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IRF4 / Interferon regulatory factor 4 · IHC design guide
Design Immunohistochemistry for IRF4
Plan IRF4 staining in paraffin sections using the selective nuclear pattern seen in immune cells (HPA tissue IHC). Use tonsil non-germinal center cells as a positive reference (HPA tissue IHC) and start the IHC-validated antibody at 0.5–1 µg/mL (datasheet: PB9222 IHC-P).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
IRF4 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Selective nuclear staining in immune cells (HPA tissue IHC) | |
| Staining pattern | Nuclear staining in a subset of immune cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image PB9222) | |
| Caveat | Only a subset of immune cells may stain (HPA tissue IHC) | |
| Regulation | Lymphoid-cell enriched; regulation unreported (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope differences unreported (UniProt) |
Section 1
Recommended IRF4 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by one published IRF4 protocol for paraffin-embedded whole-tissue sections (PMC3122134).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet PB9222); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-IRF4, 0.5-1μg/ml (datasheet PB9222) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | IRF4-positive staining in non-germinal center cells of appendix (HPA tissue IHC: Medium). HPA tissue profile: Selective nuclear expression in a subset of immune cells in bone marrow, lymphoid tissues and gastrointestinal mucosa. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 at 95–98 °C for 20 min (page retrieval rule); compare the published EDTA retrieval if needed (PMC3122134).
Section 2
What Is the Expected IRF4 Staining Pattern?
IRF4 (MUM1) is a transcriptional activator annotated in the nucleus and cytoplasm, with no transmembrane segment (UniProt Q15306). In paraffin-section IHC, expect selective nuclear staining in a subset of immune cells in bone marrow, lymphoid tissues and gastrointestinal mucosa (HPA tissue IHC). HPA rates the tissue pattern Enhanced, citing high consistency between antibody staining and RNA expression (HPA tissue IHC).
| Discrete nuclear staining in some, but not all, immune cells in a lymphoid section. | This fits HPA’s selective nuclear pattern; uniform staining of every cell is unnecessary (HPA tissue IHC). Appendix, lymph node and tonsil show medium staining in non-germinal center cells (HPA tissue IHC). Score the stained cell population and its nuclear location together, rather than judging the section as wholly positive or negative. |
| Strong, widespread cytoplasmic staining with little or no nuclear staining. | This does not match the predominant tissue IHC pattern (HPA tissue IHC). Cytoplasmic IRF4 is annotated by UniProt, but HPA’s additional cytosolic ICC-IF location is uncertain (UniProt Q15306; HPA subcellular). Review nuclear counterstain alignment, detection background and controls before calling the cytoplasmic signal specific. |
| Prominent staining in a cell population listed as negative by HPA, such as adipocytes or respiratory epithelium. | HPA reports no detection in adipocytes from adipose tissue and respiratory epithelial cells from bronchus (HPA tissue IHC). A discrepant signal can reflect cross-reactivity or endogenous detection activity; morphology alone cannot distinguish them. Check the compartment and compare the IHC-validated antibody with appropriate detection controls (general IHC practice). |
| Diffuse color across cells and surrounding tissue, obscuring individual nuclei. | This prevents assessment of HPA’s selective nuclear pattern (HPA tissue IHC). Uneven blocking, detection background or excessive chromogen development are general IHC possibilities, not established IRF4-specific effects (general IHC practice). Interpret the run only after cell boundaries and counterstained nuclei can be resolved against a low-background control. |
| No nuclear signal in bone marrow hematopoietic cells or lymph-node non-germinal center cells. | Both are reported at medium staining by HPA, so their absence warrants a run-level check (HPA tissue IHC). Because expression is selective, first confirm that the expected cells are present in the section. Then assess antibody validation, detection reagents and the control slide before treating the sample as IRF4-negative (general IHC practice). |
💡Expected IRF4 appearanceA convincing positive is medium, discrete nuclear staining in the specified subset of immune or hematopoietic cells; broad staining of HPA-negative cell types or nuclear-independent haze is suspect (HPA tissue IHC; general IHC practice).
| Tissue and cell selection | Bone marrow hematopoietic cells, lymph-node and tonsil non-germinal center cells, appendix non-germinal center cells, and splenic red-pulp cells are reported at medium staining (HPA tissue IHC). Lung macrophages are listed as low, making them a less clear visual benchmark (HPA tissue IHC). |
| Compartment and topology | IRF4 has no transmembrane segment and is annotated in the nucleus and cytoplasm (UniProt Q15306). For tissue IHC, HPA describes selective nuclear expression (HPA tissue IHC). These records support a nuclear scoring focus but do not prove that every cytoplasmic signal is an artefact. |
| Antibody validation | HPA lists Enhanced IHC validation for HPA002038, HPA002698 and CAB013508 (HPA antibodies). That status supports the reported tissue pattern; it does not validate an unlisted catalog antibody or establish its working dilution. Interpret a new antibody against suitable positive and negative controls (general IHC practice). |
| Isoforms and target-specific limits | UniProt lists 2 IRF4 isoforms and one annotated chain spanning residues 1–451 (UniProt Q15306). The supplied records give no epitope mapping or isoform-specific IHC pattern. They also give no target-specific fixation or antigen-retrieval effect, so a failed signal cannot be assigned to either from these sources. |
| IF/ICC Q: where should signal appear? | A: HPA reports mainly nucleoplasmic localization, with additional cytosolic localization marked uncertain (HPA subcellular). This is ICC-IF evidence, not a paraffin-section IHC protocol or proof that cytosolic tissue staining is expected. Use the separate IF/ICC guide for that application. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells stain weakly or remain unstained. | The selected field may lack the relevant subset, or the IHC run may have inadequate signal; HPA reports selective expression rather than positivity in every cell (HPA tissue IHC). | Confirm the cell population on the counterstained section. Compare with a bone marrow or lymphoid positive control reported by HPA, then review the catalog antibody’s IHC-P instructions and detection controls (HPA tissue IHC; general IHC practice). |
| Only diffuse cytoplasmic color appears in the expected tissue. | The pattern departs from HPA’s selective nuclear tissue signal, although UniProt also annotates cytoplasm (HPA tissue IHC; UniProt Q15306). The image alone cannot identify the source. | Check whether color tracks counterstained nuclei. Review detection-only controls and repeat under the catalog antibody’s IHC-P conditions before scoring the run (general IHC practice). |
| Adipocytes or respiratory epithelial cells stain prominently. | Those cell populations are reported as not detected by HPA; cross-reactivity or endogenous chromogenic activity is possible (HPA tissue IHC; general IHC practice). | Compare a known-positive immune-cell population, a relevant negative tissue and a detection-only control. Record the stained cell type and compartment before attributing the signal to IRF4 (general IHC practice). |
| The whole section has brown haze or patchy background. | Background obscures the cell-restricted nuclear pattern reported by HPA (HPA tissue IHC). Blocking, reagent carryover or chromogen development are general IHC variables, with no IRF4-specific cause established here (general IHC practice). | Inspect the detection-only control, wash steps and chromogen development according to the assay instructions. Rescore only when individual nuclei and negative areas remain distinguishable (general IHC practice). |
| A low-signal lung field seems negative. | HPA lists lung macrophages as low, whereas several lymphoid and bone marrow populations are medium (HPA tissue IHC). A low-signal field gives limited evidence about run performance. | Use a reported medium-staining tissue as the primary positive comparison, and identify the relevant cells before scoring the lung field (HPA tissue IHC; general IHC practice). |
| IF/ICC shows nucleoplasmic signal plus faint cytosol. | Nucleoplasm is HPA’s supported main ICC-IF location; additional cytosol is marked uncertain (HPA subcellular). This observation does not establish an IHC tissue pattern. | Interpret the two applications separately: score selective nuclear cells in paraffin IHC (HPA tissue IHC), and consult the separate IF/ICC guide for fluorescence controls and interpretation. |
Experiment evidence
IHC & IF images showing IRF4 staining
PB9222 · IHCAnti-MUM1 antibody, PB9222, IHC(P) IHC(P): Human Tonsil Tissue
A00401 · IFImmunocytochemistry of IRF4 in Jurkat cells with IRF4 antibody at 10 μg/mL.
A00401-2 · IFIF analysis of IRF4 using anti-IRF4 antibody (A00401-2). IRF4 was detected in an immunocytochemical section of A549 cells. Enzyme antigen re
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000137265-IRF4/tissue/cerebral+cortex#imid_970680
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000137265-IRF4/tissue/colon#imid_970614
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000137265-IRF4/tissue/liver#imid_970623
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000137265-IRF4/tissue/kidney#imid_970616
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA002038; green = IRF4, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA002038; green = IRF4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for IRF4 IHC & IF
🧪Run tonsil first and score non-germinal center cells for IRF4 staining (HPA: Medium in tonsil non-germinal center cells). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); cells without IRF4 staining on the tonsil slide should show counterstain without chromogenic signal.
Positive control tissue: Appendix (Non-germinal center cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show IRF4 in A-431, REH, U-251MG, HDLM-2, SK-MEL-30, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species, isotype and concentration; use an IRF4-knockout specimen as a biological negative if available. Check for endogenous peroxidase signal in tonsil and block it as needed before chromogenic detection.
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the fixative is unreported in the selected PB9222 tonsil IHC-P caption. Retrieval dependence is unreported, so optimize antigen retrieval on paraffin sections; the supplied evidence does not establish that frozen sections or IF are easier. Tonsil background from endogenous peroxidase can complicate interpretation, while nuclear staining is expected for IRF4 (UniProt Q15306: nucleus; HPA: supported nucleoplasm localization).
HPA tissue IHC evidence for IRF4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Non-germinal center cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Lymph node | Non-germinal center cells | Medium | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | Medium | Protein (IHC) | HPA → |
| Tonsil | Non-germinal center cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced IRF4 IHC Tips
Troubleshoot IRF4 chromogenic IHC by checking nuclear signal, cell context and section quality before comparing staining intensity across samples.
How should I adjust retrieval when IRF4 nuclear staining is weak?
Start with Tris-EDTA pH 9.0 heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval rule: nuclear antigen). If staining remains weak, compare a longer heating time or a citrate pH 6.0 fallback on matched sections, changing one variable at a time (standard IHC practice). Judge recovery by nuclear staining in the expected immune-cell subset, including tonsil non-germinal center cells, rather than by diffuse color across the section (HPA tissue IHC). Keep the antibody, detection and chromogen conditions constant during that comparison, and reject a retrieval change that raises background as much as specific nuclear signal (standard IHC practice).
Could fixation explain absent IRF4 staining in my paraffin sections?
Target-specific fixation sensitivity is unknown from the supplied evidence, so an absent signal alone cannot identify overfixation or underfixation as the cause (supplied evidence). The PB9222 tonsil image is labeled IHC(P), but its caption does not state a fixative or fixation duration (PB9222 tissue-IHC caption). Check documented fixation and processing records, then compare sections with matched thickness, retrieval and detection conditions before changing several steps together (standard IHC practice). Include a section with an expected immune-cell population and assess nuclear staining there; if that control also fails, troubleshoot tissue processing, retrieval, antibody incubation and detection systematically (HPA tissue IHC; standard IHC practice).
Should cytoplasmic IRF4 staining count as a positive IHC result?
Prioritize nuclear staining when scoring chromogenic IHC: IRF4 is a transcriptional activator, and HPA describes selective nuclear expression in immune-cell subsets (UniProt Q15306; HPA tissue IHC). Cytoplasmic localization is also listed, but HPA calls the additional cytosolic location uncertain, so cytoplasmic color alone needs separate validation (UniProt Q15306; HPA subcellular). Compare the signal with a nuclear counterstain and inspect whether it is confined to plausible immune cells rather than spread across unrelated structures (HPA tissue IHC; standard IHC practice). Record nuclear and cytoplasmic staining separately, especially when retrieval or detection changes alter their relative intensity (standard IHC practice).
Could IRF4 isoforms or phosphorylation change what my antibody detects?
IRF4 has 2 listed isoforms, but the supplied evidence gives no antibody epitope map or isoform-specific IHC performance (UniProt Q15306; supplied evidence). Phosphoserines at residues 447 and 448 are annotated, yet their effect on this antibody's staining is likewise unknown (UniProt Q15306; supplied evidence). If staining differs between samples, first compare section processing, retrieval and nuclear signal in an expected positive population under identical conditions (HPA tissue IHC; standard IHC practice). Interpret a persistent difference as a staining observation until an epitope map or independent assay establishes whether isoform abundance or phosphorylation contributes (standard IHC practice).
How can IF help assess an ambiguous IRF4 IHC pattern?
On a separate IF/ICC workflow, multiplex IRF4 with a marker that identifies the expected immune-cell population and a nuclear counterstain, then check whether signals coincide in the same cells (HPA tissue IHC; standard IF practice). Choose fluorophores in channels with low measured tissue autofluorescence and include single-stain controls to assess bleed-through (standard IF practice). Because IRF4 has no transmembrane segment and its supported location is nucleoplasmic, use permeabilisation sufficient for antibody access to the nuclear target and check nuclear morphology (UniProt Q15306; HPA subcellular; standard IF practice). Optimize fixation and permeabilisation within that IF workflow; the PB9222 IHC(P) caption supplies no IF fixation evidence (PB9222 tissue-IHC caption).
What should I check when brown staining obscures IRF4-positive nuclei?
Inspect a no-primary control and the section edges to separate detection background from candidate nuclear staining (standard IHC practice). For a peroxidase and DAB workflow, use a peroxidase block and assess endogenous pigment or enzyme-associated color before interpreting the chromogen (standard IHC practice). If diffuse staining persists, titrate the IHC-validated antibody, review blocking and washes, and compare matched sections without changing retrieval at the same time (standard IHC practice). A credible pattern should favor nuclei in a subset of immune cells; widespread color in unrelated structures is inconsistent with the reported selective tissue pattern (HPA tissue IHC).
How should I score IRF4 staining across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and analysis region before scoring, and count nuclear-positive cells using a consistent intensity threshold across matched slides (HPA tissue IHC; standard IHC practice). Report the percentage of positive cells and, when intensity matters, an H-score using 0–3 nuclear intensity categories (standard IHC practice). For spatial comparisons, report positive-cell density per mm² of viable analyzed tissue and exclude folds, necrosis and empty areas consistently (standard IHC practice). Normalize positive-cell counts to all evaluable cells of the same defined population, rather than to every cell in fields with different immune-cell content (HPA tissue IHC; standard IHC practice).
How do I distinguish genuine IRF4 signal from tissue artefact?
Look for discrete nuclear staining in an appropriate immune-cell subset, such as tonsil non-germinal center cells, and compare it with the nuclear counterstain (HPA tissue IHC; standard IHC practice). Treat isolated cytoplasmic color cautiously because the supported HPA subcellular location is nucleoplasmic, while its additional cytosolic location is uncertain (HPA subcellular). Check whether apparent positives cluster at section edges or necrotic areas, where uneven staining can distort interpretation (standard IHC practice). If brown deposits remain in a no-primary control, investigate endogenous enzyme activity or pigment before assigning them to IRF4 (standard IHC practice).
Boster reagents
Best IRF4 / Interferon regulatory factor 4 IHC Antibodies
Anti-IRF4 figures show human tonsil paraffin-section IHC and Jurkat and A549 cell IF/ICC; catalog reactivity also includes Mouse and Rat (product image captions; A00401 catalog reactivity).
Real IHC data
Anti-MUM1/IRF4 Antibody ®
Cat # PB9222
Real IF data
Anti-Interferon regulatory factor 4 IRF4 Antibody
Cat # A00401
Real IF data
Anti-MUM1/IRF4 Antibody ®
Cat # A00401-2
PB9222 shows human tonsil IHC(P) (PB9222 image caption), while A00401 shows Jurkat cell ICC at 10 μg/mL and lists IF/ICC applications (A00401 image caption; catalog applications). A00401-2 shows A549 cell IF/ICC at 5 μg/mL (A00401-2 image caption).
Which to pick: For tissue IHC, choose PB9222: its own human tonsil IHC(P) image supports paraffin sections; the fixative is unreported (PB9222 image caption). For IF/ICC, choose A00401-2 for its A549 cell image and polyclonal format (A00401-2 image caption; datasheet). For cross-species work, A00401 lists Human, Mouse and Rat reactivity and IF/ICC applications, though its shown cell image is from Jurkat cells (A00401 catalog reactivity and applications; image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q15306 (IRF4_HUMAN, Interferon regulatory factor 4).
- Human Protein Atlas. IRF4 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. IRF4 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol..
- Human Protein Atlas. IRF4 antibody validation summary (3 antibodies).
- Diagnostic Accuracy of MUM1 (IRF4) Immunohistochemistry in Chronic Endometritis: A Systematic Review and Bayesian Meta-Analysis. Diagnostics (Basel, Switzerland) 2026 — PMC13115080.
- Specificity of IRF4 translocations for primary cutaneous anaplastic large cell lymphoma: a multicenter study of 204 skin biopsies. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2011 — PMC3122134.
- IRF4 translocation status in pediatric follicular and diffuse large B-cell lymphoma patients enrolled in Children's Oncology Group trials. Pediatric blood & cancer 2019 — PMC6941672.
- Expression of the interferon regulatory factor 8/ICSBP-1 in human reactive lymphoid tissues and B-cell lymphomas: a novel germinal center marker. The American journal of surgical pathology 2008 — PMC9036392.
- PubMed PMID:8921401 — UniProt-cited evidence.
- PubMed PMID:9326949 — UniProt-cited evidence.
- PubMed PMID:14574404 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for IRF4 (Interferon regulatory factor 4). Expected localisation Selective nuclear staining in immune cells (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: PB9222 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Non-germinal center cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet PB9222); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-IRF4, 0.5-1μg/ml (datasheet PB9222); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image PB9222); Caveat: Only a subset of immune cells may stain (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope differences unreported (UniProt); Regulation: Lymphoid-cell enriched; regulation unreported (UniProt). Evidence: UniProt Q15306, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet PB9222, PMC3122134. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/IRF4/immunohistochemistry-ihc-guide.