Immunofluorescence Staining (5-plex): Five Marker Detection with Nuclear Context

Overview

5-plex immunofluorescence staining allows for the simultaneous visualization of five distinct protein markers plus DAPI on a single tissue slide. This level of multiplexing is ideal for exploring cellular interactions, immune landscape profiling, and spatial biomarker analysis in limited or precious tissue sections.

At iHisto, our advanced 5-plex workflow combines TSA amplification, expert antibody design, and Leica BOND RX automation to deliver clean, reproducible, and publication-grade results.

How 5-Plex Immunofluorescence Staining Works

Tissue sections are stained with five primary antibodies, followed by sequential or combined detection using fluorophore-labeled secondary antibodies. When antibodies share the same host species, TSA (Tyramide Signal Amplification) is used to ensure clean signal separation.

iHisto Fluorophore Panel:

• FITC

• CYAN

• CY3

• Texas-Red

• CY5

• CY7

• DAPI (nuclear counterstain)

Typical Visualization:

• Marker 1: Green (e.g., FITC)

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

• Marker 3: Far-red (e.g., CY5)

• Marker 4: Near-infrared (e.g., CY7)

• Marker 5: Cyan or alternate unused channel

• Nuclei: Blue (DAPI)

Step-by-Step Staining Process

1. Sectioning & Retrieval
   Tissue sections (FFPE or frozen) are prepared and antigen retrieval is applied.

2. Blocking
   Autofluorescence and nonspecific binding are minimized.

3. Primary Antibody Application
   Five validated antibodies are applied, with attention to host species and compatibility.

4. Sequential TSA Amplification or Direct Detection
   TSA is used to prevent cross-reactivity for same-species primaries; direct fluorescent detection for others.

5. DAPI Counterstain
   Adds clear nuclear contrast for spatial context.

6. Mounting
   Antifade media is used to preserve fluorophore intensity.

7. Imaging
   High-resolution imaging via Leica BOND RX or whole-slide fluorescence scanning.

iHisto’s Optimized Workflow

• ✅ Automated multiplex staining with Leica BOND RX

• ✅ TSA-based signal amplification and separation

• ✅ Full spectrum fluorophore support (CYAN to CY7)

• ✅ Panel design and antibody validation included

• ✅ Digital slide scanning with multichannel export

• ✅ Expert troubleshooting and result interpretation available

Applications of 5-Plex IF Staining

This level of multiplexing enables deep tissue phenotyping and simultaneous marker correlation.

Common use cases:

• Tumor microenvironment profiling – CD3, CD8, PD-1, FOXP3, Cytokeratin

• Macrophage subtyping – CD68, CD163, CD206, HLA-DR

• Neuroinflammation panels – NeuN, GFAP, Iba1, CD68, CD45

• Stem cell and differentiation studies – Nestin, SOX2, OCT4, Olig2

• Signal pathway analysis – pAKT, pERK, STAT3, NF-κB, Ki-67

Advantages of iHisto’s 5-Plex IF Services

• ✅ High-level multiplexing on FFPE or frozen tissue

• ✅ Clean separation of signals with TSA amplification

• ✅ Fluorophore combinations tailored to your microscope setup

• ✅ Digital overlays and channel-separated files provided

• ✅ Pathologist and AI-compatible slide formats

FAQs

• Can 5-plex be done on FFPE tissue?
  Yes. Our protocols are optimized for FFPE and frozen tissue using TSA amplification where needed.

• What if two antibodies are from the same species?
  We use TSA-based sequential detection to isolate and amplify the signals without cross-reactivity.

• Is slide scanning available?
  Absolutely — we offer fluorescent whole-slide scanning with channel merging and image overlays.

• Can I design a custom panel?
  Yes. Our team can help you build and validate a custom 5-marker antibody panel matched to your project.

Ready to Explore Tissue Complexity with 5 Markers?

At iHisto, we offer precision 5-plex immunofluorescence staining using advanced TSA technology and Leica BOND RX automation. Whether you're mapping immune interactions or tracking tumor heterogeneity, we deliver the detail your data demands.

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