Comparing IHC, ICC, and IF: Which One Fits Your Research?

Immunohistochemistry (IHC), immunocytochemistry (ICC), and immunofluorescence (IF) are three fundamental techniques widely used in life science and medical research. However, many researchers—from beginners to experienced professionals—often struggle to distinguish between them. Are you also wondering:

What are the key differences between IHC, ICC, and IF?

Which technique should I choose for my samples?

Which one is more sensitive? Which one is easier to perform?

In this article, we will provide you a detailed comparison between these 3 techniques including their principles, applications, pros/cons and tips for choosing, to help you select the best method to support your experiments.

What are IHC, ICC, and IF?

Fig. 1. Workflow of histology slide preparation, microscopic examination, and image-based analysis (Gheban-Roșca I-A, Gheban B-A, *et al.*, 2024).

IHC (Immunohistochemistry) is an immune-detection technique to detect and locate specific antigens (typically proteins) in cells or tissues using the specific binding between antibodies and antigens. It is commonly used in pathology, cancer research, and diagnostics, for example, to study the expression and distribution of specific proteins in tumor tissues. The IHC result is usually observed with a microscope. The method has high specificity and reproducibility but is prone to inter-observer variability, and only one marker can be detected in each assay, thus is not suitable for detecting multiple markers.
ICC (Immunocytochemistry) is also an immune-based staining technique similar to IHC but is used for staining cell samples, like cell smears, cell blocks or liquid-based monolayers. The fixation method of ICC is different from that of IHC, often involving wet fixation or air drying. Another difference is that it is usually more challenging to get reliable positive or negative control samples to interpret the ICC results. In summary, although the principle and application of ICC and IHC are similar, ICC is more suitable for cell-based samples, and IHC is more suitable for tissue sections.
IF (Immunofluorescence) is an immune-detection technique using fluorescent labeling, in which fluorescent dyes are conjugated to antibodies to visualize the expression of antigens in cells and tissues. It offers high sensitivity and a broad dynamic range, enabling the simultaneous detection of multiple markers. This makes it ideal for quantitative analysis and spatial distribution studies. IF is commonly used to investigate cellular functions, intercellular interactions, and the tumor microenvironment (TME).

Which Technique is Best for Your Experiment?

Technique	Sample Types	Typical Applications
IHC	Formalin-fixed paraffin-embedded (FFPE) tissues Frozen tissue sections Clinical pathology samples (e.g., tumors, inflammatory tissues)	Protein expression analysis in tissues (e.g., Ki-67 for tumor proliferation activity) * Pathological diagnosis (e.g., HER2 in breast cancer) Spatial localization studies (e.g., vascular endothelial marker CD31 distribution)
ICC	Adherent cells (e.g., HeLa, HEK293) Suspension cells (e.g., Jurkat, THP-1) Primary cells or stem cells	Intracellular protein expression analysis (e.g., β-actin cytoskeleton localization) Protein changes under drug treatment (e.g., p53 induction by chemotherapy) Cell differentiation marker detection (e.g., OCT4 in stem cells)
IF	Tissue sections (frozen/FFPE) Cultured cells (adherent/suspension) 3D spheroids or organoids	Multiplex protein co-localization (e.g., mitochondrial COX4 + microtubule protein double labeling) Subcellular structure studies (e.g., nuclear membrane protein Lamin B1 + ER marker Calnexin) Super-resolution imaging (e.g., STED, SIM microscopy)

Comparison of Advantages and Disadvantages

Technique	Advantages	Disadvantages
IHC	Compatible with archived samples (long-term FFPE storage) Visually intuitive results (DAB staining, observable with standard light microscopy) Integrates with clinical automation platforms	Typically limited to single-color detection (sequential sections needed for multiplexing) Lower sensitivity (compared to fluorescence) Complex tissue processing (dewaxing, antigen retrieval required)
ICC	Simple protocol (direct cell fixation/permeabilization/staining) Cost-effective (no specialized equipment needed) Suitable for high-throughput screening (e.g., 96-well plate assays)	Lacks tissue microenvironment context (limited to monolayer cells Restricted multiplexing (chromogenic detection usually single-color) Sensitive to cell state (e.g., apoptotic cells may cause nonspecific staining)
IF	High sensitivity (stronger signal than chromogenic methods) Simultaneous multiplex detection (e.g., Alexa Fluor 488/555/647 triple labeling) Compatible with 3D imaging	Requires fluorescence microscopy (higher equipment costs) Photobleaching issues (anti-fade mounting media needed) Autofluorescence interference (e.g., lipofuscin in tissues may cause false positives)

Still undecided? Try this universal strategy:

Start with ICC (chromogenic method) → Rapidly validate antibody efficacy.

Optimize and upgrade to IF → Obtain publication-quality images.

Supplement with IHC for clinical samples → Meet pathological diagnostic requirements.

Creative Bioarray Relevant Recommendations

Products & Services	Description
Immunohistochemistry (IHC), Immunofluorescence (IF) Service	Creative Bioarray offers a comprehensive IHC service from project design, marker selection to image completion and data analysis.
Histology Services	Creative Bioarray offers tissue processing, embedding, sectioning, and staining, along with set of histological examination services.

Reference

Gheban-Roșca I-A, Gheban B-A, et al. Immunohistochemical and Morphometric Analysis of Lung Tissue in Fatal COVID-19. Diagnostics. 2024; 14(9):914.

For research use only. Not for any other purpose.