IHC vs IF: Key Differences, Use Cases & Which to Choose
- KAMFEI WONG
- Apr 29
- 4 min read
Updated: Jun 1
Quick Summary:
IHC is ideal for simple, permanent slides with 1–2 markers. IF detects more targets per slide (up to 60) and offers spatial detail—perfect for tumor microenvironments. Choose based on your equipment, goal, and budget.
IHC uses enzymes to create a visible color stain under a regular microscope. IF uses fluorescent dyes that glow under special lighting. This lets you see more markers on a single slide.
This quick guide explains the trade-offs in plain language so you can pick the best method—or combine both—to speed up your next milestone.
At a Glance Comparison
Feature | IHC | IF (2–8‑plex) | Ultra-high-plex IF (10–60 plex*) |
Detection chemistry | Chromogenic enzyme (HRP/AP + DAB, AEC, etc.) | Direct or secondary fluorophores | Repeated dye cycles with color separation software |
Max markers/slide | 1–2 markers | 2–8 markers | 10–60 (Akoya PhenoFusion) |
Signal Stability | Permanent, archivable | Moderate (photobleaching risk) | Moderate (software‑corrected) |
Sensitivity / Dynamic Range | Moderate | High | Very high |
Equipment Needed | Brightfield scope | Fluorescence scope | Advanced scanner + AI analytics |
Best For | Diagnostic workflows, GLP archiving | Spatial biology, co‑localization | Tumor microenvironment & complex panels |
Typical turnaround** | 3–5 days | 5–7 days | 7–10 days |
* Akoya PhenoCycler-Fusion 2.0 platform.
** iHisto averages; rush service available.
What is Immunohistochemistry (IHC)?
IHC uses antibodies linked to enzymes like HRP or AP. The enzymes react with a chemical, creating a visible stain under a microscope. The slides are stable. You can store them for years—ideal for regulatory use and pathology.
Key strengths
Permanent slides—regulatory archiving ready.
Works on any brightfield microscope.
Crisp morphology aids pathologist reads.
Typical limitations
1–2 markers per slide by default.
Moderate sensitivity; low-copy targets need amplification.
Colour overlap complicates deep multiplexing.
What is immunofluorescence (IF)?
IF uses fluorescent dyes instead of colored chemicals. Special light excites the dye, which then glows and is captured by the microscope. By using different light colors or repeating dye steps, researchers can study many proteins on one slide.
Key strengths
Traditional IF offers high multiplexing potential (typically 2–8+ targets on the same slide).
Advanced platforms Platforms like PhenoFusion detect 10+ proteins and show where they are in the tissue., See details.
Superior sensitivity and dynamic range.
Helps you see tiny features inside cells
Limitations
Prone to photobleaching.
Requires fluorescence imaging systems and expertise.
Higher cost and complexity.
Quick Reference Table: IHC vs IF
Parameter | IHC | IF |
Archivable | ✅ Yes | ⚠️ Limited (digital archive recommended) |
Co‑localization | 🚫 Limited | ✅ Excellent |
Cost / Complexity | 💲 | 💲💲–💲💲💲 |
Typical Turnaround* | 3–5 days | 5–7 days |
*iHisto average
When Should You Choose IHC or IF?
Understanding the strengths of each method can help you decide based on your study goals:
Choose IHC if:
Need permanent slides for GLP or regulatory submission.
Your lab only has a regular microscope.
Require crisp morphology for pathologist review.
Choose IF if:
Need ≥ 3 markers or spatial biology data.
You’re analyzing immune cells or tumor structure.
Have access to fluorescence imaging—or plan to outsource scanning to iHisto.
Cost Breakdown:
IHC: Lower upfront cost per slide.
Multiplex IF: Lower cost per marker.
IF saves time and cost when analyzing many proteins together.
Sample-Prep Tips
Fixation: Use the right fixative for each tissue type. See our Fixation Cheat-sheet.
Antigen Retrieval: Choose citrate or Tris-EDTA depending on the antibody.
Controls: Always include a positive, negative, and “no-primary” slide for IF.
Section Thickness: Use 4 µm for IHC; 5–7 µm for IF to protect tissue.
Storage: Keep unstained slides cold and dry. Stain IF slides within 4 weeks.
Why Partner with iHisto?
Automated staining on Leica Bond RX & Akoya PhenoFusion ensures run‑to‑run consistency.
Certified multiplex assays up to 60‑plex.
Whole-slide imaging included: brightfield + fluorescence, shareable within 24 h.
Expert antibody optimization: Dilution curves, retrieval buffers and blocking tailored per project.
Secure HistoCloud™ portal keeps data accessible to your entire team.
Your research drives discovery—iHisto gets you there faster.
Related Services: Molecular Pathology | Spatial Biology | Request a Quote
In Summary:
IHC vs IF, If you need long-term storage and simple analysis, IHC is a great choice. If you’re studying multiple markers or spatial relationships, IF offers greater flexibility and data depth. Both have value—and at iHisto, we help you decide based on your specific study goals.
Frequently Asked Questions
Can I use the same antibody for IHC and IF?
Yes, if it’s tested and tagged correctly for both IHC and IF.
Which technique is better for multiplexing?
IF. Traditional IF handles up to 8 markers; our Akoya PhenoFusion workflow reaches 10–60 markers on one slide.
Does iHisto optimize antibodies?
Yes—dilution curves, retrieval buffers & blocking are tailored to maximize specificity and signal.
Ready to Accelerate Your Study?
Email info@ihisto.io or request a quote. Most projects receive a protocol draft within 24 hours.