Immunohistochemistry (IHC) and immunofluorescence (IF) localise a protein of interest directly on the tissue section, preserving the tissue architecture. Where cytometry dissolves the tissue into a suspension, IHC/IF answers a different question: where, in the tissue, is a given protein expressed, by which cells and in what neighbourhood? It is the reference tool for reading the spatial organisation of a sample.
Principle and workflow
The tissue is fixed and embedded (most often in paraffin, FFPE) or frozen, then cut into thin sections. After antigen retrieval (heat or enzyme), a primary antibody recognises the target. Detection then differs: in IHC, an enzymatic system (peroxidase/DAB) deposits a coloured precipitate visible in light microscopy; in IF, a fluorochrome is read in fluorescence or confocal microscopy, offering better resolution and the co-localisation of several targets.
As in cytometry, reliability is decided in the details: antibody specificity and titration, isotype and positive/negative controls, control of autofluorescence, and above all protocol standardisation. Reading can remain qualitative (presence, localisation) or become quantitative through digital pathology: cell densities, intensity, tumour/stroma compartments, intercellular distances.
Variants and options
Single-marker chromogenic IHC remains robust and archivable. IF allows the fine co-localisation of several targets. Multiplex — several markers on the same slide, by multiplexed immunofluorescence or tyramide-type amplification — simultaneously maps different populations and their spatial interactions. Coupled with digital pathology and image analysis, quantification becomes objective and comparable from one sample to another — this is the bedrock of reproducible indices such as the Immunoscore.
When and why this technique
IHC/IF is chosen when localisation and spatial context matter: identifying the cell type that expresses a target, mapping an immune infiltrate, distinguishing tumour expression from stromal expression, all on archival tissues (FFPE) often available retrospectively.
Its limits mirror those of cytometry. The technique depends strongly on the quality of fixation and retrieval; it is semi-quantitative by nature, and objective quantification requires strict standardisation and validated algorithms. The number of simultaneous markers is limited (especially in chromogenic mode): multiplex pushes back this boundary but complicates the analysis (autofluorescence, spectral overlap, cell segmentation). Finally, throughput remains far below that of cytometry. To count cells in suspension at high throughput, cytometry takes over; for the transcriptome in its tissue context, spatial omics extends multiplexed immunofluorescence.
Inovarion’s expertise
Inovarion puts quantitative IHC/IF at the service of characterising the tumour microenvironment. The laboratory has contributed to the standardised quantification, by immunohistochemistry and image analysis, of the densities of T lymphocytes (CD3+, CD8+) and other populations (CD45RO, FOXP3, CD20) at the centre and margin of tumours, including in a metastatic context — the approach that underpins the Immunoscore. Quantitative IHC has also served to explore therapeutic targets on tumour tissue, for example in clear-cell renal carcinoma. From antibody choice to digital quantification, Inovarion masters the full chain, from section to number.
See also: Immunoscore (clinical application of immune quantification on tissue).
Key publications
- Mlecnik et al. Comprehensive Intrametastatic Immune Quantification and Major Impact of Immunoscore on Survival. Journal of the National Cancer Institute, 2018. Record → · PubMed
- Roelants et al. Combined inhibition of PI3K and Src kinases demonstrates synergistic therapeutic efficacy in clear-cell renal carcinoma. Oncotarget, 2018. PubMed