Immunohistochemistry
The standard brightfield method: primary antibody, HRP polymer and DAB give a permanent brown precipitate on a haematoxylin counterstain, read by eye or by image analysis and often scored as an H-score or percentage of positive cells.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Chromogenic (DAB)
The standard brightfield method: primary antibody, HRP polymer and DAB give a permanent brown precipitate on a haematoxylin counterstain, read by eye or by image analysis and often scored as an H-score or percentage of positive cells.
How it differs from the standard method
- Quench endogenous peroxidase first.
- Fix the DAB time and titrate the primary on control tissue.
- Keep the counterstain light for nuclear targets.
1An antibody, an enzyme and a coloured precipitate
Immunohistochemistry shows where a protein is in a tissue section. A primary antibody binds the target; a secondary reagent, today usually a polymer carrying many secondary antibodies and many enzyme molecules, binds the primary; and the enzyme turns a soluble chromogen into an insoluble coloured precipitate exactly where it sits.
2Formalin hides epitopes; heat gives them back
The methylene bridges that make formalin a good fixative also cover or distort many epitopes, and the longer the tissue sits in formalin the more are masked. Heat-induced epitope retrieval (HIER) reverses much of this: heating sections to about 95–100 °C for 20 minutes, or briefly in a pressure cooker, in citrate pH 6 or Tris–EDTA pH 9 breaks enough cross-links for the antibody to bind.
Positive control
Tissue known to express the target at a range of levels, stained in the same run. If it fails, the run fails.
Negative control
A serial section without the primary (or with an isotype antibody) shows brown that comes from the detection system or the tissue's own enzymes.
Endogenous enzymes
Red cells and granulocytes have peroxidase; quench with hydrogen peroxide. Some tissues have alkaline phosphatase; block with levamisole.
Mouse on mouse
An anti-mouse secondary on mouse tissue also finds the mouse's own immunoglobulin; prefer non-mouse primaries or a blocking kit.
3Scoring: intensity is not linear
Many studies score IHC semi-quantitatively. The H-score multiplies the percentage of cells at each intensity by its weight, 1×%1+ + 2×%2+ + 3×%3+, giving 0 to 300.
4More than one target
Double or multiplex chromogenic IHC stains two or more targets in different colours, usually in sequential rounds. Each round's detection reagent must not find the other round's primary: use primaries from different species, or strip the first round's antibodies with heat before the second (the DAB stays).
Key terms
Practise these as flashcards →A backbone carrying many secondary antibodies and peroxidase molecules; more sensitive than avidin–biotin and free of endogenous biotin background.
3,3′-Diaminobenzidine, oxidised by HRP into a brown, alcohol-insoluble precipitate.
Heat-induced epitope retrieval: heating sections in buffer to reverse formalin masking.
Semi-quantitative score, 1×%1+ + 2×%2+ + 3×%3+, from 0 to 300.
An antibody of the same class and species as the primary but with no relevant target, used at the same concentration.
Testing a series of dilutions on control tissue to find the one that separates weak from strong without background.
When to use it
- Finding which cells in a tissue express a protein, and where in the cell it sits.
- Scoring expression of a marker across samples, for example a receptor in tumours.
- Counting cell types in situ, such as T and B cells in lymphoid tissue, with double staining.
Compared with alternatives
Better for co-localisation and several targets at once, and more linear; fades and lacks the H&E-like context.
Checks that the antibody recognises one protein of the right size, and quantifies it in bulk.
Detects the mRNA rather than the protein, when no good antibody exists.
Fixation time is the largest hidden variable in IHC: record it and keep it within 24–72 h.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
The standard brightfield method: primary antibody, HRP polymer and DAB give a permanent brown precipitate on a haematoxylin counterstain, read by eye or by image analysis and often scored as an H-score or percentage of positive cells.
- Quench endogenous peroxidase first.
- Fix the DAB time and titrate the primary on control tissue.
- Keep the counterstain light for nuclear targets.