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IRF8 / Interferon regulatory factor 8 · IHC design guide
Design Immunohistochemistry for IRF8
Use tonsil germinal center cells as a positive reference for paraffin IRF8 IHC (HPA tissue IHC). This guide covers fixation consistency, the catalog antibody’s 2–5 μg/ml IHC range (datasheet PA2209), and interpretation of nuclear staining in lymphoid cells (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
IRF8 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 | Nuclear in a subset of lymphoid cells (HPA tissue IHC) | |
| Staining pattern | Selective nuclear staining in some lymphoid cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PA2209) | |
| 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 | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Resting macrophages can retain cytoplasmic IRF8 (UniProt) | |
| Regulation | IFN-γ induces nuclear translocation (UniProt) | |
| Isoform / epitope | No annotated isoforms or processing; one 1–426 chain (UniProt) |
Section 1
Recommended IRF8 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet: PA2209) with four published IRF8 IHC protocols (PMC7992828; PMC3498170; PMC9601976; PMC2605678).
| Sample | Paraffin-embedded human tonsil tissue; fixative not specified (datasheet PA2209) |
| Fixation | Image fixative and duration unreported (datasheet PA2209); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet PA2209); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PA2209) |
| Primary antibody | Rabbit anti-IRF8, 2-5μg/ml (datasheet PA2209) |
| Primary incubation | Overnight at 4 °C (datasheet PA2209) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet PA2209) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | IRF8-positive staining in lymphoid tissue of appendix (HPA tissue IHC: High). HPA tissue profile: Selective nuclear expression in a subset a lymphoid cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet: PA2209); published citrate retrieval conditions apply to the antibodies used in those studies (PMC7992828; PMC3498170).
Section 2
What Is the Expected IRF8 Staining Pattern?
IRF8 is a nuclear transcription factor, with cytoplasmic localisation reported in resting macrophages and nuclear translocation after IFN-gamma induction (UniProt Q02556). In paraffin sections, expect selective nuclear staining in a subset of lymphoid cells, especially germinal center cells (HPA: tissue IHC). IRF8 has no transmembrane segment (UniProt Q02556 topology). HPA rates its tissue IHC reliability Enhanced, with medium consistency between antibody staining and RNA expression (HPA: tissue IHC).
| Distinct nuclear staining in a subset of lymphoid cells, strongest in germinal centers. | This matches the selective tissue pattern: germinal center cells in lymph node and tonsil are High, and appendix lymphoid tissue is High (HPA: tissue IHC). Judge the labelled cell population and compartment together; a positive section need not show staining in every cell (HPA: tissue IHC). |
| Predominantly cytoplasmic staining, with little convincing nuclear signal. | In lymphoid tissue, this departs from the selective nuclear IHC pattern and calls for a compartment check (HPA: tissue IHC). Cytoplasm alone is not conclusive evidence of artefact in macrophages: resting macrophages can contain cytoplasmic IRF8, which moves to the nucleus after IFN-gamma induction (UniProt Q02556). |
| Strong staining across respiratory epithelium or other cells expected to be negative. | HPA reports IRF8 as Not detected in bronchial respiratory epithelial cells, so widespread staining there warrants a specificity check (HPA: tissue IHC). Cross-reactivity or endogenous chromogenic detection activity are possible explanations (general IHC practice); the pattern alone does not identify which one occurred. |
| A diffuse chromogen haze obscures nuclei and tissue boundaries. | The slide cannot be scored confidently for the selective nuclear pattern while background masks cell identity (HPA: tissue IHC; general IHC practice). Review blocking, washing, detection controls and counterstain intensity before deciding whether the apparent staining belongs to IRF8-positive cells (general IHC practice). |
| No signal in lymph node or tonsil germinal centers. | Both are reported High in germinal center cells, making them useful known-positive tissue checks (HPA: tissue IHC). An entirely blank section may reflect assay failure, unsuitable antibody conditions or detection failure (general IHC practice); it does not by itself establish biological absence of IRF8. |
💡Expected IRF8 appearanceCall a paraffin-section result positive when a subset of lymphoid-cell nuclei shows distinct staining, with High staining reported in lymph node and tonsil germinal centers; widespread bronchial epithelial staining is suspect because those cells are Not detected (HPA: tissue IHC).
| Which tissue and cells are being scored? | HPA reports High staining in appendix lymphoid tissue and lymph node and tonsil germinal center cells; bone marrow hematopoietic cells, lung macrophages and splenic red-pulp cells are Medium (HPA: tissue IHC). Compare each observed population with its own reference level rather than applying one intensity threshold across tissues (general IHC interpretation). |
| Does cellular state affect the compartment? | UniProt places IRF8 in nucleus and cytoplasm, specifying cytoplasmic localisation in resting macrophages and nuclear translocation after IFN-gamma induction (UniProt Q02556). Record cell type and experimental state when interpreting macrophage cytoplasm; HPA's selective nuclear tissue profile remains the reference for routine lymphoid-section scoring (HPA: tissue IHC). |
| How strong is the validation evidence? | HPA lists tissue IHC reliability as Enhanced, while describing only medium consistency between antibody staining and RNA expression (HPA: tissue IHC). Three listed antibodies have Enhanced IHC validation: HPA002267, HPA002531 and CAB013480 (HPA: antibodies). Treat an unexpected pattern as a reason to verify specificity, not as proof of a new expression site. |
| What molecular features guide the compartment check? | IRF8 is a 426-residue protein with no transmembrane segment, signal peptide, propeptide or annotated alternative isoforms in the supplied record (UniProt Q02556 topology and processing). These facts fit an intracellular staining assessment; they do not predict antigen retrieval requirements or fixation sensitivity (UniProt Q02556; source-scope limitation). |
| IF/ICC Q&A: where should fluorescence appear? | HPA reports enhanced nucleoplasmic localisation in ICC-IF (HPA: subcellular). UniProt also reports cytoplasmic IRF8 in resting macrophages and nuclear translocation after IFN-gamma induction (UniProt Q02556). Use cell identity and state when interpreting fluorescence; the ICC-IF localisation record does not define an IHC-P protocol (HPA: subcellular). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive germinal centers are blank. | A failed primary-antibody or detection step is possible; lymph node and tonsil germinal center cells are High in the reference profile (HPA: tissue IHC; general IHC practice). | Repeat with a known-positive section and review the IHC-validated antibody's specified conditions, reagent integrity and chromogenic detection controls before interpreting a negative sample (general IHC practice). |
| Signal is mostly cytoplasmic in lymphoid cells. | This differs from HPA's selective nuclear lymphoid IHC profile; macrophage cytoplasm is a documented state-dependent exception (HPA: tissue IHC; UniProt Q02556). | Identify the stained cells, inspect nuclear boundaries with the counterstain and compare a known-positive lymphoid section; record macrophage activation state when known (general IHC practice; UniProt Q02556). |
| Bronchial respiratory epithelium stains strongly. | Those epithelial cells are Not detected in the HPA tissue profile; cross-reactivity or endogenous chromogenic activity may contribute (HPA: tissue IHC; general IHC practice). | Compare the pattern with a matched no-primary detection control and a known-positive lymphoid section; review blocking and antibody dilution if the unexpected signal persists (general IHC practice). |
| Brown haze covers both positive and expected-negative areas. | Nonspecific background or endogenous detection activity may obscure the selective nuclear pattern (general IHC practice; HPA: tissue IHC). | Check no-primary controls, washing and blocking; adjust chromogenic development and counterstain so individual nuclei remain readable (general IHC practice). |
| A Medium-reference population stains less than germinal centers. | The reference levels differ: bone marrow hematopoietic cells, lung macrophages and splenic red-pulp cells are Medium, while lymph node and tonsil germinal center cells are High (HPA: tissue IHC). | Score the correct cell type against its tissue-specific HPA level before changing assay conditions; compare sections using the same staining run where possible (HPA: tissue IHC; general IHC practice). |
| Staining appears in nearly every cell of a lymphoid section. | Uniform staining conflicts with HPA's selective expression in a subset of lymphoid cells and may reflect background or antibody cross-reactivity (HPA: tissue IHC; general IHC practice). | Check whether stained nuclei belong to the expected population, then compare a no-primary control and an expected-negative cell population before calling the section positive (general IHC practice; HPA: tissue IHC). |
Experiment evidence
IHC & IF images showing IRF8 staining
PA2209 · IHCIHC analysis of IRF8 using anti-IRF8 antibody (PA2209). IRF8 was detected in a paraffin-embedded section of human tonsil tissue. Heat mediat
PA2209 · IHCIHC analysis of IRF8 using anti-IRF8 antibody (PA2209). IRF8 was detected in a paraffin-embedded section of mouse spleen tissue. Heat mediat
PA2209 · IFIF analysis of IRF8 using anti-IRF8 antibody (PA2209) and anti-Tubulin Alpha antibody (M03989-3). IRF8 was detected in immunocytochemical se
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000140968-IRF8/tissue/cerebral+cortex#imid_977384
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000140968-IRF8/tissue/colon#imid_977302
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000140968-IRF8/tissue/liver#imid_977303
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000140968-IRF8/tissue/kidney#imid_977320
HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA002267; green = IRF8, blue = nucleus, red = microtubules.
HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA002267; green = IRF8, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for IRF8 IHC & IF
🧪Run human tonsil first and look for IRF8 staining in germinal center cells (HPA: High in tonsil germinal center cells). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the tonsil slide, treat cells outside the germinal centers as internal comparators only where they are unstained, rather than assuming every such cell is IRF8-negative.
Positive control tissue: Appendix (Lymphoid tissue, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show IRF8 in CACO-2, U-251MG, U2OS, A-431, THP-1, with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a nonimmune control matched to the primary antibody’s rabbit host species, isotype, and monoclonal or polyclonal format; and an IRF8 knockout biological negative (selected IHC caption: rabbit anti-IRF8 antibody; standard IHC controls). For tonsil chromogenic IHC, block endogenous peroxidase before HRP/DAB detection (selected IHC caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected PA2209 paraffin-section caption does not state the fixative (selected IHC caption: fixative unreported). That caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required under other conditions (selected IHC caption: EDTA pH 8.0); the evidence does not establish whether frozen sections or IF would be easier. Tonsil immune cells can contribute endogenous peroxidase signal in HRP/DAB IHC, so interpret staining against the blocked no-primary control (selected IHC caption: tonsil and HRP/DAB; standard IHC practice).
HPA tissue IHC evidence for IRF8
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium 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 | Lymphoid tissue | High | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | High | Protein (IHC) | HPA → |
| Tonsil | Germinal center cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Lung | Macrophages | 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 IRF8 IHC Tips
Troubleshoot IRF8 chromogenic IHC in paraffin sections by checking retrieval, nuclear staining, cell identity, and matched controls (datasheet PA2209; HPA tissue IHC).
What retrieval conditions should I try when IRF8 staining is weak?
Start with heat-mediated EDTA retrieval at pH 8.0 for paraffin-section IRF8 IHC (datasheet PA2209). Include a tonsil section in each retrieval comparison; germinal center cells are a reported high-expression reference (HPA tissue IHC). Keep section thickness, heating equipment, cooling, and antibody incubation constant while adjusting retrieval duration in small steps (standard IHC practice). The selected antibody was used at 2 μg/ml overnight at 4°C, providing a documented starting point after retrieval (datasheet PA2209). If staining remains weak, compare an alternative buffer only as a fallback and assess nuclear signal alongside tissue damage and background (standard IHC practice; HPA tissue IHC).
Could fixation explain weak or uneven IRF8 staining?
Target-specific IRF8 sensitivity to fixation is unknown from the supplied evidence; the selected tonsil caption identifies a paraffin section but does not report its fixative (datasheet PA2209). Record fixative, time in fixative, tissue thickness, and processing history for each specimen before comparing staining (standard IHC practice). For a controlled pilot, process comparable samples under two documented fixation durations while holding retrieval at EDTA pH 8.0 and detection constant (standard IHC practice; datasheet PA2209). Review morphology and staining together, since poor preservation or processing variation can confound an apparent loss of antigen (standard IHC practice). Do not infer an IRF8 fixation requirement from its reported localisation or tissue distribution (UniProt Q02556; HPA tissue IHC).
Should IRF8 staining appear in nuclei or cytoplasm?
Expect selective nuclear staining in a subset of lymphoid cells when evaluating tissue sections (HPA tissue IHC). IRF8 is also reported in the cytoplasm of resting macrophages and translocates to nuclei after IFN-gamma induction, so record cell context before rejecting cytoplasmic signal (UniProt Q02556). Use the counterstain to assign chromogen to individual nuclei, and examine a documented positive compartment such as tonsil germinal center cells (HPA tissue IHC; standard IHC practice). Score nuclear and cytoplasmic staining separately rather than combining them into one intensity value (standard IHC practice). Diffuse signal across every cell compartment warrants review of retrieval, blocking, and detection controls (standard IHC practice).
How do I troubleshoot discordant results between IRF8 antibodies?
The supplied IRF8 record annotates one chain spanning residues 1–426, with no annotated isoforms or glycosylation sites (UniProt Q02556). Those annotations do not identify the catalog antibody's epitope or establish that another antibody recognizes the same accessible region (UniProt Q02556; datasheet PA2209). Compare each antibody on adjacent sections with the same EDTA pH 8.0 retrieval and matched detection settings before attributing disagreement to biology (datasheet PA2209; standard IHC practice). If immunogen information is available, check whether the recognized region could be obscured by tissue processing, then test retrieval systematically (standard IHC practice). Use a second validated reagent and a positive tissue control to assess whether the staining pattern reproduces (standard IHC practice; HPA tissue IHC).
How should I assess IRF8 in a multiplex IF experiment?
Treat IF/ICC as a separate assay: the selected PA2209 tissue caption documents chromogenic staining of a paraffin section, without reporting IF performance or a fixative (datasheet PA2209). Pair IRF8 with a marker identifying the expected cell population, and use a nuclear counterstain to assess colocalisation within cells (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and consider a far-red channel for a weak target when tissue autofluorescence affects shorter wavelengths (standard IF practice). Because IRF8 can localize to nuclei and cytoplasm and has no transmembrane segment, assess permeabilisation for access to intracellular epitopes (UniProt Q02556; standard IF practice). Check single-stain and secondary-only controls before interpreting apparent overlap (standard IF practice).
What should I check when DAB staining is widespread?
First compare a no-primary control and a secondary-only control with the stained section to locate background from the detection system (standard IHC practice). The selected tissue caption used 10% goat serum, a peroxidase-linked secondary, and DAB; these are documented conditions for PA2209 (datasheet PA2209). Check endogenous peroxidase blocking and DAB development time as general chromogenic workflow variables, especially if deposits appear outside plausible cells (standard IHC practice). Titrate primary concentration around the documented 2 μg/ml condition while keeping retrieval at EDTA pH 8.0 (datasheet PA2209; standard IHC practice). Judge improvements against selective lymphoid nuclear staining rather than an overall reduction in color (HPA tissue IHC).
How should I quantify IRF8 staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define tissue regions and eligible cell populations before scoring, since IRF8 staining is selective among lymphoid cells (HPA tissue IHC; standard IHC practice). Report the percentage of positive nuclei using all evaluable nuclei in the chosen population as the denominator, or report positive nuclei per mm² of viable tissue (standard IHC practice). For intensity, calculate an H-score from the percentages at 0, 1+, 2+, and 3+, yielding a 0–300 scale (standard IHC practice). Record cytoplasmic staining separately when evaluating macrophages, whose localisation may vary with activation state (UniProt Q02556; standard IHC practice). Keep retrieval, DAB development, imaging thresholds, and region selection consistent across the comparison (standard IHC practice).
How can I distinguish true IRF8 signal from artefact?
Look for staining in an expected cell population and compartment: tonsil germinal center cells show high expression with selective nuclear staining (HPA tissue IHC). Do not classify every cytoplasmic macrophage signal as artefact, because resting macrophages can contain cytoplasmic IRF8 (UniProt Q02556). Distrust uniform staining across unrelated cells, section-edge intensification, necrotic deposits, or color persisting in a no-primary control (standard IHC practice). Review endogenous peroxidase blocking when DAB appears in cells or structures beyond the expected distribution (standard IHC practice). Interpret a questionable field alongside its morphology, matched control section, and the reported absence of staining in adipocytes of adipose tissue (standard IHC practice; HPA tissue IHC).
Boster reagents
Best IRF8 / Interferon regulatory factor 8 IHC Antibodies
PA2209 has IHC data from human tonsil and mouse spleen paraffin sections (catalog IHC captions), plus IF/ICC data from U2OS cells (catalog IF caption).
Real IHC data
Anti-Interferon regulatory factor 8 IRF8 Antibody ®
Cat # PA2209
PA2209 was tested by IHC on human tonsil and mouse spleen paraffin sections (catalog IHC captions). PA2209 also has IF/ICC data from U2OS cells (catalog IF caption).
Which to pick: Choose PA2209 for paraffin-section IHC: its captions document EDTA retrieval at pH 8.0 and primary antibody at 2 μg/ml (PA2209 IHC captions); the fixative is unreported (PA2209 IHC captions). For IF/ICC, PA2209 has a U2OS-cell image at 5 μg/ml (PA2209 IF caption). For human and mouse tissue work, PA2209 lists reactivity with both species and has IHC images from each (PA2209 catalog; PA2209 IHC captions); clonality is unreported (PA2209 catalog).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q02556 (IRF8_HUMAN, Interferon regulatory factor 8).
- Human Protein Atlas. IRF8 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. IRF8 subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. IRF8 antibody validation summary (3 antibodies).
- High IRF8 expression correlates with CD8 T cell infiltration and is a predictive biomarker of therapy response in ER-negative breast cancer. Breast cancer research : BCR 2021 — PMC7992828.
- IFN regulatory factor 8 is a key constitutive determinant of the morphological and molecular properties of microglia in the CNS. PloS one 2012 — PMC3498170.
- Loss of a Negative Feedback Loop between IRF8 and AR Promotes Prostate Cancer Growth and Enzalutamide Resistance. Cancer research 2020 — PMC9601976.
- DNA methylation represses IFN-gamma-induced and signal transducer and activator of transcription 1-mediated IFN regulatory factor 8 activation in colon carcinoma cells. Molecular cancer research : MCR 2008 — PMC2605678.
- PubMed PMID:1460054 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:10991940 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for IRF8 (Interferon regulatory factor 8). Expected localisation Nuclear in a subset of lymphoid cells (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PA2209). Validated IHC antibody: PA2209 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Lymphoid tissue); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissue; fixative not specified (datasheet PA2209); 2. Fixation: Image fixative and duration unreported (datasheet PA2209); 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 retrieval: EDTA pH 8.0 (datasheet PA2209); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PA2209); 8. Primary antibody: Rabbit anti-IRF8, 2-5μg/ml (datasheet PA2209); 9. Primary incubation: Overnight at 4 °C (datasheet PA2209). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Resting macrophages can retain cytoplasmic IRF8 (UniProt); Isoform / epitope: No annotated isoforms or processing; one 1–426 chain (UniProt); Regulation: IFN-γ induces nuclear translocation (UniProt). Evidence: UniProt Q02556, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoplasm., Datasheet PA2209, PMC7992828, PMC3498170. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/IRF8/immunohistochemistry-ihc-guide.