Immunofluorescence Staining (6-plex): Six Marker Multiplexing with Nuclear Counterstain

Overview

6-plex immunofluorescence staining allows visualization of six protein targets plus DAPI in a single tissue section, offering exceptional power for spatial biology, immune profiling, and high-content biomarker studies.

At iHisto, we deliver reliable 6-plex IF staining using TSA (Tyramide Signal Amplification), expert panel design, and Leica BOND RX automation — enabling precise, high-throughput detection with minimal signal overlap.

How 6-Plex Immunofluorescence Staining Works

Tissue is stained with six primary antibodies. Where antibodies are from the same species, TSA amplification is used to isolate each target’s signal. DAPI is included for nuclear contrast, allowing accurate interpretation of tissue architecture.

Fluorophore Options at iHisto:

• FITC

• CYAN

• CY3

• Texas-Red

• CY5

• CY7

• DAPI (nuclei)

Note: When multiple antibodies share a host species, TSA is used for sequential amplification without cross-reactivity, enabling confident marker distinction.

Step-by-Step Staining Process

1. Tissue Sectioning & Retrieval
   FFPE or frozen sections are processed and antigens exposed with optimized conditions.

2. Blocking & Autofluorescence Control
   Ensures clean, high-contrast signals.

3. Sequential Antibody Application
   Six primary antibodies are applied sequentially or in species-compatible groups.

4. TSA Amplification & Fluorophore Detection
   TSA enables strong signal with clear channel separation, especially for same-species primaries.

5. DAPI Nuclear Counterstain
   Provides clear nuclear landmarks for context.

6. Antifade Mounting
   Slides are sealed with long-lasting media.

7. Multichannel Imaging
   Scanning and imaging conducted using Leica BOND RX or fluorescence slide scanners.

Our Optimized Workflow

• ✅ Leica BOND RX automation for precision multiplexing

• ✅ TSA amplification ensures clean separation of overlapping primaries

• ✅ Supported across human, mouse, rat, NHP, and xenograft tissues

• ✅ Digital whole-slide scanning with channel overlays available

• ✅ Expert panel design and fluorophore matching included

Applications of 6-Plex IF Staining

Ideal for projects requiring deep spatial profiling and co-expression analysis of complex cell populations.

Common use cases:

• Tumor microenvironment deconvolution – CD3, CD8, FOXP3, PD-1, PD-L1, Cytokeratin

• Neuroimmune interfaces – GFAP, Iba1, NeuN, CD68, CD45, Olig2

• Stem cell differentiation tracking – SOX2, Nestin, OCT4, GFAP, Olig2, Ki-67

• Signal pathway visualization – pSTAT3, pERK, pAKT, NF-κB, Ki-67, HIF1α

Advantages of iHisto’s 6-Plex IF Services

• ✅ High-complexity panel support (up to 6 targets + DAPI)

• ✅ Signal amplification and separation via TSA

• ✅ Fully automated workflow using Leica BOND RX

• ✅ Compatible with image analysis software and AI tools

• ✅ Optional quantification and expert pathologist review

FAQs

• Can 6-plex IF be done on FFPE tissues?
  Yes, our workflow is optimized for FFPE and frozen sections, with TSA for high-efficiency signal detection.

• How do you avoid cross-reactivity in high-plex panels?
  We use TSA amplification and sequential antibody application to resolve markers from the same species.

• Do you offer slide scanning and export?
  Yes. We provide whole-slide imaging with separated channels and merged overlays.

• Can I send my own antibody panel?
  Absolutely — we can validate and integrate your antibodies or assist in panel design using our in-house stock.

Ready for High-Definition Spatial Biology?

iHisto’s 6-plex immunofluorescence staining delivers unmatched precision for spatial mapping and biomarker analysis. From immune profiling to multiplex biomarker discovery, our combination of TSA technology, Leica BOND RX automation, and expert support ensures results you can trust.

📩 Contact us to request a quote or consultation.