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HECTD3 / E3 ubiquitin-protein ligase HECTD3 · IHC design guide
Design Immunohistochemistry for HECTD3
Plan chromogenic paraffin IHC for HECTD3 using the catalog antibody’s documented tissue protocol (datasheet A11560). Compare staining with the cytoplasmic tissue profile and use controls when scoring, as presumed off-target binding was observed (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HECTD3 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 most tissues (HPA tissue IHC) | |
| Staining pattern | Most tissues: cytoplasmic; breast glandular cells: medium (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A11560) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+1 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 | Presumed off-target binding can confound scoring (HPA tissue IHC) | |
| Regulation | No expression regulator annotated in the record (UniProt) | |
| Isoform / epitope | 2 isoforms; cytoplasmic target, epitope map unspecified (UniProt) |
Section 1
Recommended HECTD3 IHC & IF Protocols
Use the catalog antibody’s IHC-P protocol as the starting point, then compare the published HECTD3 IHC methods from three PMC articles (PMC8994759; PMC3847339; PMC7904838).
| Sample | Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A11560) |
| Fixation | Image fixative and duration unreported (datasheet A11560); 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 A11560) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A11560) |
| Primary antibody | Rabbit anti-HECTD3, 1μg/ml (datasheet A11560) |
| Primary incubation | Overnight at 4 °C (datasheet A11560) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A11560) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | HECTD3-positive staining in glandular cells of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate retrieval at pH 6 (datasheet A11560); the cited articles do not specify a different retrieval buffer.
Section 2
What Is the Expected HECTD3 Staining Pattern?
HECTD3 is a cytoplasmic protein with a perinuclear location and no transmembrane segment (UniProt Q5T447). In paraffin sections, expect cytoplasmic staining in many tissues, including glandular cells, marrow hematopoietic cells and selected brain cells (HPA: cytoplasmic expression in most tissues; Medium in listed cells). Treat the pattern as a guide: HPA rates tissue IHC Approved, with medium staining–RNA consistency, presumed off-target staining disregarded and external verification pending (HPA: reliability description).
| Cytoplasmic signal, sometimes concentrated near nuclei, in glandular cells or marrow hematopoietic cells. | This fits UniProt’s cytoplasmic, perinuclear location and HPA’s Medium staining in glandular cells of breast, appendix and adrenal gland and in bone marrow hematopoietic cells (UniProt Q5T447; HPA: tissue IHC). Judge the stained cells within their tissue architecture. |
| Predominantly nuclear or sharply cell-surface signal, with little cytoplasmic staining. | That distribution conflicts with the reported cytoplasmic, perinuclear location and lack of a transmembrane segment (UniProt Q5T447). Check morphology and controls before calling it HECTD3; a compartment mismatch alone cannot identify the source of the signal. |
| Prominent staining in adipocytes or splenic red-pulp cells. | HPA reports HECTD3 as Not detected in those cells (HPA: adipocytes; spleen red pulp). Consider cross-reactivity or endogenous detection activity, and compare a no-primary control. A negative HPA observation is a reference pattern, not proof that every specimen must be unstained. |
| A uniform haze covers cells and surrounding tissue, obscuring their boundaries. | A field-wide haze cannot establish the cell-restricted cytoplasmic pattern described by HPA (HPA: cytoplasmic expression in most tissues). Assess background with a no-primary control, then review blocking, washes and detection conditions as general IHC troubleshooting steps. |
| No signal in breast glandular cells or bone marrow hematopoietic cells. | Both are reported at Medium staining in HPA tissue IHC (HPA: breast glandular cells; bone marrow hematopoietic cells). Confirm the expected cells are present, then check assay controls and reagent performance. The HPA rating is a reference, not a guarantee for every section. |
💡Expected HECTD3 appearanceCall a result consistent when staining is mainly cytoplasmic, with possible perinuclear emphasis (UniProt Q5T447), in HPA-listed cells at approximately Medium intensity (HPA: tissue IHC); dominant nuclear, cell-surface or field-wide signal is discordant with that reference pattern.
| Cellular location and topology | UniProt places HECTD3 in the cytoplasm and perinuclear region and lists no transmembrane segment (UniProt Q5T447). Use compartment as a pattern check; these annotations do not predict antigen-retrieval conditions or fixation sensitivity. |
| Strength and limits of the tissue reference | HPA describes cytoplasmic expression in most tissues but rates antibody staining–RNA consistency Medium, notes disregarded presumed off-target binding and awaits external verification (HPA: reliability description). Interpret unusual staining with controls. |
| Isoforms and unknown epitope coverage | UniProt lists two HECTD3 isoforms (UniProt Q5T447). The supplied record does not locate the antibody epitope or establish isoform-specific staining, so do not assign a cell pattern to one isoform from IHC alone. |
| IF/ICC: is a validated cell-image pattern available? | No main subcellular location or ICC/IF cell-line images are supplied, and the listed antibody has no ICC validation entry (HPA: subcellular record; HPA027467). UniProt’s cytoplasmic, perinuclear location can guide interpretation, but IF/ICC needs its own validation (UniProt Q5T447). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected HPA-positive cells are unstained. | The relevant cells may be absent from the section, or the IHC assay may have failed; HPA reports Medium staining, not assured positivity in every specimen (HPA: breast; bone marrow). | Locate the expected cells on the counterstained section and check a suitable positive control. As general IHC practice, review retrieval, primary-antibody conditions and detection reagents without assuming a HECTD3-specific fixation effect. |
| Signal is mainly nuclear or outlines cell borders. | That pattern disagrees with cytoplasmic, perinuclear HECTD3 and its lack of a transmembrane segment (UniProt Q5T447); the underlying cause remains undetermined. | Recheck cell boundaries and compare the positive and no-primary controls. Treat the compartment call as unresolved until reproducible cytoplasmic staining is demonstrated. |
| Adipocytes or splenic red-pulp cells stain prominently. | Those cell groups are Not detected in the HPA tissue reference (HPA: adipocytes; spleen red pulp). Cross-reactivity or endogenous detection activity is possible. | Compare a no-primary control and nearby tissue compartments. If staining persists without primary antibody, investigate endogenous detection activity using controls appropriate to the chromogenic system. |
| The whole section has diffuse color. | Broad background can obscure the cell-specific cytoplasmic pattern reported by HPA (HPA: tissue IHC); excessive detection signal or inadequate washing are general IHC possibilities. | Inspect a no-primary control and review wash and blocking steps. Adjust assay conditions only after confirming that the tissue’s morphology and expected cell populations remain assessable. |
| Only one unexpected cell population shows a strong signal. | HPA reports a broad cytoplasmic profile but also notes presumed off-target binding was observed and disregarded (HPA: profile; reliability description). The new pattern needs independent checking. | Compare tissue morphology and HPA-listed cell populations, then test specificity with an independent antibody or an appropriate orthogonal control when available; do not infer a new tissue restriction from one slide. |
| Perinuclear accentuation is unclear despite visible cytoplasmic signal. | UniProt reports a perinuclear location, while HPA’s tissue-level summary states cytoplasmic expression (UniProt Q5T447; HPA: tissue IHC). Neither source requires a perinuclear ring in every cell. | Score the reproducible cytoplasmic pattern and HPA-listed cell type first. Record whether perinuclear emphasis is visible, without making it a mandatory criterion for positivity. |
Experiment evidence
IHC & IF images showing HECTD3 staining
A11560 · IHCIHC analysis of HECTD3 using anti-HECTD3 antibody (A11560). HECTD3 was detected in paraffin-embedded section of human lung cancer tissue. He
A11560 · IHCIHC analysis of HECTD3 using anti-HECTD3 antibody (A11560). HECTD3 was detected in paraffin-embedded section of human lung cancer tissue. He
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000126107-HECTD3/tissue/cerebral+cortex#imid_7135904
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000126107-HECTD3/tissue/colon#imid_7135838
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000126107-HECTD3/tissue/liver#imid_7135839
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000126107-HECTD3/tissue/kidney#imid_7135832
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HECTD3 IHC & IF
🧪Run breast first: glandular cells should stain for HECTD3 (HPA: Breast glandular cells, Medium). Use adipose tissue as the negative tissue (HPA: adipocytes, Not detected); on the breast slide, assess any adipocytes present for background-only staining as a putative internal negative (HPA: adipose tissue adipocytes, Not detected).
Positive control tissue: Adrenal gland (Glandular cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for HECTD3; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include no-primary (secondary-only) and matched nonimmune rabbit IgG controls, plus HECTD3 knockout material if available to test antibody specificity (caption: rabbit primary antibody). Quench endogenous peroxidase and check for endogenous biotin before interpreting DAB signal (caption: biotinylated secondary, streptavidin-biotin complex and DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A11560 paraffin-section caption does not state a fixative (caption: fixative unreported). The caption uses heat retrieval in citrate buffer at pH 6 for 20 min, but does not establish retrieval dependence or whether frozen sections or IF/ICC are easier (caption: citrate retrieval). In breast, distinguish glandular staining from signal in adipose areas when scoring (HPA: Breast glandular cells, Medium; HPA: adipose tissue adipocytes, Not detected).
HPA tissue IHC evidence for HECTD3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded. 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | 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 → |
| Caudate | Glial cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced HECTD3 IHC Tips
Troubleshoot HECTD3 staining in paraffin sections by checking retrieval, cellular localisation, controls and scoring before interpreting differences between specimens.
Which retrieval condition should I try first for HECTD3 paraffin sections?
Start with heat-mediated retrieval in citrate buffer at pH 6 for 20 minutes (datasheet A11560). That condition produced HECTD3 staining in paraffin-embedded human lung cancer tissue with 1 μg/ml primary antibody incubated overnight at 4°C (datasheet A11560). The image caption does not state the fixative, so match its retrieval conditions without assuming how the specimen was fixed (datasheet A11560). If staining is weak, check heating uniformity and compare retrieval times on adjacent sections while holding detection conditions constant (standard IHC practice). Try another buffer or pH only as a fallback, using matched positive and primary-omission controls to assess signal and background (standard IHC practice).
How should I investigate weak staining after fixation?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not identify a fixative (datasheet A11560). Record the fixative, tissue thickness and time before processing for each specimen, then compare sections with different fixation histories under the same staining conditions (standard IHC practice). As a general starting point, assess routine 10% neutral buffered formalin processing and document the actual exposure time; do not treat that as a validated HECTD3 condition (standard IHC practice). Include a consistently processed positive section in every run and compare its cytoplasmic signal with the test section (HPA tissue IHC; standard IHC practice). Adjust retrieval and antibody exposure one variable at a time if the control stains but the test section remains weak (standard IHC practice).
What staining pattern is plausible for HECTD3 in tissue?
Expect predominantly cytoplasmic staining, with possible perinuclear enrichment, when evaluating the chromogenic signal (UniProt Q5T447 subcellular location; HPA tissue IHC). HECTD3 has no transmembrane segment, so a crisp plasma-membrane rim alone needs independent validation before it is scored as target staining (UniProt Q5T447 topology; standard IHC practice). HPA reports cytoplasmic expression in most tissues but also flags presumed off-target binding in its antibody assessment (HPA tissue IHC). Examine intact cells at higher magnification and compare staining with an adjacent section lacking primary antibody (standard IHC practice). Record compartment and cell type separately from intensity so diffuse DAB deposit is not mistaken for perinuclear enrichment (standard IHC practice).
Could isoforms or epitope accessibility explain inconsistent staining?
HECTD3 has 2 annotated isoforms, but the supplied product caption does not map the antibody epitope to either one (UniProt Q5T447 isoforms; datasheet A11560). Its DOC domain spans residues 219–397 and HECT domain spans 512–857; neither range establishes where this antibody binds (UniProt Q5T447 domains; datasheet A11560). Do not attribute differing tissue staining to isoform abundance without epitope mapping or an independent isoform-specific assay (standard IHC practice). N-terminal acetylation at residue 2 and phosphorylation at residue 12 are annotated, but their effect on this antibody is unknown (UniProt Q5T447 modified residues). Compare retrieval conditions and replicate sections before assigning a biological explanation to a weak signal (standard IHC practice).
How can I check HECTD3 by IF alongside a cell-type marker?
Treat IF as a separate assay requiring its own antibody and fixation controls; the selected product evidence describes chromogenic staining of paraffin sections (datasheet A11560). For a glandular region, multiplex HECTD3 with a cell-type marker such as cytokeratin and check whether cytoplasmic signal falls in the expected cells (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and place the weaker target in a channel with low tissue autofluorescence, after inspecting unstained and single-stain controls (standard IF practice). Because HECTD3 is cytoplasmic and has no transmembrane segment, permeabilise sufficiently to expose the intracellular epitope, then titrate detergent against cell preservation (UniProt Q5T447 location and topology; standard IF practice). Assess fixation and permeabilisation together rather than transferring the paraffin-section conditions directly (standard IF practice).
What should I change when DAB background obscures HECTD3?
Run a primary-omission section to separate secondary or detection background from antibody-dependent staining (standard IHC practice). The illustrated workflow used 10% goat serum blocking, a biotinylated secondary and avidin–biotin detection with DAB; use those details to identify where background enters this workflow (datasheet A11560). Include a peroxidase block before DAB and consider endogenous biotin when using avidin–biotin detection, especially if signal persists without primary antibody (standard IHC practice). Titrate primary antibody below the illustrated 1 μg/ml starting concentration and shorten chromogen development on matched sections if diffuse deposit remains (datasheet A11560; standard IHC practice). Preserve a consistently processed positive control while changing only one step per comparison (standard IHC practice).
How should I quantify HECTD3 staining across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic compartment before scoring, consistent with the reported HECTD3 distribution (UniProt Q5T447 subcellular location; HPA tissue IHC). For chromogenic sections, record the percentage of positive cells and intensity categories, then calculate an H-score from the percentage in each category (standard IHC practice). Alternatively, measure positive-cell density per mm² of viable tissue when cell counts per area answer the study question (standard IHC practice). Normalise to the eligible cell population or viable tissue area, and apply the same threshold, counterstain and image settings across slides (standard IHC practice). Keep cell-type strata separate because HPA reports medium staining in several populations and no detected staining in others (HPA tissue IHC).
How can I distinguish true HECTD3 staining from artefact?
Prioritise reproducible cytoplasmic or perinuclear staining in intact cells because those compartments agree with the annotated localisation (UniProt Q5T447 subcellular location). Compare cell identity with the section anatomy: HPA reports medium signal in several glandular cell populations, while adipocytes are listed as not detected (HPA tissue IHC). Treat isolated nuclear or membrane-only staining, section-edge enrichment and staining concentrated in necrotic areas as reasons to review controls and morphology before assigning positivity (UniProt Q5T447 topology and location; standard IHC practice). Check a primary-omission section for endogenous enzyme or detection-system signal, particularly when DAB deposit lacks clear cellular boundaries (standard IHC practice). Interpret modest differences cautiously because the HPA assessment reports medium RNA–staining consistency, presumed off-target binding and pending external verification (HPA tissue IHC).
Boster reagents
Best HECTD3 / E3 ubiquitin-protein ligase HECTD3 IHC Antibodies
A11560 has paraffin-section IHC images from human lung cancer and mouse gaster tissue (catalog IHC captions). IHC-F and ICC are listed, but no IF image is supplied (catalog applications/images).
Real IHC data
Anti-HECTD3 Antibody ®
Cat # A11560
A11560 is shown in paraffin-section IHC of human lung cancer and mouse gaster tissue (catalog IHC captions). A11560 lists IHC, IHC-F, and ICC applications and human, mouse, and rat reactivity (catalog applications/reactivity).
Which to pick: Choose A11560 for paraffin-section tissue IHC: its caption documents citrate retrieval at pH 6 for 20 minutes and 1 μg/ml primary antibody overnight at 4°C; the fixative is unreported (A11560 IHC caption). For IF/ICC, A11560 lists IHC-F and ICC, but supplies no IF image or IF dilution (catalog applications/IF images/dilution). For cross-species work, A11560 is polyclonal and lists human, mouse, and rat reactivity, with IHC images from human and mouse tissue (catalog dilution_raw/reactivity; A11560 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q5T447 (HECD3_HUMAN, E3 ubiquitin-protein ligase HECTD3).
- Human Protein Atlas. HECTD3 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. HECTD3 subcellular location (ICC-IF): Highest expression in HUVEC/TERT2: 46.6 nTPM.
- Human Protein Atlas. HECTD3 antibody validation summary (1 antibodies).
- HECTD3 promotes gastric cancer progression by mediating the polyubiquitination of c-MYC. Cell death discovery 2022 — PMC8994759.
- The HECTD3 E3 ubiquitin ligase facilitates cancer cell survival by promoting K63-linked polyubiquitination of caspase-8. Cell death & disease 2013 — PMC3847339.
- Hypothermic oxygenated perfusion inhibits HECTD3-mediated TRAF3 polyubiquitination to alleviate DCD liver ischemia-reperfusion injury. Cell death & disease 2021 — PMC7904838.
- Identification of a Biomarker Panel in Extracellular Vesicles Derived From Non-Small Cell Lung Cancer (NSCLC) Through Proteomic Analysis and Machine Learning. Journal of extracellular vesicles 2025 — PMC12077270.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16710414 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HECTD3 (E3 ubiquitin-protein ligase HECTD3). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A11560). Validated IHC antibody: A11560 at 1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A11560); 2. Fixation: Image fixative and duration unreported (datasheet A11560); 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: Citrate pH 6 , 20 min (datasheet A11560); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A11560); 8. Primary antibody: Rabbit anti-HECTD3, 1μg/ml (datasheet A11560); 9. Primary incubation: Overnight at 4 °C (datasheet A11560). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Presumed off-target binding can confound scoring (HPA tissue IHC); Isoform / epitope: 2 isoforms; cytoplasmic target, epitope map unspecified (UniProt); Regulation: No expression regulator annotated in the record (UniProt). Evidence: UniProt Q5T447, HPA tissue IHC · Approved, HPA cell-line RNA · Highest expression in HUVEC/TERT2: 46.6 nTPM, Datasheet A11560, PMC8994759, PMC3847339. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HECTD3/immunohistochemistry-ihc-guide.