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IRF5 / Interferon regulatory factor 5 · IHC design guide
Design Immunohistochemistry for IRF5
Plan IRF5 staining in paraffin sections using spleen red-pulp cells as a positive reference (HPA tissue IHC). Start with the catalog antibody at 0.5–1 μg/ml (datasheet PB9646), and assess nuclear staining in the context of activation-linked translocation (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
IRF5 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 | Cytoplasmic staining in selected tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic stain in spleen red-pulp cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB9646) | |
| 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 conditions consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Activation can shift IRF5 into nuclei (UniProt) | |
| Regulation | Expression-level regulation unreported (UniProt) | |
| Isoform / epitope | 6 isoforms; check epitope coverage (UniProt) |
Section 1
Recommended IRF5 IHC & IF Protocols
Start with the catalog antibody’s IHC-P protocol, then compare the published IRF5 IHC methods for HCC and adjacent tissues and spleen (PMC10412693; PMC4969615).
| Sample | Paraffin-embedded mouse spleen tissue; fixative not specified (datasheet PB9646) |
| Fixation | Image fixative and duration unreported (datasheet PB9646); 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 PB9646); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9646) |
| Primary antibody | Rabbit anti-IRF5, 0.5-1μg/ml (datasheet PB9646) |
| Primary incubation | Overnight at 4 °C (datasheet PB9646) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9646) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | IRF5-positive staining in lymphoid tissue of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in selected tissues. No signal in the no-primary control. |
💡Decision noteTry heat-mediated EDTA at pH 8.0 first for the catalog antibody (datasheet: PB9646); use the published retrieval conditions when reproducing the spleen protocol (PMC4969615).
Section 2
What Is the Expected IRF5 Staining Pattern?
IRF5 is a cytoplasmic and nuclear protein that can move into the nucleus after MYD88-dependent activation and phosphorylation (UniProt Q13568: subcellular location). In paraffin-section IHC, expect chiefly cytoplasmic staining in selected tissues, including lymphoid and hematopoietic cell populations reported as High by HPA (HPA: tissue IHC). IRF5 has no transmembrane segment, so a membrane-only pattern is unexpected (UniProt Q13568: topology). HPA rates its tissue IHC profile Enhanced, with medium consistency between staining and RNA data (HPA: tissue IHC).
| Distinct cytoplasmic staining in appendix lymphoid tissue, bone marrow hematopoietic cells, or lymph node non-germinal center cells (HPA: High in each population). | This fits HPA's reported cytoplasmic tissue profile. Compare the stained cells with adjacent structures and a same-run control; a High HPA level identifies a useful reference population, not a required intensity for every section (HPA: tissue IHC; general IHC practice). |
| Nuclear staining appears alongside, or instead of, cytoplasmic staining in otherwise plausible cells (UniProt Q13568: subcellular location). | IRF5 can translocate to the nucleus after MYD88-dependent activation and phosphorylation, so nuclear signal alone does not prove an artefact (UniProt Q13568: subcellular location). Check cell morphology and controls before interpreting activation; HPA's tissue summary describes cytoplasmic expression (HPA: tissue IHC). |
| A sharp membrane-only or extracellular outline dominates the section (UniProt Q13568: topology and subcellular location). | That compartment conflicts with the reported cytoplasmic and nuclear locations and absence of a transmembrane segment (UniProt Q13568). Treat it as suspect staining; assess the detection controls and whether signal follows tissue edges or deposits (general IHC practice). |
| Strong signal appears in adipocytes of adipose tissue or glandular cells of adrenal gland (HPA: Not detected in those cells). | Those cell-specific HPA observations make the result worth checking for cross-reactivity or endogenous detection activity (HPA: tissue IHC; general IHC practice). They do not establish that every cell in either tissue must be negative; inspect morphology and controls before assigning the signal to IRF5. |
| The whole section has diffuse color, or a known HPA-positive cell population has no discernible signal (HPA: tissue IHC). | Diffuse color obscures compartment and cell-type scoring; an absent reference signal leaves the run uninterpretable for IRF5 distribution (general IHC practice). Evaluate background and positive controls separately before calling test cells negative, because HPA reports selected High populations rather than uniform staining (HPA: tissue IHC). |
💡Expected IRF5 appearanceCall a positive IHC result when staining is discernible mainly in the cytoplasm of an HPA-reported High cell population, with possible nuclear signal consistent with activation; diffuse color or membrane-only outlines are suspect (HPA: tissue IHC; UniProt Q13568: location and topology; general IHC practice).
| Activation and compartment (UniProt Q13568: subcellular location) | IRF5 shuttles between cytoplasm and nucleus; MYD88-dependent activation followed by phosphorylation promotes nuclear translocation (UniProt Q13568). Record cytoplasmic and nuclear staining separately when scoring, and use the biological context before assigning meaning to a shift (general IHC practice). |
| Choice of reference cells (HPA: tissue IHC) | HPA reports High staining in appendix lymphoid tissue, bone marrow hematopoietic cells, spleen red-pulp cells, and tonsil germinal-center cells (HPA: tissue IHC). Use the named cell population, not the whole organ, as the comparison; HPA also reports no detection in several specific populations (HPA: tissue IHC). |
| Antibody evidence and isoforms (HPA: antibodies; UniProt Q13568: isoforms) | One listed rabbit polyclonal antibody has Enhanced IHC validation and another has Approved IHC validation (HPA: HPA046700, HPA076024). UniProt lists 6 isoforms, but the supplied record does not map either antibody's epitope or establish isoform-specific staining (UniProt Q13568: isoforms; HPA: antibodies). |
| Processing and antigen retrieval (UniProt Q13568: processing; general IHC practice) | UniProt reports a single 1–498 chain and no signal peptide or propeptide; the record supplies no basis for a cleaved-product staining pattern (UniProt Q13568: processing). Antigen retrieval is a general paraffin-IHC workflow variable, but target-specific fixation sensitivity and retrieval effects are unreported here (general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain in appendix lymphoid tissue or bone marrow hematopoietic cells reported High by HPA (HPA: tissue IHC). | A failed positive reference may reflect an unsuccessful IHC run or an unsuitable assay setting; HPA's staining level does not identify which step failed (HPA: tissue IHC; general IHC practice). | Check the same-run positive control, reagent application, antigen retrieval conditions, and detection sequence under the antibody's IHC-P instructions before interpreting the test section (general IHC practice). |
| Diffuse chromogen makes cellular boundaries and compartments hard to score (general IHC practice). | Background can arise from nonspecific antibody binding or endogenous detection activity; the supplied HPA profile does not diagnose the mechanism in a particular section (HPA: tissue IHC; general IHC practice). | Compare a no-primary control, inspect blocking and washing, and optimize the IHC-validated antibody's working dilution within its instructions; score IRF5 only where cellular staining resolves (general IHC practice). |
| Membrane-only outlines are stronger than cytoplasmic or nuclear signal (UniProt Q13568: topology and location). | IRF5 has no transmembrane segment, and its reported locations are cytoplasm and nucleus, making the outline inconsistent with the supplied target record (UniProt Q13568). | Compare the no-primary control and tissue morphology, then review detection deposits and antibody specificity before recording an IRF5-positive cell (general IHC practice). |
| Nuclear staining seems to conflict with HPA's cytoplasmic tissue summary (HPA: tissue IHC). | IRF5 can enter the nucleus after activation and phosphorylation; HPA's summary does not define the activation state of every cell on a new slide (UniProt Q13568: subcellular location; HPA: tissue IHC). | Score nuclear and cytoplasmic signal separately, verify that staining belongs to intact cells, and interpret a nuclear shift only with appropriate biological context and controls (general IHC practice). |
| Unexpected staining occurs in adipocytes or adrenal glandular cells reported Not detected by HPA (HPA: tissue IHC). | Cross-reactivity or endogenous detection activity is possible, but a negative HPA observation alone cannot establish either cause in the current assay (HPA: tissue IHC; general IHC practice). | Confirm the cell identity, inspect a no-primary control, and compare an HPA-reported High population stained in the same run before accepting the unexpected signal (HPA: tissue IHC; general IHC practice). |
| Can an IF/ICC result be judged against this IHC pattern? (HPA: tissue IHC and subcellular records). | HPA supplies no ICC-IF images or main ICC-IF location for IRF5; its IHC tissue observations do not establish an IF/ICC reference pattern (HPA: subcellular record; HPA: tissue IHC). | Use the separate IF/ICC guide and its own controls for that application. Keep this section's cell-type and intensity expectations tied to paraffin-section IHC (HPA: tissue IHC; general IF/ICC practice). |
Experiment evidence
IHC & IF images showing IRF5 staining
PB9646 · IHCIHC analysis of IRF5 using anti-IRF5 antibody (PB9646). IRF5 was detected in a paraffin-embedded section of mouse spleen tissue. Heat mediat
PB9646 · IHCIHC analysis of IRF5 using anti-IRF5 antibody (PB9646). IRF5 was detected in a paraffin-embedded section of rat spleen tissue. Heat mediated
A00958-1 · IFIF analysis of IRF5 using anti-IRF5 antibody (A00958-1). IRF5 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/ENSG00000128604-IRF5/tissue/cerebral+cortex#imid_13200783
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000128604-IRF5/tissue/colon#imid_13200709
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000128604-IRF5/tissue/liver#imid_13200710
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000128604-IRF5/tissue/kidney#imid_13200703
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for IRF5 IHC & IF
🧪Run spleen first and score IRF5 staining in red-pulp cells (HPA: High in spleen red-pulp cells); the selected antibody caption also shows staining in mouse spleen (caption: PB9646). Use adipose tissue adipocytes as the biological negative (HPA: Not detected in adipocytes); on the spleen slide, cells outside the scored red-pulp population can provide a background reference, but their IRF5-negative status is unreported (HPA: spleen row specifies red-pulp cells only).
Positive control tissue: Appendix (Lymphoid tissue, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for IRF5; derive a cell-line control from the positive tissue's cell type (Lymphoid tissue) and confirm it by RNA or western blot first.
Technical controls: Include no-primary (secondary-only) and primary host- and clonality-matched isotype controls, plus IRF5-knockout tissue as a target-specific negative where available (standard IHC practice; caption: rabbit primary). Quench endogenous peroxidase and check for endogenous biotin background in spleen because the selected assay uses biotin-based detection and DAB (standard IHC practice; caption: SABC and DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state the fixative (caption: fixative unreported). The demonstrated paraffin-section staining used heat-mediated EDTA retrieval at pH 8.0; retrieval dependence beyond that example is unreported (caption: PB9646). Whether frozen sections or IF are easier is unreported; spleen red-pulp background should be checked with the peroxidase and biotin controls used for this detection system (HPA: red-pulp cells; caption: SABC and DAB; standard IHC practice).
HPA tissue IHC evidence for IRF5
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Antibody staining in cells/structures not annotated, view images.). 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 → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Fallopian tube | Ciliated cells (tip of cilia) | High | Protein (IHC) | HPA → |
| Lymph node | Non-germinal center cells | High | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | High | 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 → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced IRF5 IHC Tips
Troubleshoot IRF5 staining in paraffin sections by checking retrieval, compartment, cell identity, background, and scoring against matched controls.
What retrieval should I try when IRF5 staining is weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet PB9646). The catalog antibody’s mouse spleen image used that condition before incubation at 1 μg/ml overnight at 4°C (datasheet PB9646). If staining is weak, compare a longer heating interval with the original interval on adjacent sections, while keeping antibody concentration and detection constant (standard IHC practice). Check whether staining improves in plausible cells without a parallel rise in diffuse tissue background (standard IHC practice). Record heating time, cooling, and section retention so any improvement can be reproduced (standard IHC practice).
Could fixation explain weak or uneven IRF5 staining?
Target-specific fixation sensitivity is unknown because the selected paraffin-section caption does not state a fixative (datasheet PB9646). Compare sections with documented fixation histories using the same EDTA pH 8.0 retrieval and 1 μg/ml primary concentration before changing antibody conditions (datasheet PB9646; standard IHC practice). Uneven fixation can complicate antigen access and staining comparisons, so inspect tissue preservation and compare equivalent cell populations away from damaged edges (standard IHC practice). Include a same-run positive control to distinguish a processing problem from weak staining in the study section (standard IHC practice). Do not assign a fixation effect to IRF5 from tissue expression patterns alone (HPA tissue IHC).
How should I assess cytoplasmic versus nuclear IRF5 staining?
Score cytoplasmic and nuclear signal separately: IRF5 shuttles between these compartments and can enter the nucleus after MYD88-dependent activation and phosphorylation (UniProt Q13568). Predominantly cytoplasmic tissue staining is reported, so cytoplasmic signal alone does not establish pathway inactivity (HPA tissue IHC; UniProt Q13568). Inspect the counterstain at high magnification and require chromogen within a clearly bounded nucleus before calling a cell nuclear-positive (standard IHC practice). Compare nuclear fractions across matched cell populations and processing batches, using the same exposure and scoring rules (standard IHC practice). A nuclear shift is consistent with activation but does not independently prove transcriptional output (UniProt Q13568).
Could splice variants or epitope accessibility change the pattern?
IRF5 has 6 annotated isoforms, so an antibody’s recognized sequence matters when interpreting differences among samples (UniProt Q13568). Check the catalog antibody’s stated immunogen or epitope against the isoforms before claiming that a stained or unstained population expresses every variant (standard IHC practice; UniProt Q13568). IRF5 also has annotated phosphorylation sites, including TBK1-associated residues 158 and 293, but the supplied IHC caption does not establish phospho-specific detection (UniProt Q13568; datasheet PB9646). Keep retrieval and detection fixed while comparing sections, and interpret a changed pattern as staining behavior until epitope coverage and specificity are established (standard IHC practice).
How can I examine IRF5 and cell identity by IF?
For a separate IF experiment, pair IRF5 with a validated marker for the cell population being examined; spleen red-pulp cells and bone-marrow hematopoietic cells are high-staining IHC populations (HPA tissue IHC; standard IF practice). Choose fluorophores after checking unstained tissue for autofluorescence, and reserve a well-separated channel for the weaker signal (standard IF practice). IRF5 has no transmembrane segment and is reported in cytoplasm and nucleus, so use permeabilisation compatible with access to intracellular epitopes and preservation of nuclear boundaries (UniProt Q13568; standard IF practice). Include single-stain and secondary-only controls to assess bleed-through and nonspecific fluorescence before comparing compartments (standard IF practice).
How can I reduce diffuse brown staining without losing IRF5 signal?
First compare a no-primary section with the stained section to identify signal from detection reagents or endogenous enzyme activity (standard IHC practice). The catalog image used 10% goat serum, a biotinylated secondary, a streptavidin–biotin complex, and DAB, so background from that detection route warrants a matched control (datasheet PB9646; standard IHC practice). For an enzymatic IHC workflow, check peroxidase blocking and shorten chromogen development if the no-primary control is brown (standard IHC practice). Then titrate primary antibody around the reported 1 μg/ml condition, comparing expected cellular staining with adjacent background under identical development conditions (datasheet PB9646; standard IHC practice).
What is a reproducible way to quantify IRF5 IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because IRF5 can occupy cytoplasm or nucleus (UniProt Q13568; standard IHC practice). For comparable sections, report the percentage of positive cells or an H-score from intensity categories, and give nuclear-positive and cytoplasmic-positive results separately (standard IHC practice). If the question concerns infiltrating cells, report positive-cell density per mm² of viable tissue and normalize counts to the relevant cell population or sampled area (standard IHC practice). Apply one positivity threshold across batches, exclude folds and necrotic regions, and document fields sampled (standard IHC practice). Interpret between-sample differences alongside cell composition, since high staining occurs in several hematopoietic compartments (HPA tissue IHC).
Which staining patterns would make an IRF5 result doubtful?
A credible result should occupy identifiable cells and a plausible cytoplasmic or nuclear compartment, with any nuclear shift interpreted in the context of IRF5 activation (UniProt Q13568; standard IHC practice). Compare the pattern with known high-staining populations, such as spleen red-pulp cells, and with a no-primary control (HPA tissue IHC; standard IHC practice). Be cautious when brown signal follows section edges, necrosis, or damaged areas, or appears equally in the no-primary control; these patterns suggest processing or detection artefact (standard IHC practice). Endogenous enzyme signal can also mimic DAB positivity, so verify blocking and inspect cellular boundaries before calling a result positive (standard IHC practice).
Boster reagents
Best IRF5 / Interferon regulatory factor 5 IHC Antibodies
Anti-IRF5 IHC images cover paraffin sections of mouse and rat spleen and human mammary cancer (PB9646 image captions); IF imaging covers A549 cells (A00958-1 image caption).
Real IHC data
Anti-IRF5 Antibody ®
Cat # PB9646
Real IF data
Anti-IRF5 Antibody ®
Cat # A00958-1
PB9646 renders with mouse spleen IHC; additional captions show rat spleen and human mammary cancer paraffin sections (PB9646 image captions). A00958-1 renders with IF in A549 cells (A00958-1 image caption).
Which to pick: Choose PB9646 for paraffin-section tissue IHC at 0.5–1 μg/ml (PB9646 catalog IHC dilution); its image captions do not report a fixative (PB9646 image captions). Choose A00958-1 for IF/ICC in human cells at 5 μg/ml (A00958-1 catalog application and dilution listing), supported by its A549 IF image (A00958-1 image caption). For cross-species tissue IHC, PB9646 has direct images from human, mouse and rat samples (PB9646 image captions); both rendered antibodies are rabbit antibodies with no clone listed (catalog host and clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q13568 (IRF5_HUMAN, Interferon regulatory factor 5).
- Human Protein Atlas. IRF5 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. IRF5 subcellular location (ICC-IF): Highest expression in HDLM-2: 112.9 nTPM.
- Human Protein Atlas. IRF5 antibody validation summary (2 antibodies).
- Dysregulation of PLOD2 Promotes Tumor Metastasis and Invasion in Hepatocellular Carcinoma. Journal of clinical and translational hepatology 2023 — PMC10412693.
- Interferon Regulator Factor 5: A Novel Inflammatory Marker and Promising Therapeutic Target in Ulcerative Colitis. Biomedicines 2025 — PMC12467263.
- Specific detection of interferon regulatory factor 5 (IRF5): A case of antibody inequality. Scientific reports 2016 — PMC4969615.
- Securidaca inappendiculata-Derived Xanthones Protected Joints from Degradation in Male Rats with Collagen-Induced Arthritis by Regulating PPAR-γ Signaling. Journal of inflammation research 2021 — PMC7896782.
- PubMed PMID:11303025 — UniProt-cited evidence.
- PubMed PMID:15695821 — UniProt-cited evidence.
- PubMed PMID:15805103 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for IRF5 (Interferon regulatory factor 5). Expected localisation Cytoplasmic staining in selected tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9646). Validated IHC antibody: PB9646 at 0.5-1μg/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 mouse spleen tissue; fixative not specified (datasheet PB9646); 2. Fixation: Image fixative and duration unreported (datasheet PB9646); 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 PB9646); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9646); 8. Primary antibody: Rabbit anti-IRF5, 0.5-1μg/ml (datasheet PB9646); 9. Primary incubation: Overnight at 4 °C (datasheet PB9646). Watch for: Fixation: Keep fixation conditions consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Activation can shift IRF5 into nuclei (UniProt); Isoform / epitope: 6 isoforms; check epitope coverage (UniProt); Regulation: Expression-level regulation unreported (UniProt). Evidence: UniProt Q13568, HPA tissue IHC · Enhanced, HPA cell-line RNA · Highest expression in HDLM-2: 112.9 nTPM, Datasheet PB9646, PMC10412693, PMC4969615. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/IRF5/immunohistochemistry-ihc-guide.