Single Immunofluorescence Staining: Target-Specific Fluorescent Labeling for Tissue Analysis

Overview

Single immunofluorescence staining is a high-sensitivity method used to detect and localize a single protein target within tissue sections using a fluorescent-labeled antibody. This technique offers exceptional signal clarity, low background, and is ideal for biomarker validation, expression profiling, and spatial localization studies in research or preclinical models.

At iHisto, our single-marker immunofluorescence services combine validated antibodies, Leica BOND RX automation, optimized detection protocols, and clean imaging for reproducible, publication-ready results.

How Single Immunofluorescence Staining Works

The staining process involves applying a primary antibody specific to the target antigen, followed by a fluorescent-conjugated secondary antibody (or a directly labeled primary antibody). Upon excitation under a fluorescence microscope, the fluorophore emits a visible signal, revealing the location of the protein of interest.

Fluorophores Available at iHisto:

• FITC

• CYAN

• CY3

• Texas-Red

• CY5

• CY7

• DAPI (for nuclear counterstaining)

Typical Visualization:

• Target protein: Green, red, or far-red fluorescence depending on fluorophore

• Nuclei: Blue (DAPI)

• Background: Minimal due to optimized blocking and washing

Step-by-Step Staining Process

1. Sectioning & Antigen Retrieval
   FFPE or frozen tissues are sectioned and treated to unmask antigens.

2. Blocking
   Reduces non-specific binding and autofluorescence.

3. Primary Antibody Incubation
   A target-specific antibody is applied and incubated.

4. Secondary Antibody Detection
   A fluorophore-labeled secondary antibody binds to the primary.

5. Nuclear Counterstaining
   DAPI or Hoechst is used to highlight nuclei.

6. Mounting
   Slides are mounted using antifade medium and coverslipped.

Our Optimized Workflow

At iHisto, we ensure reliable, high-quality single-marker IF staining with:

✅ Automated staining available using Leica BOND RX platform

✅ Expert antibody titration and validation
✅ Precise blocking and washing for low background
✅ Compatibility with FFPE and frozen tissue types
✅ Fluorescence whole-slide scanning available upon request
✅ Guidance on fluorophore selection and panel design

Applications o

f Single Immunofluorescence Staining

Single IF staining is ideal when specificity, clarity, or limited tissue availability is a concern.

Common use cases include:

• Tumor biomarker detection (e.g., Ki-67, PD-L1)

• Immune profiling – CD3, CD8, CD68, FOXP3

• Neural/glial markers – NeuN, GFAP, Iba1

• Phospho-protein detection – pSTAT3, pAKT

• Stem cell lineage tracing – SOX2, OCT4, Nestin

Advantages of iHisto’s IF Staining Services

• ✅ Clean, high signal-to-noise visualization

• ✅ Ideal for limited or archived tissue

• ✅ Compatible with human, rodent, and xenograft models

• ✅ Optional high-resolution slide scanning and image export

• ✅ Custom antibody validation and consultation included

We deliver precise, reproducible results tailored for research, therapeutic development, or regulatory submission.

FAQs

• What’s the difference between single and multiplex IF?
  Single IF detects one target, maximizing clarity. Multiplex IF visualizes 2 or more markers on the same slide.

• Which tissue types can be used?
  We support FFPE and frozen tissue sections from human, mouse, rat, NHP, and more.

• Can I supply my own antibody?
  Yes. Client-supplied antibodies are welcome, and we offer validation support as needed.

• Is imaging included?
  Yes. We provide fluorescence slide scanning, image processing, and optional overlays.

Ready to Visualize a Single Target with Clarity?

At iHisto, we deliver reliable single immunofluorescence staining with expert support, validated reagents, and precise imaging. Whether you’re mapping protein expression or validating a therapeutic target, we help you get clear, actionable data — every time.

📩 Contact us to request a quote or consultation.