Technique hub
Immunohistochemistry (IHC) protocols — versioned, with troubleshooting and fixes.
IHC localises a protein in tissue using an antibody and an enzyme readout (usually HRP/DAB). Most failures cluster in a few places: antigen retrieval that under- or over-exposes the epitope, endogenous peroxidase that paints the whole section brown, and a weak block that lets nonspecific staining through. The immunohistochemistry protocols below keep those fixes visible — version by version.
Step by step
The IHC workflow — and where each step fails.
A standard FFPE IHC run is six stages. Most technique guides stop at the recipe; this one names the failure mode that lives at each step, because that's where reproducibility actually leaks.
- 1
Fixation
Formalin / PFA cross-links proteins to preserve morphology. Over-fixation masks epitopes; under-fixation lets tissue degrade.
Where it fails: Most “no staining” traces back here — epitopes locked up by hours of extra formalin.
- 2
Embedding & sectioning
Paraffin (FFPE) or frozen sections cut at 4–6 µm and mounted on charged slides.
Where it fails: Sections lifting off mid-protocol → patchy or absent signal; thick sections trap reagents and raise background.
- 3
Antigen retrieval
Heat-induced (HIER, citrate/EDTA) or enzymatic retrieval re-exposes epitopes masked by fixation.
Where it fails: Skipped or wrong-pH retrieval is the single most common cause of weak or no staining in FFPE.
- 4
Block & peroxidase quench
Serum/protein block reduces nonspecific binding; H₂O₂ quenches endogenous peroxidase before a DAB readout.
Where it fails: Skip the quench and endogenous peroxidase paints the whole section brown — the classic “high background”.
- 5
Primary & detection
Primary antibody, then a labelled secondary or polymer-HRP system, developed with DAB chromogen.
Where it fails: Over-concentrated primary or too-long DAB development swamps specific signal in nonspecific stain.
- 6
Counterstain & mount
Haematoxylin counterstain for context, dehydrate, then mount with a permanent medium.
Where it fails: Incomplete dehydration clouds the section; the result reads as diffuse background under the scope.
Troubleshooting
IHC troubleshooting, grouped by symptom.
High background, no staining, weak signal — start from what you see down the scope. Each block lists the likely causes and the fix that usually clears it.
High background / nonspecific staining
- Endogenous peroxidase not quenched (DAB methods)
- Block too short, too dilute, or wrong serum species
- Primary or secondary antibody too concentrated
- Sections dried out between steps
No staining
- Antigen retrieval skipped or wrong buffer/pH
- Over-fixation masking the epitope
- Secondary doesn't match the primary host species
- Primary degraded or used below working concentration
Weak or uneven signal
- Incomplete retrieval or thick/uneven sections
- Detection system not sensitive enough for a low-abundance target
- DAB development cut short
- Reagent not covering the whole section
The content moat
Every fix stays in the record.
A vendor protocol tells you the steps. It never tells you that v2 raised the peroxidase quench, or that switching retrieval buffer is what recovered the nuclear signal. On jnar that knowledge lives next to the step it fixed — version by version, attributed by ORCID — so the protocol gets more reproducible instead of forking into copies.
- ✓What broke, who fixed it, and why — kept together
- ✓Negative results recorded inline, not lost
- ✓Contributions converge on one canonical method
Have an IHC protocol as a PDF?
A PDF can't tell you why a step changed. Import your method into jnar, turn it into a structured protocol, and start recording the fixes. Your first AI conversion is free.
FAQ
IHC troubleshooting, answered.
Why is my IHC staining showing high background?
The usual culprits are endogenous peroxidase that wasn't quenched (for DAB methods), a block that's too short or the wrong serum species, an over-concentrated antibody, or sections that dried out between steps. Add a 3% H₂O₂ quench, block for at least an hour in serum matched to your secondary host, titrate the primary down, and keep sections in a humidity chamber.
Why is there no staining on my IHC slides?
Most no-staining results come from antigen retrieval — either skipped or run with the wrong buffer/pH — or from over-fixation masking the epitope. Confirm the retrieval method validated for your antibody (citrate pH 6 vs EDTA pH 9), shorten fixation time, and check that your secondary actually targets the host species of your primary.
What does a step-by-step IHC protocol look like?
Fixation → embedding and sectioning → antigen retrieval → block and peroxidase quench → primary antibody and detection → counterstain and mount. On jnar each step also carries the failure mode that lives there and the fix that resolved it in a previous version, so the protocol teaches what to watch for, not just what to do.
Do you have an IHC protocol PDF?
If you already have a method as a PDF or a Word file, you can turn it into a structured, versioned IHC protocol on jnar instead of leaving it static. Import it and start recording the fixes — your first AI conversion is free. A living protocol beats a PDF that can't tell you why a step changed.
What's the difference between IHC and immunofluorescence?
IHC and IF use the same antibody-targeting principle; the readout differs. IHC typically uses an enzyme (HRP/DAB) for a permanent, brightfield-visible chromogen, while immunofluorescence uses fluorophore-conjugated antibodies imaged on a fluorescence microscope. Many failure modes overlap, but IF adds autofluorescence and photobleaching as its own troubleshooting axes — see our immunofluorescence hub.
Have an IHC method of your own?
Bring it into jnar and start recording the fixes — antigen retrieval, blocking, detection. Your first AI conversion is free.