This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
STEAP3 / Metalloreductase STEAP3 · IHC design guide
Design Immunohistochemistry for STEAP3
Use glandular cells in appendix as a medium-staining reference and oral squamous cells as a no-detection comparator (HPA tissue IHC). Interpret cytoplasmic tissue staining alongside STEAP3’s endosomal membrane location and check which side of the membrane the antibody recognizes (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
STEAP3 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 | Mainly cytoplasmic in tissue sections (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in glandular and other cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A30509) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | Cerebral cortex+2 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 | Staining has low agreement with RNA expression (HPA tissue IHC) | |
| Regulation | Lower in hepatocellular carcinoma (UniProt) | |
| Isoform / epitope | 4 isoforms; check cytoplasmic vs vesicular epitope (UniProt) |
Section 1
Recommended STEAP3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by one published STEAP3 tissue microarray protocol with sufficient method details (PMC9686107).
| Sample | Paraffin-embedded human Colon cancer tissue; fixative not specified (datasheet A30509) |
| Fixation | Image fixative and duration unreported (datasheet A30509); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 9.0 (datasheet A30509); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-STEAP3, 1:100 - 1:300 (datasheet A30509) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | STEAP3-positive staining in adipocytes of adipose tissue (HPA tissue IHC: Medium). HPA tissue profile: Mainly cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated Tris-EDTA pH 9.0 retrieval (datasheet A30509); the published tissue microarray protocol used EDTA buffer (PMC9686107).
Section 2
What Is the Expected STEAP3 Staining Pattern?
In paraffin-section IHC, expect mainly cytoplasmic STEAP3 staining; adipocytes and several glandular or epithelial cell populations show medium staining (HPA tissue IHC). STEAP3 is a 6-pass endosome-membrane protein also found in vesicular-like structures near the plasma membrane and nucleus (UniProt Q658P3 topology and subcellular location). Treat the tissue pattern as provisional: HPA rates its IHC antibody Approved but reports low agreement between staining and RNA expression (HPA tissue IHC).
| Cytoplasmic staining in adipocytes or glandular cells of breast, appendix, cervix, duodenum, endometrium or epididymis (HPA tissue IHC). | This matches HPA's mainly cytoplasmic pattern and medium staining in those cells (HPA tissue IHC). Vesicular or perinuclear emphasis is compatible with UniProt's membrane-localisation annotation, but HPA IHC alone cannot identify the stained organelle (UniProt Q658P3; HPA tissue IHC). |
| A predominantly nuclear or sharply uniform cell-surface signal replaces the expected cytoplasmic pattern (HPA tissue IHC). | Treat this as a compartment mismatch and investigate staining artefact or antibody specificity (HPA tissue IHC; general IHC practice). HPA reports nucleolar localisation in ICC-IF, so an IF nucleolar signal alone should not be called an IHC failure (HPA subcellular ICC-IF). |
| Strong stain appears in cerebral-cortex endothelial cells, oral-mucosa squamous cells or smooth-muscle cells (HPA tissue IHC). | HPA reports STEAP3 as not detected in those cell populations; investigate cross-reactivity or endogenous chromogen-generating activity before calling them positive (HPA tissue IHC; general IHC practice). A negative HPA observation is a comparator, not proof that every specimen must be negative (HPA tissue IHC). |
| Diffuse chromogen covers tissue, including spaces or structures without a clear cellular pattern (general IHC practice). | The result cannot be scored confidently as STEAP3 because HPA describes a mainly cytoplasmic cellular pattern (HPA tissue IHC). Excess detection background, incomplete blocking or inadequate washing are general IHC possibilities; compare with a no-primary control (general IHC practice). |
| No cellular signal appears in an adipose, breast or appendix section expected to provide a positive comparator (HPA tissue IHC). | Check the run before interpreting absence as biology: HPA reports medium staining in adipocytes and the specified glandular cells (HPA tissue IHC). Verify that those cells are present, then review antibody dilution, retrieval and detection controls as general IHC steps (general IHC practice). |
💡Expected STEAP3 appearanceA credible positive is medium, mainly cytoplasmic staining in HPA-listed adipocytes or glandular or epithelial cells; strong staining in HPA-listed undetected cell populations or acellular diffuse chromogen warrants investigation (HPA tissue IHC; general IHC practice).
| Membrane topology: 6 transmembrane segments with cytoplasmic and vesicular loops (UniProt Q658P3 topology). | The location of the antibody's epitope is not supplied, so topology cannot predict whether a specific retrieval or permeabilisation condition will expose it (UniProt Q658P3 topology; epitope information not supplied). Optimise those steps empirically under the antibody's IHC protocol (general IHC practice). |
| Tissue and cell selection: HPA lists medium staining in selected adipocytes and glandular or epithelial cells, with undetected cells elsewhere (HPA tissue IHC). | Include a listed positive cell population and a listed undetected population when assessing a run (HPA tissue IHC; general IHC practice). Preserve cell-level context: a tissue name alone does not specify which cells should stain (HPA tissue IHC). |
| Evidence strength: antibody HPA050510 is IHC Approved; HPA notes low consistency between antibody staining and RNA expression, with retesting pending (HPA tissue IHC; HPA antibodies). | Use the reported staining pattern as a working expectation, not definitive validation of an unexpected result (HPA tissue IHC). Confirm surprising compartment or cell-type staining with appropriate controls and independent evidence where available (general IHC practice). |
| Isoforms: UniProt lists 4 STEAP3 isoforms; the antibody epitope and isoform coverage are not supplied (UniProt Q658P3). | An absent or altered IHC signal cannot be assigned to a particular isoform from these records (UniProt Q658P3; epitope information not supplied). Interpret cell staining against the HPA IHC pattern and the run controls (HPA tissue IHC; general IHC practice). |
| Application-specific localisation: HPA ICC-IF reports mainly cytosol with additional nucleoli in imaged PC-3 and U2OS cells (HPA subcellular ICC-IF). | That IF observation can inform an IF/ICC question, but its cell lines and nucleolar annotation do not establish a nucleolar expectation in paraffin-section IHC (HPA subcellular ICC-IF; HPA tissue IHC). Use the separate IF/ICC guide for that application. |
| Situation | Likely cause | Next action |
|---|---|---|
| The positive comparator has no stain (HPA tissue IHC). | The expected cells may be absent, or the staining run may have failed; HPA reports medium signal only for specified cell populations (HPA tissue IHC; general IHC practice). | Confirm the relevant cells on the section, then check the catalog antibody's IHC-P instructions, retrieval step, dilution, detection reagents and run control (general IHC practice). Do not infer STEAP3-specific fixation sensitivity; none is supplied. |
| Every tissue compartment looks brown, including areas without defined cells (general IHC practice). | Diffuse detection background can obscure the mainly cytoplasmic pattern reported by HPA (general IHC practice; HPA tissue IHC). | Inspect a no-primary control, review blocking and washing, and adjust detection conditions according to the assay controls (general IHC practice). Score only interpretable cellular staining against the HPA pattern (HPA tissue IHC). |
| A listed undetected cell population stains strongly (HPA tissue IHC). | Cross-reactivity or endogenous detection activity is possible; HPA reports no detection in the specified cerebral-cortex endothelial, oral-mucosa squamous or smooth-muscle cells (general IHC practice; HPA tissue IHC). | Compare adjacent sections and a no-primary control, check endogenous peroxidase blocking for chromogenic IHC, and review whether the signal tracks a plausible cell compartment (general IHC practice). |
| Signal is mainly nuclear or surface-rimmed instead of cytoplasmic (HPA tissue IHC). | This differs from HPA's tissue-IHC profile; compartment-specific artefact or antibody specificity needs assessment (HPA tissue IHC; general IHC practice). | Check morphology, detection controls and repeatability across sections (general IHC practice). Do not transfer HPA's nucleolar ICC-IF annotation to an IHC result without separate support (HPA subcellular ICC-IF). |
| Staining varies between two tissues selected as positives (HPA tissue IHC). | HPA reports staining by cell population, and its Approved IHC record has low consistency with RNA expression (HPA tissue IHC). Different proportions of the reported cells can change the apparent field-wide signal (HPA tissue IHC; general IHC interpretation). | Compare the same annotated cell types at matched staining conditions, record their intensity separately, and treat a disagreement as unresolved until controls support interpretation (HPA tissue IHC; general IHC practice). |
| An IF/ICC image shows nucleolar signal while the IHC section looks cytoplasmic (HPA subcellular ICC-IF; HPA tissue IHC). | HPA reports additional nucleolar localisation for ICC-IF and mainly cytoplasmic staining for tissue IHC; these are different application observations (HPA subcellular ICC-IF; HPA tissue IHC). | Evaluate each result against its own HPA application record and controls; use the dedicated IF/ICC guide for IF interpretation (HPA subcellular ICC-IF; HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing STEAP3 staining
A30509 · IHCImmunohistochemical analysis of paraffin-embedded human Colon cancer. 1, Antibody was diluted at 1:200(4° overnight). 2, Tris-EDTA,pH9.0 was
A30509 · IFImmunofluorescence analysis of HeLa cells, using STEA3 Antibody. The picture on the right is blocked with the synthesized peptide.
A05568-1 · IHCImmunohistochemistry of STEAP3 in mouse liver tissue with STEAP3 antibody at 2.5 μg/mL.
A05568-1 · IFImmunofluorescence of STEAP3 in Mouse Liver cells with STEAP3 antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000115107-STEAP3/tissue/cerebral+cortex#imid_13802025
HPA · RectumHuman Protein Atlas IHC, Rectum. Source: https://www.proteinatlas.org/ENSG00000115107-STEAP3/tissue/rectum#imid_13801927
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000115107-STEAP3/tissue/liver#imid_13801952
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000115107-STEAP3/tissue/kidney#imid_13801937
HPA · ICC-IF PC-3Human Protein Atlas immunofluorescence, PC-3, antibody HPA050510; green = STEAP3, blue = nucleus, red = microtubules.
HPA · ICC-IF PC-3Human Protein Atlas immunofluorescence, PC-3, antibody HPA050510; green = STEAP3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for STEAP3 IHC & IF
🧪Run breast first and assess glandular cells for STEAP3 staining (HPA: Medium in breast glandular cells). Use oral mucosa squamous epithelial cells as the negative tissue (HPA: Not detected); on the breast slide, treat unstained neighboring non-glandular cells as internal negatives only after confirming their identity and lack of signal.
Positive control tissue: Adipose tissue (Adipocytes, HPA Medium)
Negative control tissue: Cerebral cortex (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show STEAP3 in PC-3, U2OS, with annotated localisation: Cytosol (approved) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host-species- and clonality-matched isotype controls, plus STEAP3-knockout tissue or a validated immunizing-peptide block as a biological specificity control (standard IHC practice). Quench endogenous peroxidase for chromogenic detection and check breast glandular lumens for nonspecific chromogen deposits (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state a fixative (datasheet: A30509). The caption uses Tris-EDTA at pH 9.0 for retrieval and a primary dilution of 1:200, but does not establish that this retrieval condition is required (datasheet: A30509). No matched evidence establishes that frozen sections or IF are easier than paraffin IHC; assess cellular staining separately from possible luminal deposits in breast glands (standard IHC practice).
HPA tissue IHC evidence for STEAP3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data. Pending retesting.). 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 → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Medium | Protein (IHC) | HPA → |
Section 3
Advanced STEAP3 IHC Tips
Troubleshoot STEAP3 staining in paraffin sections by checking retrieval, cellular pattern, controls and scoring before interpreting chromogenic signal.
What should I change when STEAP3 staining is weak after antigen retrieval?
Use heat-mediated Tris-EDTA, pH 9.0, for STEAP3 retrieval in paraffin sections (datasheet A30509). The selected image used this retrieval with primary antibody at 1:200 overnight at 4°C (datasheet A30509). If signal is weak, first check that sections reached the intended retrieval temperature and that slides remained covered by buffer throughout heating (standard IHC practice). Compare a repeat section with a modestly longer heating period while keeping the primary dilution and detection steps constant (standard IHC practice). Excessive heating can damage morphology, so judge signal together with preserved cellular detail (standard IHC practice).
How can I troubleshoot variable staining between differently fixed sections?
The selected paraffin-section caption does not state a fixative, so STEAP3-specific fixation sensitivity is unknown (datasheet A30509). Record each specimen’s fixative, fixation duration and processing history before comparing staining intensity across blocks (standard IHC practice). Run matched sections in the same batch using Tris-EDTA, pH 9.0, retrieval and the documented 1:200 primary dilution (datasheet A30509). Check whether weak signal accompanies poor morphology or uneven staining, and repeat processing only when a matched specimen is available (standard IHC practice). Do not attribute a difference to STEAP3 fixation sensitivity without a controlled comparison.
Is diffuse cytoplasmic or nuclear staining plausible for STEAP3?
STEAP3 is annotated as an endosome membrane protein with vesicular structures near the plasma membrane and nucleus (UniProt Q658P3 localisation). Tissue IHC shows mainly cytoplasmic expression, while separate ICC/IF data report cytosol and additional nucleolar localisation (HPA tissue IHC; HPA subcellular). Examine staining at high magnification for cytoplasmic or punctate distribution within intact cells, using the counterstain to define nuclear boundaries (standard IHC practice). A predominantly nuclear chromogenic pattern needs independent validation; nucleolar ICC/IF localisation alone does not establish that pattern in paraffin sections (HPA subcellular). Score compartments separately if both patterns remain reproducible.
Could the antibody miss a STEAP3 isoform or a processed epitope?
STEAP3 has 4 annotated isoforms, but the supplied caption does not identify the catalog antibody’s epitope or establish isoform coverage (UniProt Q658P3 isoforms; datasheet A30509). Its 6 transmembrane segments separate cytoplasmic and vesicular regions, so epitope position matters when assessing accessibility (UniProt Q658P3 topology). Glycosylation sites at residues 256 and 344 are annotated in vesicular regions; their effect on this antibody’s staining is unknown (UniProt Q658P3 topology and glycosylation). Obtain the epitope sequence and map it across isoforms before interpreting a negative section as absence of STEAP3. Use an independently validated antibody against a different region if isoform coverage is critical.
How should I check STEAP3 localisation by multiplex immunofluorescence?
For a separate IF experiment, pair STEAP3 with a marker identifying the expected cell type; glandular cells show medium tissue-IHC staining in several listed tissues (HPA tissue IHC). Choose spectrally separated fluorophores, favouring a far-red channel when tissue autofluorescence compromises shorter wavelengths, and include single-colour controls (standard IF practice). STEAP3 has cytoplasmic and vesicular membrane-facing regions, so select permeabilisation conditions after establishing which side contains the antibody epitope (UniProt Q658P3 topology). Compare punctate, cytosolic and nucleolar signals with the documented ICC/IF localisation, without assuming that those images validate this catalog antibody (HPA subcellular). Evaluate bleed-through and background before calling colocalisation (standard IF practice).
How do I separate STEAP3 signal from chromogenic background?
The selected paraffin-section image used primary antibody at 1:200 overnight at 4°C and secondary antibody at 1:200 for 45 minutes at room temperature (datasheet A30509). If staining is widespread, compare serial sections with and without primary antibody while holding detection conditions constant (standard IHC practice). Apply an appropriate peroxidase block before an enzyme-based DAB workflow, and inspect the no-primary section for endogenous enzyme signal (standard IHC practice). Check whether deposits cluster at section edges, folds or damaged tissue rather than within intact cells (standard IHC practice). Titrate primary antibody only after these controls identify antibody-dependent background.
What is a defensible way to quantify STEAP3 tissue staining? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring because tissue IHC reports mainly cytoplasmic STEAP3 staining (HPA tissue IHC). For chromogenic sections, record the percentage of viable cells at each intensity level and calculate an H-score from 0–300 (standard IHC practice). If cells are sparse, report positive-cell density per mm² of evaluable tissue; otherwise report percentage positive among the specified viable cell population (standard IHC practice). Apply identical segmentation, colour thresholds and exposure-independent brightfield acquisition settings across comparison groups (standard IHC practice). Exclude folds and necrosis, and keep cell-type scores separate when tissue composition differs (standard IHC practice).
What evidence distinguishes true STEAP3 staining from an artefact?
Judge a positive result by reproducible staining in intact cells, appropriate cytoplasmic distribution and a clean no-primary control (HPA tissue IHC; standard IHC practice). STEAP3 is annotated at endosome membranes, while tissue IHC is mainly cytoplasmic, so a punctate component is plausible but not required (UniProt Q658P3 localisation; HPA tissue IHC). Recheck staining confined to section edges, necrotic areas or unexpected cell populations against morphology and a matched control section (standard IHC practice). Exclude endogenous enzyme signal with the peroxidase control before assigning DAB deposits to STEAP3 (standard IHC practice). Interpret modest differences cautiously because HPA labels its tissue-IHC evidence Approved with low staining-to-RNA consistency pending retesting (HPA tissue IHC).
Boster reagents
Best STEAP3 / Metalloreductase STEAP3 IHC Antibodies
Two STEAP3 antibodies have IHC images from human colon cancer paraffin sections and mouse liver tissue, plus IF images from cells (catalog image captions).
Real IHC data
Anti-STEA3 Antibody
Cat # A30509
Real IHC data
Anti-Metalloreductase STEAP3 STEAP3 Antibody
Cat # A05568-1
A30509 lists IHC and IF applications, with images from human colon cancer paraffin sections and HeLa cells (A30509 applications and image captions). A05568-1 lists IHC-P and IF applications, with images from mouse liver tissue and mouse liver cells (A05568-1 applications and image captions).
Which to pick: For tissue IHC, choose A30509 for human colon cancer paraffin sections (A30509 IHC image caption: 1:200; fixative unreported) or A05568-1 for mouse liver tissue (A05568-1 IHC image caption: 2.5 μg/mL). For IF/ICC, A30509 lists both applications and has a HeLa cell IF image; A05568-1 lists IF and has a mouse liver cell IF image (catalog applications and IF image captions). For work across species, both list Human, Mouse and Rat reactivity; the supplied IHC images show human tissue for A30509 and mouse tissue for A05568-1 (catalog reactivity and IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q658P3 (STEA3_HUMAN, Metalloreductase STEAP3).
- Human Protein Atlas. STEAP3 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. STEAP3 subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the nucleoli..
- Human Protein Atlas. STEAP3 antibody validation summary (1 antibodies).
- Transcriptomic Characterization of Copper-Binding Proteins for Predicting Prognosis in Glioma. Brain sciences 2023 — PMC10605646.
- Six-transmembrane epithelial antigen of prostate 3 (STEAP3) is a potential prognostic biomarker in clear cell renal cell carcinoma that correlates with M2 macrophage infiltration and epithelial-mesenchymal. Cancer reports (Hoboken, N.J.) 2023 — PMC10432435.
- STEAP3 can predict the prognosis and shape the tumor microenvironment of clear cell renal cell carcinoma. BMC cancer 2022 — PMC9686107.
- High expression of six-transmembrane epithelial antigen of prostate 3 promotes the migration and invasion and predicts unfavorable prognosis in glioma. PeerJ 2023 — PMC10065001.
- PubMed PMID:12606722 — UniProt-cited evidence.
- PubMed PMID:15897894 — UniProt-cited evidence.
- PubMed PMID:10969787 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for STEAP3 (Metalloreductase STEAP3). Expected localisation Mainly cytoplasmic in tissue sections (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A30509). Validated IHC antibody: A30509 at 1:100 - 1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative Cerebral cortex. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human Colon cancer tissue; fixative not specified (datasheet A30509); 2. Fixation: Image fixative and duration unreported (datasheet A30509); 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: Tris-EDTA pH 9.0 (datasheet A30509); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-STEAP3, 1:100 - 1:300 (datasheet A30509); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining has low agreement with RNA expression (HPA tissue IHC); Isoform / epitope: 4 isoforms; check cytoplasmic vs vesicular epitope (UniProt); Regulation: Lower in hepatocellular carcinoma (UniProt). Evidence: UniProt Q658P3, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the…, Datasheet A30509, PMC9686107. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/STEAP3/immunohistochemistry-ihc-guide.