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- Table of Contents
Plan paraffin-section IRAK1 staining with the catalog antibody at 0.5–1 μg/mL (datasheet A01021-1). Compare cytoplasmic signal across cell types (HPA tissue IHC) and score staining against control sections (standard IHC practice).
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
| Expected localisation | Cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Widespread cytoplasmic staining across cell types (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A01021-1) | |
| Positive control | Breast+4 more · see all | |
| Negative control | Adipose tissue+2 more · see all |
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Adipocytes and myocytes may lack staining (HPA tissue IHC) | |
| Regulation | Expression induction is unreported (UniProt) | |
| Isoform / epitope | 4 isoforms; epitope coverage is unspecified (UniProt) |
Compare the catalog antibody’s IHC-P protocol (datasheet: A01021-1) with three published IRAK1 tissue IHC methods (PMC5367901; PMC12521082; PMC6438138).
| Sample | Paraffin-embedded human Ovarian cancer tissues; fixative not specified (datasheet A01021-1) |
| Fixation | Image fixative and duration unreported (datasheet A01021-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet A01021-1) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01021-1) |
| Primary antibody | Rabbit anti-IRAK1, 0.5-1μg/ml (datasheet A01021-1) |
| Primary incubation | Overnight at 4 °C (datasheet A01021-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A01021-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | IRAK1-positive staining in glandular cells of breast (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression. No signal in the no-primary control. |
IRAK1 should show predominantly cytoplasmic staining in many cell types (HPA: ubiquitous cytoplasmic expression; IHC reliability Supported, with medium consistency against RNA data). Strong examples include breast and colon glandular cells, cortical neurons, and placental trophoblastic cells (HPA: High). Nuclear localization can occur under specific conditions (UniProt P51617: nuclear translocation when sumoylated). A membrane rim is not the expected pattern (UniProt P51617: no transmembrane segment).
| Cytoplasmic signal in breast or colon glandular cells, cortical neurons, or placental trophoblastic cells. | This matches the main compartment and cell populations reported for IRAK1 (HPA: ubiquitous cytoplasmic expression; High in these cells). Compare cells within the same section because widespread expression does not mean equal intensity in every cell (HPA: variable tissue and cell staining levels). |
| Predominantly sharp cell-surface rims, with little cytoplasmic signal. | A membrane-rim pattern conflicts with the reported cytoplasmic pattern and lack of a transmembrane segment (HPA: cytoplasmic expression; UniProt P51617 topology). Review morphology and detection background before assigning it to IRAK1 (general IHC practice). |
| Strong signal in adipocytes, skeletal myocytes, or smooth muscle cells. | These cells are reported as not detected in tissue IHC (HPA: adipocytes, skeletal myocytes, and smooth muscle cells Not detected). Cross-reactivity or endogenous detection activity is possible; inspect negative controls and nearby cell types before interpreting the signal (general IHC practice). |
| Broad, poorly bounded color over tissue and empty areas. | Diffuse deposition cannot establish cellular IRAK1 localization (general IHC practice). Check the no-primary control, blocking, washes, and chromogenic detection steps; excess background can obscure the cytoplasmic pattern reported by HPA (HPA: ubiquitous cytoplasmic expression). |
| No signal in an adequately preserved breast or colon glandular-cell region. | That conflicts with the High staining reported for those cells (HPA: breast and colon glandular cells High). First assess whether the IHC-validated antibody, detection reagents, and retrieval conditions worked on a control section (general IHC practice); one absent stain does not establish absent IRAK1. |
| Compartment and activation state | Cytoplasm is the principal tissue-IHC expectation (HPA: ubiquitous cytoplasmic expression). Nuclear staining has biological precedent when IRAK1 is sumoylated (UniProt P51617: nuclear translocation), but nuclear color alone does not demonstrate that modification (general IHC interpretation). |
| Cell population | Tissue context affects the expected intensity: ovarian follicle cells and pancreatic exocrine glandular cells are High, while lung macrophages and kidney glomerular cells are Low (HPA: tissue IHC). Avoid scoring all cell types against one intensity threshold (general IHC practice). |
| Isoform coverage | Isoforms 1 and 2 occur in all tissues examined, with isoform 1 more strongly expressed (UniProt P51617: tissue specificity). Four isoforms are listed (UniProt P51617: isoforms 1–4). The payload does not establish which isoforms the IHC antibodies recognize. |
| Antibody evidence | HPA marks IHC staining as Supported for HPA054476 and CAB004461 (HPA: antibody validation). Overall tissue reliability is Supported with medium agreement to RNA (HPA: tissue IHC). Treat these classifications as support for interpretation, not proof that every positive cell is specific. |
| Antigen retrieval | Retrieval conditions are a routine IHC optimization variable (general IHC practice). Neither HPA nor UniProt supplies an IRAK1-specific retrieval condition or fixation-sensitivity result here; compare a known-positive section when optimizing, without predicting a target-specific fixation effect. |
| IF/ICC Q: Where should IRAK1 appear? | A: Mainly in the cytosol, with additional nucleoplasmic localization (HPA: ICC-IF, both supported). HPA lists ICC-IF images for MCF-7, U-251MG, U2OS, RPTEC/TERT1, and serum-starved hTERT-RPE1 (HPA: subcellular). This is a localization reference, not an IF protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive glandular cells remain unstained. | An unsuccessful antibody, retrieval, or detection step is possible (general IHC practice); breast and colon glandular cells are reported High (HPA: tissue IHC). | Run the IHC-validated antibody with a positive control section; check retrieval, dilution, and detection against its validated IHC-P instructions (general IHC practice). Do not infer tissue absence from a failed control. |
| Most tissue structures carry diffuse chromogen. | Background from insufficient blocking or washing, excessive antibody concentration, or endogenous detection activity is possible (general IHC practice). | Review no-primary and detection controls, blocking and washes; adjust dilution using the validated IHC-P instructions (general IHC practice). Reassess whether cytoplasmic signal remains in expected cells (HPA: cytoplasmic expression). |
| Staining forms a dominant membrane rim. | This disagrees with the reported cytoplasmic distribution and lack of a transmembrane segment (HPA: cytoplasmic expression; UniProt P51617 topology). | Compare with the no-primary control and tissue morphology; repeat with an IHC-supported antibody if needed (general IHC practice; HPA: HPA054476 and CAB004461 IHC Supported). |
| Adipocytes or muscle cells stain strongly. | Those populations are reported as Not detected, so nonspecific staining or endogenous detection activity is possible (HPA: tissue IHC; general IHC practice). | Check negative controls and the chromogenic detection steps, then compare staining in an expected High population on the run (general IHC practice; HPA: breast glandular cells High). |
| Nuclear staining appears alongside cytoplasmic staining. | Nuclear IRAK1 is plausible under specified conditions (UniProt P51617: sumoylation-associated translocation); HPA also reports supported nucleoplasmic localization in ICC-IF (HPA: subcellular). | Record nuclear and cytoplasmic scores separately and check controls (general IHC practice). Do not call nuclear IHC staining evidence of sumoylation without independent evidence (UniProt P51617: conditional localization). |
| A Low population is weaker than a High population. | The difference can reflect reported cell-specific staining levels: lung macrophages and kidney glomerular cells are Low, while ovarian follicle cells are High (HPA: tissue IHC). | Score each cell population against its reported level and inspect a High control before changing staining conditions (HPA: tissue IHC; general IHC practice). |
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.
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Cerebellum | Processes in granular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | High | Protein (IHC) | HPA → |
Troubleshoot IRAK1 staining in paraffin sections using the catalog antibody’s tissue IHC conditions, with IF/ICC considerations kept to a separate entry.
The catalog shows IRAK1 IHC images from human ovarian cancer paraffin sections and rat liver, and IF images from A549 and HeLa cells (catalog figure captions).
A01021-1 has an IHC image of human ovarian cancer paraffin sections, while M01021-1 has an IHC image of rat liver (respective IHC captions). A01021 has an IF image of A549 cells, and A01021-2 has an IF image of paraformaldehyde-fixed HeLa cells (respective IF captions).
Which to pick: For human tissue IHC, choose polyclonal A01021-1: its paraffin-section image uses citrate retrieval at pH 6, but the fixative is unreported (A01021-1 catalog and IHC caption); monoclonal M01021-1 is another IHC-P option with a rat-liver image, although its listed reactivity is Human and Mouse (M01021-1 catalog and IHC caption). For IF/ICC, A01021 has an A549 IF image and A01021-2 has a paraformaldehyde-fixed HeLa IF image (respective catalogs and IF captions). For cross-species planning, A01021-1 lists Human, Mouse and Rat and both IHC and IF applications, but its stated IHC dilution and pictured tissue IHC are specific to human samples (A01021-1 catalog and IHC caption).