Written and maintained by CASRAI Editorial Board
Last updated
Immunostaining is a method of detecting a protein or target antigen in a sample using an antibody. The antibody is then made visible, either by a chemical reaction that produces a colour change or by a fluorophore seen under a fluorescence microscope. The words immunohistochemistry (IHC), immunocytochemistry (ICC) and immunofluorescence (IF) all fall under this umbrella, and they describe different things.
Definition and scope
StainsFile defines immunostaining as a method of detecting a protein or target antigen in a sample. The common thread is an antibody that recognises the target. What differs between the named techniques is what the sample is and how the bound antibody is revealed.
Two axes are enough to keep the vocabulary straight:
- Sample type: tissue (IHC) or individual cells (ICC).
- Readout: a chemical reaction giving a colour change (chromogenic) or a fluorophore (fluorescent).
How immunostaining is used in practice: direct and indirect detection
Fluorescent immunostaining comes in two arrangements, both described by StainsFile.
Direct detection
In direct immunofluorescence, the antibody that binds to the antigen (the primary antibody) is also conjugated to the fluorophore.
Indirect detection
In indirect immunofluorescence, a fluorophore-tagged secondary antibody binds to the primary antibody. The fluorophore lets the antibody-antigen complex be visualised with an appropriate microscope. For how primary and secondary antibodies relate, see the primary and secondary antibody guide, and for the choice of antibody type see polyclonal vs monoclonal antibodies.
Types: IHC, ICC and IF compared
| Term | What it names | Details |
|---|---|---|
| Immunohistochemistry (IHC) | A sample type: tissue | Detects the target in tissue; performed on tissue sections, paraffin-embedded or frozen, which keep the extracellular matrix and tissue architecture. The antibody is visualised by a chemical reaction giving a colour change. |
| Immunocytochemistry (ICC) | A sample type: individual cells | The same approach on individual cells such as cultured cells or smears, usually without extracellular matrix. |
| Immunofluorescence (IF) | A readout: fluorophore | Visualisation with a fluorophore. It is a detection method and does not state what the sample is. |
Step-by-step bench detail lives in the site’s protocol guides: immunohistochemistry protocol, controls and troubleshooting and immunofluorescence protocol, controls and troubleshooting. This page does not repeat them.
Where it matters for labs and supply decisions
Because the terms overlap, a request, a methods section or a catalogue entry that says only “IF” or only “IHC” leaves something unstated. Based on the distinctions above, a reader needs these items stated to know what was done or what is being asked for:
- Sample type and preparation: tissue section (paraffin-embedded or frozen) or individual cells.
- Readout: chromogenic colour change or fluorophore.
- Direct or indirect detection: whether the primary antibody carries the fluorophore or a labelled secondary antibody does.
- Controls included: see the checklist below.
Controls checklist
| Control | What it is | What it measures |
|---|---|---|
| Secondary-only control | Sample exposed to the fluorophore-conjugated secondary antibody but not the primary | Non-specific binding |
| Isotype control | Sample exposed to primary antibodies that lack target specificity but match the target antibody’s class and type | Background from the antibody class rather than the target |
Both controls are described by StainsFile for immunofluorescence.
Common misunderstandings
“Immunofluorescence is a type of sample.”
It is not. IF names a detection method, so a tissue section and a dish of cultured cells can both be stained by immunofluorescence.
“IHC always means a colour reaction.”
StressMarq notes that both IHC and ICC can use colorimetric or fluorescent detection, and suggests the terms immunohistofluorescence and immunocytofluorescence for the fluorescent versions. Sources do not all agree on where the boundaries sit, and some define IHC as enzyme or chromogenic detection only. When reporting work, state the sample type and the readout in words rather than relying on the acronym alone.
Frequently asked questions
What is immunostaining?
A method of detecting a protein or target antigen in a sample with an antibody that is then visualised by a colour reaction or a fluorophore.
Is immunostaining the same as immunohistochemistry?
Immunohistochemistry is immunostaining applied to tissue. Immunostaining is the broader term and also covers work on individual cells (ICC).
What is the difference between direct and indirect immunofluorescence?
In direct detection the primary antibody carries the fluorophore. In indirect detection a fluorophore-tagged secondary antibody binds the primary antibody.
What does a secondary-only control show?
It shows non-specific binding of the fluorophore-conjugated secondary antibody, because the sample receives no primary antibody.
Can immunofluorescence be used on tissue as well as cells?
Yes. IF names the readout, not the sample, so a tissue section or cultured cells can both be stained by immunofluorescence (StressMarq).
Sources
- StainsFile (copyright STEMCELL Technologies), “Immunostaining / immunofluorescence”, stainsfile.com/theory/methods/immunostaining-immunofluorescence. Date checked: 2026-10-29.
- StressMarq Biosciences, “Immunohistochemistry vs immunocytochemistry”, stressmarq.com/blog/immunohistochemistry-vs-immunocytochemistry. Date checked: 2026-10-29.
Related reading: H&E staining.
