
   TSA amplification is used when same-species antibodies are involved. Otherwise, traditional fluorescent secondaries are applied.

5. Nuclear Counterstaining
   DAPI is used for nuclear visualization.

6. Mounting
   Slides are mounted with antifade media and coverslipped.

7. Imaging
   Slides are scanned via multi-channel fluorescence or viewed under a confocal/widefield microscope.

iHisto’s Optimized 3-Plex Workflow

• ✅ 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 3-Plex IF Staining

3-plex IF staining is ideal for visualizing complex cellular interactions, tissue heterogeneity, and spatial biomarker localization.

Common use cases:

• CD8 + PD-L1 + Cytokeratin (immune infiltration + tumor ID)

• CD68 + CD163 + Ki-67 (macrophage phenotyping and proliferation)

• NeuN + Iba1 + GFAP (neural cell population mapping)

• FOXP3 + CD3 + DAPI (regulatory T cell profiling)

• Stem cell paneling (e.g., SOX2 + Nestin + Olig2)

Advantages of iHisto’s 3-Plex IF Services

• ✅ True triple-marker visualization with nuclear context

• ✅ TSA-based sequential detection for same-species antibodies

• ✅ Custom fluorophore selection and optimization

• ✅ Human, rodent, and xenograft samples supported

• ✅ Optional whole-slide scanning and image overlays

• ✅ Quantification and pathologist annotation available

FAQs

• What is 3-plex IF staining?
  It detects three distinct protein markers plus DAPI on the same tissue section using fluorescent antibodies.

• What if two antibodies are from the same species?
  We use a TSA amplification method to enable sequential staining without cross-reactivity.

• Do you offer slide scanning and analysis?
  Yes. We provide multichannel fluorescent slide scanning and optional co-localization analysis.

• What tissues are accepted?
  We support FFPE and frozen samples from human, mouse, rat, NHP, and xenograft models.

Ready to Visualize Complex Biology in One Slide?

At iHisto, we deliver precision 3-plex immunofluorescence staining to help you explore co-expression, cellular interactions, and biomarker localization — with expert protocols, TSA-based flexibility, and digital imaging support.

📩 Contact us to request a quote or schedule a consultation.