This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
ERBB4 / Receptor tyrosine-protein kinase erbB-4 · IHC design guide
Design Immunohistochemistry for ERBB4
Plan ERBB4 paraffin-section IHC using the nuclear and cytoplasmic tissue pattern as a starting point (HPA tissue IHC). This guide covers fixation consistency, compartment scoring, and how isoforms and cleavage affect epitope interpretation (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ERBB4 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 cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Widespread nuclear and cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A00296-1) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | Cerebral cortex+3 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 | Cleaved 4ICD may change staining by epitope location (UniProt) | |
| Regulation | NRG1 activation drives internalization (UniProt) | |
| Isoform / epitope | 4 isoforms; JM-A cleavage makes epitope location matter (UniProt) |
Section 1
Recommended ERBB4 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet A00296-1) with 4 published ERBB4 chromogenic IHC protocols (PMC3382207; PMC4103820; PMC2864910; PMC2811086).
| Sample | Paraffin-embedded human colon adenocarcinoma tissue; fixative not specified (datasheet A00296-1) |
| Fixation | Image fixative and duration unreported (datasheet A00296-1); 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 A00296-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A00296-1) |
| Primary antibody | Rabbit anti-ERBB4, 2-5 μg/ml (datasheet A00296-1) |
| Primary incubation | Overnight at 4 °C (datasheet A00296-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A00296-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ERBB4-positive staining in adipocytes of adipose tissue (HPA tissue IHC: Medium). HPA tissue profile: Ubiquitous nuclear and cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A00296-1). Retrieval conditions are unreported in these published excerpts (PMC3382207; PMC4103820; PMC2864910; PMC2811086).
Section 2
What Is the Expected ERBB4 Staining Pattern?
ERBB4 is a cell-surface receptor with one transmembrane segment; its intracellular domain can also localise to the nucleus or mitochondrion (UniProt Q15303 topology; UniProt Q15303 function). In paraffin-section IHC, expect nuclear and cytoplasmic staining in many cell types, including breast glandular cells and lung alveolar cells at medium intensity (HPA: ubiquitous nuclear and cytoplasmic profile; HPA: Medium in these cells). HPA rates its tissue IHC reliability Enhanced, with external verification pending (HPA: reliability).
| Nuclear and cytoplasmic signal in breast glandular cells, with a medium-strength example (HPA: Breast glandular cells Medium). | This agrees with the reported tissue IHC profile (HPA: ubiquitous nuclear and cytoplasmic expression). Nuclear signal is biologically plausible because a processed ERBB4 intracellular domain can enter the nucleus (UniProt Q15303 function). |
| Predominantly membranous signal in an otherwise plausible positive cell (UniProt Q15303 topology). | Membrane localisation fits the full-length receptor, while HPA tissue IHC reports a broader nuclear and cytoplasmic profile (UniProt Q15303 subcellular location; HPA: tissue IHC profile). Judge the distribution alongside a validated positive section; membrane-only staining does not by itself prove an artefact (general IHC practice). |
| Staining appears in a cell population reported as undetected, such as heart cardiomyocytes (HPA: Heart muscle cardiomyocytes Not detected). | Check cell identity and staining controls before calling ERBB4 positive: nonspecific antibody binding or endogenous chromogen activity can mimic signal (general IHC practice). A tissue-level UniProt expression statement does not establish staining in each constituent cell type (UniProt Q15303 tissue specificity; HPA: cardiomyocytes Not detected). |
| Uniform colour coats the section, including tissue spaces, with little cell-specific pattern (general IHC practice). | Treat this as background until controls clarify it; excess primary antibody, incomplete blocking, or insufficient washing are general IHC causes (general IHC practice). It does not resemble the reported nuclear and cytoplasmic cellular profile (HPA: tissue IHC profile). |
| No signal appears in breast glandular cells that serve as the positive comparator (HPA: Breast glandular cells Medium). | First investigate the IHC workflow and antibody performance using an appropriate positive section (general IHC practice). A negative result in one section cannot establish ERBB4 absence, especially given HPA's medium RNA–staining consistency and pending external verification (HPA: reliability description). |
💡Expected ERBB4 appearanceCall the section positive when interpretable cells show nuclear and cytoplasmic staining, exemplified by medium signal in breast glandular or lung alveolar cells; uniform acellular colour or control-matched staining is suspect (HPA: tissue IHC profile; HPA: Breast and Lung Medium; general IHC practice).
| Epitope location and receptor topology (UniProt Q15303 topology). | The extracellular region spans residues 26–651, the membrane segment 652–675, and the cytoplasmic region 676–1308 (UniProt Q15303 topology). Interpret nuclear signal in light of the antibody's stated epitope, if known (UniProt Q15303 processing; general IHC practice). |
| Proteolytic processing (UniProt Q15303 function). | JM-A isoforms can yield a soluble intracellular domain that enters the nucleus or mitochondrion (UniProt Q15303 function). An antibody to an extracellular epitope may not report that intracellular fragment; verify epitope coverage before attributing nuclear staining to it (UniProt Q15303 topology and processing; general IHC practice). |
| Alternative splicing and activation (UniProt Q15303 function). | ERBB4 has four listed JM/CYT isoforms, and activated receptor internalises after NRG1 treatment (UniProt Q15303 isoforms; UniProt Q15303 subcellular note). Thus, localisation can depend on isoform and signalling context, but the supplied IHC data do not assign a specific pattern to either (HPA: tissue IHC profile). |
| Cell-level tissue selection (HPA: tissue IHC). | Use the recorded cell population when selecting comparators: breast glandular and lung alveolar cells are Medium; heart cardiomyocytes and skeletal myocytes are Not detected (HPA: tissue IHC). UniProt lists expression in heart and skeletal muscle, so organ-level expression alone is an unreliable cell-level IHC control (UniProt Q15303 tissue specificity; HPA: tissue IHC). |
| Antibody evidence (HPA: antibody validation). | Three listed antibodies have Enhanced IHC validation, while the tissue profile still has medium consistency with RNA and awaits external verification (HPA: HPA012016, CAB000276, CAB025522 IHC Enhanced; HPA: reliability description). Interpret unexpected compartments against controls and antibody-specific evidence (general IHC practice). |
| IF/ICC cross-check: what pattern can this page support? (HPA: subcellular). | HPA summarises ERBB4 as membrane-localised in its subcellular record, but gives no main location, ICC-IF image cell line, or ICC validation for the listed antibodies (HPA: subcellular record; HPA: antibody validation). Use the separate IF/ICC guide for its experimental design (application scope). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells are blank (HPA: Breast glandular cells Medium). | The detection run or primary antibody may have failed; a single negative section is inconclusive (general IHC practice). | Run a recorded medium-staining cell population as a positive comparator and check reagent order, detection chemistry, and primary-antibody instructions (HPA: tissue IHC; general IHC practice). |
| A membranous-only pattern seems to conflict with the HPA slide profile (HPA: tissue IHC profile). | Full-length ERBB4 is membrane-bound, whereas processing permits intracellular-domain localisation (UniProt Q15303 topology and processing). | Check the antibody epitope and compare with the recorded nuclear and cytoplasmic tissue pattern before deciding whether the result is discrepant (UniProt Q15303 topology; HPA: tissue IHC profile). |
| Unexpected signal appears in cardiomyocytes or skeletal myocytes (HPA: both Not detected). | Nonspecific binding or endogenous detection activity may produce misleading chromogen (general IHC practice); cell-level staining is not predicted by organ-level expression (HPA: tissue IHC; UniProt Q15303 tissue specificity). | Confirm cell identity; inspect a no-primary control and an appropriate positive section, then reassess whether signal follows cellular boundaries (general IHC practice). |
| Diffuse colour obscures nuclei and cytoplasm (general IHC practice). | Primary antibody concentration, blocking, washing, or chromogen development may contribute to background (general IHC practice). | Check a no-primary control, then optimise antibody concentration, blocking, washing, and development time within the antibody's IHC instructions (general IHC practice). |
| Nuclear signal is strong but membrane signal is weak (HPA: nuclear and cytoplasmic tissue profile; UniProt Q15303 topology). | A soluble intracellular domain can enter the nucleus, but the supplied data cannot identify the stained molecular species in this section (UniProt Q15303 function; HPA: tissue IHC profile). | Verify that the antibody epitope lies in the intracellular region and compare an independent IHC-validated antibody where available (UniProt Q15303 topology; HPA: antibody validation; general IHC practice). |
| Two antibodies yield different distributions (HPA: multiple IHC Enhanced antibodies). | Different epitope coverage or nonspecific staining can produce discordance; the supplied record does not map antibody epitopes (UniProt Q15303 processing; HPA: antibody validation; general IHC practice). | Compare epitope documentation, positive-cell comparators, and controls before assigning a biological difference; record the discrepancy because external verification remains pending (general IHC practice; HPA: reliability description). |
Experiment evidence
IHC & IF images showing ERBB4 staining
A00296-1 · IHCIHC analysis of ERBB4 using anti-ERBB4 antibody (A00296-1). ERBB4 was detected in a paraffin-embedded section of human colon adenocarcinoma
A00296-1 · IHCIHC analysis of ERBB4 using anti-ERBB4 antibody (A00296-1). ERBB4 was detected in a paraffin-embedded section of human colon adenocarcinoma
A00296 · IHCIHC analysis of ErbB 4 using anti-ErbB 4 antibody (A00296). ErbB 4 was detected in paraffin-embedded section of human mammary cancer tissue.
A00296 · IHCIHC analysis of ErbB 4 using anti-ErbB 4 antibody (A00296). ErbB 4 was detected in paraffin-embedded section of human colon cancer tissue. H
M00296 · IFImmunofluorescent analysis of A673 cells, using ErbB4 (HER4) Antibody .
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000178568-ERBB4/tissue/cerebral+cortex#imid_4070708
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000178568-ERBB4/tissue/colon#imid_4070626
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000178568-ERBB4/tissue/liver#imid_4070635
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000178568-ERBB4/tissue/kidney#imid_4070628
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ERBB4 IHC & IF
🧪Run breast first and expect staining in glandular cells (Medium; HPA: breast glandular cells); use cerebral cortex endothelial cells as the negative tissue (Not detected; HPA: cerebral cortex endothelial cells). On the breast slide, cells outside the glandular compartment should provide a background reference only after their ERBB4-negative status is verified; HPA supplies no validated internal negative cell type for breast (HPA: breast glandular cells).
Positive control tissue: Adipose tissue (Adipocytes, HPA Medium)
Negative control tissue: Cerebral cortex (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for ERBB4; derive a cell-line control from the positive tissue's cell type (Adipocytes) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) slide, a concentration-matched nonimmune rabbit IgG isotype control, and ERBB4-knockout tissue as a biological negative if available (caption: rabbit primary; standard IHC practice). Quench endogenous peroxidase before HRP/DAB detection, especially when assessing blood-containing areas of breast tissue (caption: HRP/DAB; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the fixative is unreported in the selected A00296-1 tissue-IHC caption; its paraffin section does not establish fixation (selected tissue-IHC caption). The caption uses heat retrieval in EDTA at pH 8.0, but supplies no comparison establishing retrieval dependence or that frozen sections or IF are easier (selected tissue-IHC caption). In breast, blood-associated endogenous peroxidase can produce misleading DAB signal, so interpret glandular staining against the quenched control (standard IHC practice; HPA: breast glandular cells). The selected A00296-1 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image A00296-1).
HPA tissue IHC evidence for ERBB4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Pending external verification.). 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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Cerebral cortex | Endothelial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ERBB4 IHC Tips
Troubleshoot ERBB4 staining in paraffin sections by checking retrieval, controls, and the cellular compartment before comparing signal across samples.
How should I adjust retrieval when ERBB4 staining is weak in paraffin sections?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for this paraffin-section assay (datasheet A00296-1). Keep the documented 2 μg/ml primary antibody incubation overnight at 4°C while comparing retrieval conditions, so the comparison changes one variable (datasheet A00296-1; standard IHC practice). If staining remains weak, check deparaffinisation and heating consistency, then titrate retrieval time on matched sections (standard IHC practice). Assess improvement by morphology and compartment-specific staining rather than overall DAB intensity, since ERBB4 is a membrane receptor whose processed intracellular domain can enter nuclei (UniProt Q15303 topology and processing).
Could fixation explain variable ERBB4 staining between paraffin blocks?
The selected paraffin-section image does not state its fixative, so target-specific fixation sensitivity is unknown for this antibody (datasheet A00296-1). Record fixative, fixation duration, block age, and processing history for each specimen before attributing differences to ERBB4 abundance (standard IHC practice). Compare matched blocks using the documented EDTA pH 8.0 retrieval and 2 μg/ml primary concentration, keeping detection conditions constant (datasheet A00296-1; standard IHC practice). If staining varies with processing, assess preserved morphology and an appropriate tissue control before adjusting retrieval or antibody concentration (standard IHC practice). Do not infer a fixation effect from tissue-expression patterns alone (standard IHC practice).
Should ERBB4 staining appear at the membrane, in cytoplasm, or in nuclei?
Score membrane, cytoplasmic, and nuclear staining separately: ERBB4 is a transmembrane receptor, while its processed intracellular domain can move to nuclei or mitochondria (UniProt Q15303 topology, processing, and localisation). Activated ERBB4 can internalise after NRG1 treatment, making intracellular signal biologically plausible in the relevant context (UniProt Q15303 localisation). The HPA tissue profile reports widespread nuclear and cytoplasmic staining, but its Enhanced reliability has medium agreement with RNA data and awaits external verification (HPA tissue IHC). Use matched controls and preserved cell morphology to assess each compartment; diffuse colour across unrelated structures should prompt a background check (standard IHC practice).
Can this antibody distinguish ERBB4 isoforms or the cleaved intracellular domain?
Do not assign an isoform from this stain alone: ERBB4 has 4 listed JM-A/JM-B and CYT-1/CYT-2 combinations, and the supplied caption does not map this antibody's epitope (UniProt Q15303 isoforms; datasheet A00296-1). An extracellular epitope within residues 26–651 and a cytoplasmic epitope within 676–1308 would support different interpretations of nuclear staining (UniProt Q15303 topology and processing). Cleavage of JM-A isoforms can release an intracellular domain that enters nuclei, but epitope mapping is needed to know whether this antibody detects it (UniProt Q15303 function; standard IHC practice). Interpret compartment patterns alongside an independently mapped reagent or orthogonal assay when isoform or cleavage status matters (standard IHC practice).
How can I investigate ERBB4 localisation by IF alongside this IHC guide?
Use a separate IF validation workflow; the supplied antibody evidence documents paraffin-section chromogenic IHC, not an IF staining condition (datasheet A00296-1). For multiplexing, pair ERBB4 with a marker identifying the expected cell population, such as breast glandular cells in a suitable specimen, and include single-stain controls (HPA tissue IHC; standard IF practice). Choose a fluorophore channel away from the specimen's strongest autofluorescence and measure an unstained section before interpreting faint signal (standard IF practice). Because the antibody epitope is unmapped, determine whether it faces the extracellular 26–651 region or cytoplasmic 676–1308 region before selecting permeabilisation conditions (UniProt Q15303 topology; datasheet A00296-1; standard IF practice).
What should I check if ERBB4 DAB staining is diffuse or widespread?
Begin with a no-primary control and inspect whether colour follows tissue edges, damaged areas, or the expected cells (standard IHC practice). The documented section was blocked with 10% goat serum, followed by a peroxidase-conjugated anti-rabbit secondary and DAB detection (datasheet A00296-1). Check endogenous peroxidase blocking, wash adequacy, and DAB development time as general chromogenic IHC controls; their use is not established by the selected caption (standard IHC practice; datasheet A00296-1). Reduce primary concentration or development time only after controls identify the source of background, then verify that cell-specific staining remains (standard IHC practice).
How should I quantify ERBB4 staining when compartments and cell populations differ? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and record membrane, cytoplasmic, and nuclear signal separately before comparing sections (standard IHC practice; UniProt Q15303 localisation). For intensity-based scoring, use an H-score from 0–300, calculated as the sum of each intensity grade multiplied by its percentage of positive cells (standard IHC practice). Alternatively, report percentage positive cells or positive-cell density per mm², with identical thresholds and sampling rules across specimens (standard IHC practice). Normalise to viable cells of the prespecified type or to evaluated tissue area, and exclude necrotic or damaged regions (standard IHC practice). Record controls and staining batch because DAB intensity depends on processing and development (standard IHC practice).
How can I distinguish genuine ERBB4 staining from a tissue artefact?
Look for reproducible signal in intact cells and a plausible compartment: ERBB4 spans the cell membrane, while its processed intracellular domain can enter nuclei or mitochondria (UniProt Q15303 topology, processing, and localisation). Interpret cell identity cautiously; HPA reports medium staining in breast glandular cells but no detected staining in heart cardiomyocytes in its tissue images (HPA tissue IHC). Edge-restricted colour, necrotic deposits, or signal in a no-primary control favours processing or detection artefact (standard IHC practice). Check endogenous peroxidase when DAB colour appears broadly, and confirm any unusual nuclear pattern with an independently mapped epitope or orthogonal assay (standard IHC practice; UniProt Q15303 processing).
Boster reagents
Best ERBB4 / Receptor tyrosine-protein kinase erbB-4 IHC Antibodies
Catalog antibodies show ERBB4 staining in human paraffin tissue sections, mouse and rat brain sections, and A673 cells by IF (catalog image captions).
Real IHC data
Anti-ERBB4 Antibody ®
Cat # A00296-1
Real IHC data
Anti-ErbB 4/ERBB4 Antibody ®
Cat # A00296
Real IF data
Anti-ErbB (HER4) ERBB4 Rabbit Monoclonal Antibody
Cat # M00296
A00296-1 has IHC data from human colon adenocarcinoma and pancreatic ductal adenocarcinoma paraffin sections; A00296 has IHC data from human mammary and colon cancer and mouse and rat brain paraffin sections (catalog IHC captions). M00296 is listed for IF/ICC and has an IF image of A673 cells; the caption does not report the cells’ species (catalog applications and IF caption).
Which to pick: For human tissue IHC, choose A00296-1 for the EDTA pH 8.0 retrieval example or A00296 for the citrate pH 6 example; both captions describe paraffin sections but do not report the fixative (respective catalog IHC captions). For IF/ICC, choose rabbit monoclonal M00296, clone HC-5, which lists both applications and shows IF staining in A673 cells (catalog applications, clone and IF caption). For IHC across the listed species, A00296 has the strongest image support: its rabbit polyclonal antibody has paraffin-section examples from human, mouse and rat, whereas A00296-1 lists Human, Mouse and Rat reactivity but provides human IHC images only (catalog host, dilution details, reactivity and IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q15303 (ERBB4_HUMAN, Receptor tyrosine-protein kinase erbB-4).
- Human Protein Atlas. ERBB4 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ERBB4 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. ERBB4 antibody validation summary (3 antibodies).
- Proteolytic processing of ErbB4 in breast cancer. PloS one 2012 — PMC3382207.
- ERBB4 promoter polymorphism is associated with poor distant disease-free survival in high-risk early breast cancer. PloS one 2014 — PMC4103820.
- Expressions of neuregulin 1beta and ErbB4 in prefrontal cortex and hippocampus of a rat schizophrenia model induced by chronic MK-801 administration. Journal of biomedicine & biotechnology 2010 — PMC2864910.
- The ErbB4 growth factor receptor is required for colon epithelial cell survival in the presence of TNF. Gastroenterology 2009 — PMC2811086.
- PubMed PMID:8383326 — UniProt-cited evidence.
- PubMed PMID:9334263 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ERBB4 (Receptor tyrosine-protein kinase erbB-4). Expected localisation Nuclear and cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A00296-1). Validated IHC antibody: A00296-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative Cerebral cortex. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon adenocarcinoma tissue; fixative not specified (datasheet A00296-1); 2. Fixation: Image fixative and duration unreported (datasheet A00296-1); 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 A00296-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A00296-1); 8. Primary antibody: Rabbit anti-ERBB4, 2-5 μg/ml (datasheet A00296-1); 9. Primary incubation: Overnight at 4 °C (datasheet A00296-1). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Cleaved 4ICD may change staining by epitope location (UniProt); Isoform / epitope: 4 isoforms; JM-A cleavage makes epitope location matter (UniProt); Regulation: NRG1 activation drives internalization (UniProt). Evidence: UniProt Q15303, HPA tissue IHC · Enhanced, HPA ICC-IF · Membrane, Datasheet A00296-1, PMC3382207, PMC4103820. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ERBB4/immunohistochemistry-ihc-guide.