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IRF1 / Interferon regulatory factor 1 · IHC design guide
Design Immunohistochemistry for IRF1
Plan chromogenic paraffin-section IHC for IRF1 using the catalog antibody’s tissue staining evidence (datasheet A00580-1). Assess nuclear and nucleolar staining, with placental endothelial cells and testis pachytene spermatocytes as high-staining references (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
IRF1 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 and nucleolar tissue staining (HPA tissue IHC) | |
| Staining pattern | Nuclear/nucleolar; high in placental endothelial cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A00580-1) | |
| Positive control | Placenta+4 more · see all | |
| Negative control | Cerebellum+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across samples. (standard IHC practice; not target-specific) | |
| Caveat | MYD88 association can increase nuclear migration (UniProt) | |
| Regulation | No intensity regulator specified (UniProt) | |
| Isoform / epitope | No isoforms or processing variants annotated (UniProt) |
Section 1
Recommended IRF1 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published IRF1 staining methods for breast tissue (PMC4359953), human and mouse tissue (PMC13472745), and gastric tissue (PMC12799679).
| Sample | Paraffin-embedded human Lung cancer tissue; fixative not specified (datasheet A00580-1) |
| Fixation | Image fixative and duration unreported (datasheet A00580-1); 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 A00580-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A00580-1) |
| Primary antibody | Rabbit anti-IRF1, 0.5-1μg/ml (datasheet A00580-1) |
| Primary incubation | Overnight at 4 °C (datasheet A00580-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A00580-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | IRF1-positive staining in endothelial cells of placenta (HPA tissue IHC: High). HPA tissue profile: Nuclear and nucleolar expression in several different tissue types. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet: A00580-1); the published gastric protocol reports retrieval without specifying its conditions (PMC12799679).
Section 2
What Is the Expected IRF1 Staining Pattern?
IRF1 is expected mainly in nuclei, with nuclear and nucleolar staining reported across several tissues (HPA tissue IHC). Cytoplasmic localization is also recorded (UniProt P10914; HPA subcellular ICC-IF). For IHC controls, placental endothelial cells and testicular pachytene spermatocytes show high staining (HPA tissue IHC). The tissue pattern has Supported reliability, with medium consistency between staining and RNA data (HPA tissue IHC). IRF1 has no transmembrane segment (UniProt P10914 topology).
| Strong nuclear staining in placental endothelial cells or testicular pachytene spermatocytes (HPA tissue IHC). | This matches HPA's high-staining cell populations. Nuclear and nucleolar staining is reported in tissue IHC (HPA tissue IHC). Judge the named cells, rather than every cell in the section, when assessing a positive control. |
| Predominantly cytoplasmic staining with little nuclear staining in an otherwise strong section. | This differs from the reported nuclear and nucleolar tissue pattern (HPA tissue IHC). IRF1 can also be cytoplasmic (UniProt P10914), so compartment alone cannot prove artefact. Check the pattern against a known-positive section and detection controls (standard IHC practice). |
| Strong staining in a cell population listed as not detected by HPA. | For example, HPA did not detect staining in liver cholangiocytes or lymph-node germinal-center cells (HPA tissue IHC). Unexpected signal warrants checks for cross-reactivity or endogenous detection activity (standard IHC practice); an HPA 'not detected' entry is cell-specific, not a claim about the entire tissue. |
| Diffuse color over tissue and empty areas, with weak cellular boundaries. | This is difficult to interpret as the reported nuclear and nucleolar pattern (HPA tissue IHC). Excess primary antibody, incomplete blocking, inadequate washing, or detection background are general possibilities; assess a no-primary control (standard IHC practice). |
| No visible staining in placental endothelial cells or testicular pachytene spermatocytes. | Both are reported as high-staining populations (HPA tissue IHC), so a blank result raises a workflow concern. Confirm that those cells are present, then review retrieval, antibody dilution, detection, and counterstain against the assay controls (standard IHC practice). |
💡Expected IRF1 appearanceCall a control positive when the expected cell population shows clear nuclear, potentially nucleolar, staining—high in placental endothelial cells or testicular pachytene spermatocytes (HPA tissue IHC); diffuse color without a cell-specific pattern is suspect (standard IHC practice).
| Cell population and tissue | HPA reports high staining in placental endothelial cells and testicular pachytene spermatocytes, medium staining in several other listed populations, and no detection in specific cells such as liver cholangiocytes (HPA tissue IHC). Select and score controls by cell type. |
| Compartment | Tissue IHC reports nuclear and nucleolar expression (HPA tissue IHC). UniProt also lists cytoplasm and notes more efficient nuclear migration for MYD88-associated IRF1 (UniProt P10914). Cytoplasmic signal therefore needs context; it does not by itself establish specificity. |
| Validation limits | HPA rates the tissue pattern Supported and describes medium consistency with RNA expression (HPA tissue IHC). Its listed IHC antibodies HPA063131 and CAB011662 are Supported; that status supports interpretation but does not validate every antibody or staining condition (HPA antibodies; standard IHC practice). |
| Isoforms and processing | UniProt annotates no isoforms, signal peptide, or propeptide and identifies one chain spanning residues 1–325 (UniProt P10914). The supplied record provides no basis for predicting a distinct compartment-specific isoform or cleavage-product staining pattern. |
| IF/ICC Q&A | Q: What should IF/ICC show? A: Mainly nucleoplasmic staining, with additional cytosolic localization (HPA subcellular ICC-IF). This is a cross-check for localization; IF/ICC assay design belongs on its own guide page. |
| Fixation evidence | Target-specific fixation sensitivity is unreported in the supplied UniProt and HPA evidence. If staining fails, assess retrieval and detection as general IHC workflow variables (standard IHC practice), without attributing the result to a demonstrated IRF1 fixation effect. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive cells are blank. | The expected cells may be absent from the section, or retrieval, antibody concentration, or detection may be inadequate (HPA tissue IHC; standard IHC practice). | Locate placental endothelial cells or testicular pachytene spermatocytes, then verify the antibody's IHC conditions, retrieval step, and detection controls (HPA tissue IHC; standard IHC practice). |
| Most color is cytoplasmic. | IRF1 can occur in cytoplasm, but tissue IHC describes nuclear and nucleolar staining (UniProt P10914; HPA tissue IHC). The observed pattern needs a specificity check. | Compare nuclear and cytoplasmic signal in a known-positive cell population; inspect a no-primary control for detection background (HPA tissue IHC; standard IHC practice). |
| Unexpected cells stain strongly. | Cross-reactivity or endogenous detection activity is possible (standard IHC practice). HPA 'not detected' calls apply to the named cells, not every cell in the tissue (HPA tissue IHC). | Identify the stained cell type, compare its HPA entry, and examine no-primary and detection controls before assigning IRF1 positivity (HPA tissue IHC; standard IHC practice). |
| Color is diffuse across cells or empty areas. | Non-specific antibody binding, incomplete blocking, insufficient washing, or detection background can obscure the reported cellular pattern (standard IHC practice; HPA tissue IHC). | Inspect a no-primary control; review blocking, washing, primary-antibody dilution, and chromogen development using the assay instructions (standard IHC practice). |
| A presumed negative tissue shows focal staining. | A tissue-wide negative assumption may be wrong: HPA reports staining by cell population, including cells scored not detected within a tissue (HPA tissue IHC). | Match each stained cell to the named HPA population and use a cell-specific comparator; investigate signal outside that population with controls (HPA tissue IHC; standard IHC practice). |
| Nuclear staining is hard to distinguish from counterstain. | Heavy counterstain or weak chromogen can make nuclear localization difficult to score (standard IHC practice). | Review chromogen and counterstain balance alongside a known-positive section, and score only nuclei with distinguishable specific signal (standard IHC practice; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing IRF1 staining
A00580-1 · IHCIHC analysis of IRF1 using anti-IRF1 antibody (A00580-1). IRF1 was detected in paraffin-embedded section of human Lung cancer tissue. Heat m
A00580-1 · IHCIHC analysis of IRF1 using anti-IRF1 antibody (A00580-1). IRF1 was detected in paraffin-embedded section of mouse intestine tissue. Heat med
A00580-1 · IFIF analysis of IRF1 using anti-IRF1 antibody (A00580-1). IRF1 was detected in immunocytochemical section of A431 cells. Enzyme antigen retri
M00580 · IHCImmunohistochemical analysis of paraffin-embedded rat kidney, using IRF1 Antibody.
M00580 · IFImmunofluorescent analysis of Hela cells, using IRF1 Antibody .
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000125347-IRF1/tissue/cerebral+cortex#imid_3056912
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000125347-IRF1/tissue/colon#imid_3051878
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000125347-IRF1/tissue/liver#imid_3051863
HPA · GallbladderHuman Protein Atlas IHC, Gallbladder. Source: https://www.proteinatlas.org/ENSG00000125347-IRF1/tissue/gallbladder#imid_3051855
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB011662; green = IRF1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB011662; green = IRF1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for IRF1 IHC & IF
🧪Run placenta first and look for staining in endothelial cells (HPA: High in placental endothelial cells). Use cerebellum as the negative tissue and assess its granular-layer cells (HPA: Not detected in cerebellar granular-layer cells); on the placenta slide, compare endothelial staining with background in cells outside the identified endothelial compartment, without assuming those cells are biologically negative (HPA: High in placental endothelial cells).
Positive control tissue: Placenta (Endothelial cells, HPA High)
Negative control tissue: Cerebellum (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show IRF1 in A-431, U-251MG, HaCaT, RT-4, U2OS, with annotated localisation: Nucleoplasm (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a rabbit immunoglobulin-matched isotype control (caption: rabbit anti-IRF1 primary and goat anti-rabbit secondary). A validated IRF1 knockout sample can provide a biological specificity control (standard IHC practice). Check endogenous peroxidase and biotin background in placenta when using the caption’s biotin-based, DAB detection system (caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected SKU’s paraffin-section caption does not state the fixative (caption: fixative not stated). The demonstrated IHC procedure uses heat retrieval in EDTA at pH 8.0, so start with that retrieval condition (caption: EDTA pH 8.0); no supplied evidence establishes whether frozen sections or IF are easier. For placenta, verify that any vascular signal exceeds endogenous peroxidase or biotin background before scoring endothelial cells (caption: biotinylated secondary, SABC and DAB; HPA: High in placental endothelial cells).
HPA tissue IHC evidence for IRF1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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 |
|---|---|---|---|---|
| Placenta | Endothelial cells | High | Protein (IHC) | HPA → |
| Testis | Pachytene spermatocytes | High | Protein (IHC) | HPA → |
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Prostate | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced IRF1 IHC Tips
Troubleshoot IRF1 staining in paraffin sections by checking retrieval, cell type and nuclear signal before comparing chromogenic scores across samples.
What should I adjust when IRF1 staining is weak after antigen retrieval?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A00580-1). The caption does not specify heating time or fixative, so compare adjacent sections using a controlled retrieval-time series while keeping cooling and detection conditions constant (datasheet A00580-1; standard IHC practice). Judge recovery by nuclear staining in an expected positive cell population, such as placental endothelial cells, alongside a no-primary control (HPA: High in placental endothelial cells; standard IHC practice). If staining remains weak, assess an alternative retrieval buffer as a fallback, changing only one condition at a time (standard IHC practice).
Can fixation explain weak or uneven IRF1 staining in paraffin sections?
Target-specific sensitivity of IRF1 staining to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (datasheet A00580-1). Record fixation conditions for each specimen and compare sections processed together before attributing a weak result to fixation (standard IHC practice). Check whether uneven staining follows tissue edges or poorly preserved regions, then repeat staining on a well-preserved section with the same EDTA pH 8.0 retrieval and detection settings (datasheet A00580-1; standard IHC practice). Do not infer a fixation effect from the reported tissue staining pattern or IRF1’s annotated protein features (HPA: tissue IHC profile; UniProt P10914).
Should I count cytoplasmic IRF1 staining as a positive result?
Score nuclear staining separately because IRF1 is annotated in the nucleus and cytoplasm, and tissue IHC reports nuclear and nucleolar expression (UniProt P10914; HPA: tissue IHC profile). Cytoplasmic staining is biologically plausible, with nucleoplasm listed as the main location and cytosol as an additional location in cell imaging (HPA: subcellular profile). However, diffuse cytoplasmic color alone should prompt comparison with nuclear signal, morphology and a no-primary control before it is scored as specific (standard IHC practice). Report the proportion of positive nuclei and cytoplasmic staining separately; MYD88 association can affect nuclear migration, but staining alone cannot establish that mechanism (UniProt P10914).
Could an unrecognized epitope or isoform explain discordant IRF1 staining?
The supplied record lists 0 annotated isoforms and one IRF1 chain spanning residues 1–325; it provides no antibody epitope map (UniProt P10914). An annotated N6-acetyllysine occurs at residue 78, but its effect on this antibody’s tissue staining cannot be determined without an epitope assignment (UniProt P10914). If staining differs between preparations, compare matched sections under the same EDTA pH 8.0 retrieval and detection conditions, then assess whether signal follows expected cellular compartments (datasheet A00580-1; UniProt P10914; standard IHC practice). Do not assign a staining discrepancy to a particular isoform or modification from chromogenic appearance alone (UniProt P10914; standard IHC practice).
How can IF help resolve ambiguous IRF1 staining in tissue sections?
Use IF as a separate localisation check while retaining chromogenic paraffin-section IHC as this page’s primary assay (standard IHC/IF practice). For multiplexing, pair IRF1 with an endothelial-cell marker in placenta, where endothelial IRF1 staining is reported as High, and include a nuclear counterstain (HPA: High in placental endothelial cells; standard IF practice). Choose fluorophores after examining unstained tissue for autofluorescence, and reserve a spectrally distinct channel for the expected nuclear signal (UniProt P10914 localisation; standard IF practice). Permeabilise sufficiently for antibody access to the nuclear epitope, because IRF1 has no annotated transmembrane segment; validate IF staining independently since the selected caption documents IHC only (UniProt P10914 topology; datasheet A00580-1; standard IF practice).
How do I reduce diffuse brown background without losing nuclear IRF1?
Begin with the caption’s 10% goat-serum block and 1 μg/ml primary antibody incubated overnight at 4°C as a documented IHC starting point (datasheet A00580-1). Compare a no-primary section and titrate the primary antibody if diffuse color persists while retaining a positive tissue control (standard IHC practice). Because the documented detection uses a biotinylated secondary, streptavidin-biotin complex and DAB, check endogenous peroxidase and endogenous biotin contributions with appropriate blocking and controls (datasheet A00580-1; standard IHC practice). Keep washes and chromogen development consistent, and inspect whether residual color concentrates at folds, edges or damaged tissue rather than within nuclei (standard IHC practice).
How should I quantify IRF1 when cell types and staining intensity vary? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, since tissue IHC reports nuclear and nucleolar IRF1 across several tissues and cell types (HPA: tissue IHC profile). For nuclear staining, record percent positive cells and intensity, then calculate an H-score from intensity categories 0–3 and their percentages, yielding 0–300 (standard IHC practice). Alternatively, report positive-cell density per mm² when the question concerns the abundance of IRF1-positive cells within a defined region (standard IHC practice). Normalise to the number of eligible cells or analysed tissue area, exclude damaged regions consistently, and compare sections stained in the same run (standard IHC practice).
What distinguishes credible IRF1 positivity from chromogenic staining artefact?
Credible staining should occur in morphologically intact cells and include nuclear signal where expected; placental endothelial cells and testicular pachytene spermatocytes are reported as High (HPA: tissue IHC profile). Cytoplasmic signal alone is not automatically false because cytosolic IRF1 is annotated, but it needs compartment-specific reporting and control comparison (UniProt P10914; HPA: subcellular profile; standard IHC practice). Treat staining restricted to section edges, necrotic areas or folds as suspect, and use a no-primary section to assess nonspecific detection (standard IHC practice). Investigate endogenous peroxidase or biotin if brown signal survives that control, given the documented streptavidin-biotin and DAB workflow (datasheet A00580-1; standard IHC practice).
Boster reagents
Best IRF1 / Interferon regulatory factor 1 IHC Antibodies
IHC images show IRF1 staining in human lung cancer, mouse intestine, and rat kidney paraffin sections; IF images show staining in A431 and Hela cells (catalog image captions).
Real IHC data
Anti-IRF1 Antibody ®
Cat # A00580-1
Real IHC data
Anti-IRF1 Rabbit Monoclonal Antibody
Cat # M00580
A00580-1 has IHC images from human lung cancer and mouse intestine paraffin sections and an IF image from A431 cells (A00580-1 image captions). M00580 has an IHC image from a rat kidney paraffin section and an IF image from Hela cells (M00580 image captions).
Which to pick: For human or mouse tissue IHC, choose A00580-1 because its own captions document both species in paraffin sections; choose M00580 for rat tissue IHC, as shown in its rat kidney paraffin-section caption (A00580-1 and M00580 IHC image captions). For IF/ICC, both list those applications, with A00580-1 shown in A431 cells and M00580 shown in Hela cells; M00580 is a rabbit monoclonal and lists human, mouse, and rat reactivity, making it the broader cross-species option (catalog applications, reactivity, clone, and IF image captions). Neither IHC caption reports the fixative (A00580-1 and M00580 IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P10914 (IRF1_HUMAN, Interferon regulatory factor 1).
- Human Protein Atlas. IRF1 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. IRF1 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol..
- Human Protein Atlas. IRF1 antibody validation summary (3 antibodies).
- Interferon regulatory factor-1 signaling regulates the switch between autophagy and apoptosis to determine breast cancer cell fate. Cancer research 2015 — PMC4359953.
- A novel role of the TNF-α/p65/IRF1 axis in aggravating rheumatoid arthritis through the induction of fibroblast-like synoviocytes pyroptosis. Frontiers in pharmacology 2026 — PMC13472745.
- IRF1 inhibits the proliferation and metastasis of colorectal cancer by suppressing the RAS-RAC1 pathway. Cancer management and research 2019 — PMC6317468.
- IRF1 suppresses gastric tumorigenesis via dual PI3K/AKT-ERK pathway modulation and functional antagonism of oncogenic MX2. Cellular oncology (Dordrecht, Netherlands) 2026 — PMC12799679.
- PubMed PMID:2726461 — UniProt-cited evidence.
- PubMed PMID:3409321 — UniProt-cited evidence.
- PubMed PMID:1382447 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for IRF1 (Interferon regulatory factor 1). Expected localisation Nuclear and nucleolar tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A00580-1). Validated IHC antibody: A00580-1 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Placenta (Endothelial cells); negative Cerebellum. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human Lung cancer tissue; fixative not specified (datasheet A00580-1); 2. Fixation: Image fixative and duration unreported (datasheet A00580-1); 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 A00580-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A00580-1); 8. Primary antibody: Rabbit anti-IRF1, 0.5-1μg/ml (datasheet A00580-1); 9. Primary incubation: Overnight at 4 °C (datasheet A00580-1). Watch for: Fixation: Keep fixation consistent across samples. (standard IHC practice; not target-specific); Caveat: MYD88 association can increase nuclear migration (UniProt); Isoform / epitope: No isoforms or processing variants annotated (UniProt); Regulation: No intensity regulator specified (UniProt). Evidence: UniProt P10914, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A00580-1, PMC4359953, PMC13472745. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/IRF1/immunohistochemistry-ihc-guide.