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FZD6 / Frizzled-6 · IHC design guide
Design Immunohistochemistry for FZD6
Plan FZD6 paraffin IHC using high-staining colon glandular cells as a positive control (HPA tissue IHC). Compare the observed cytoplasmic pattern (HPA tissue IHC) with membrane localization (UniProt), and start within the catalog antibody’s 1:100–1:300 IHC range (datasheet: A04241-1).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
FZD6 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 tissue staining (HPA tissue IHC); cell membrane localization (UniProt) | |
| Staining pattern | Cytoplasmic signal in most tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Lymph node |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent. (standard IHC practice; not target-specific) | |
| Caveat | Cytoplasmic IHC differs from membrane localization (HPA tissue IHC; UniProt) | |
| Regulation | No staining-intensity regulator specified (UniProt) | |
| Isoform / epitope | 2 isoforms; check epitope coverage and membrane side (UniProt) |
Section 1
Recommended FZD6 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by four published FZD6 IHC methods (PMC5248601; PMC4578624; PMC8883113; PMC12311012).
| Sample | Paraffin-embedded human brain tissue; fixative not specified (datasheet A04241-1) |
| Fixation | Image fixative and duration unreported (datasheet A04241-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| 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-FZD6, 1:100-1:300 (datasheet A04241-1) |
| 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 | FZD6-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval rule); compare the published retrieval conditions where stated (PMC5248601; PMC8883113).
Section 2
What Is the Expected FZD6 Staining Pattern?
FZD6 is a seven-pass membrane receptor with cell-surface, apical-membrane and intracellular-membrane localizations (UniProt O60353 topology and subcellular location). In paraffin-section IHC, HPA reports predominantly cytoplasmic staining across tissues, including high staining in several glandular and epithelial cell populations (HPA tissue IHC). Its IHC profile is Approved, with medium agreement between staining and RNA expression and pending external verification (HPA tissue IHC).
| Cytoplasmic staining in colon, duodenum or breast glandular cells, or bronchial respiratory epithelial cells. | These are HPA-reported high-staining cell populations (HPA tissue IHC). Record the stained cell type and distribution as well as intensity. Cytoplasmic staining is compatible with the reported IHC profile; membrane accentuation is biologically plausible from FZD6 localization, but is not required to match HPA tissue IHC (HPA tissue IHC; UniProt O60353 subcellular location). |
| Membrane-enriched signal in an appropriate epithelial cell population. | FZD6 has seven transmembrane segments and is annotated at the cell surface and apical membrane (UniProt O60353 topology and subcellular location). Interpret membrane staining alongside the tissue's cell identity and cytoplasmic IHC pattern. HPA's plasma-membrane localization is supported by ICC-IF, a separate application (HPA subcellular ICC-IF; HPA tissue IHC). |
| Predominantly nuclear or extracellular-deposit staining, without a coherent cellular pattern. | These compartments do not match the supplied FZD6 localization annotations or HPA tissue IHC summary (UniProt O60353 subcellular location; HPA tissue IHC). Treat the result as suspect and review controls and morphology before scoring it as FZD6. An isolated unusual compartment cannot establish a new localization from this evidence (general IHC interpretation practice). |
| Strong signal in an unexpected cell population, such as lymph-node non-germinal-center cells. | HPA reports FZD6 as not detected in that specific lymph-node population (HPA tissue IHC). If signal persists there, consider cross-reactivity or endogenous chromogen-generating activity and inspect the antibody-omission control (general chromogenic IHC practice). The HPA observation does not make every lymph-node cell a negative control (HPA tissue IHC). |
| Diffuse staining across cells and surrounding tissue, or no staining in a selected HPA high-staining population. | A uniform haze weakens cell-level interpretation and may reflect background from detection reagents or insufficient blocking or washing (general chromogenic IHC practice). Absence of signal in a suitable positive population warrants a run-level check before a biological negative call; HPA's Approved rating still has medium staining–RNA consistency and awaits external verification (HPA tissue IHC). |
💡Expected FZD6 appearanceCall a section positive when identifiable HPA high-staining glandular or epithelial cells show convincing cytoplasmic signal, with possible membrane accentuation, above local background; isolated nuclear, extracellular or indiscriminate tissue-wide color is suspect (HPA tissue IHC; UniProt O60353 subcellular location; general IHC interpretation practice).
| Tissue and cell selection | Use a named HPA high-staining population, such as colon glandular or bronchial respiratory epithelial cells, to judge whether a run can reveal the expected pattern (HPA tissue IHC). Salivary glandular cells, fibroblasts in soft tissue and adipocytes are listed as low, so a faint result in those cells is less informative (HPA tissue IHC). |
| Compartment and antibody epitope | FZD6 contains extracellular, transmembrane and cytoplasmic regions (UniProt O60353 topology). The supplied evidence gives no epitope for the IHC antibody, so it cannot predict which cellular face its stain represents or justify an FZD6-specific retrieval condition. Evaluate retrieval using the antibody's actual instructions and controls (general IHC practice). |
| Processing, glycosylation and isoforms | UniProt lists a cleaved signal peptide at residues 1–18, a mature chain at 19–706, glycosylation sites at 38 and 352, and two isoforms (UniProt O60353 processing, glycosylation and isoforms). Without an antibody epitope or isoform-specific IHC evidence, these features do not establish a distinct staining pattern or a reason to score weak tissue as negative. |
| Strength of tissue evidence | HPA labels the tissue IHC profile Approved and reports medium consistency with RNA plus pending external verification; its listed antibody, HPA017991, has IHC Approved status (HPA tissue IHC; HPA antibody validation). Use those data as a reference pattern, while requiring appropriate run controls for each experiment (general IHC practice). |
| IF/ICC interpretation — can it confirm localization? | On its separate guide page, IF/ICC can assess the HPA-supported plasma-membrane localization; primary-cilium and basal-body localizations are marked uncertain (HPA subcellular ICC-IF). These ICC-IF findings can inform interpretation but do not supply an IHC-P protocol or override the cytoplasmic tissue-IHC profile (HPA subcellular ICC-IF; HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in colon or duodenal glandular cells. | The selected cells are reported as high staining, so the run or section may have failed (HPA tissue IHC; general IHC practice). | Confirm cell identity and section quality; inspect a known-positive run control, primary-antibody application and detection steps before calling the tissue negative (general IHC practice). |
| Weak signal in salivary glandular cells, fibroblasts or adipocytes. | HPA lists these populations as low staining (HPA tissue IHC). | Check a named HPA high-staining cell population in the same run before changing conditions; record the low result by cell type and avoid treating it alone as assay failure (HPA tissue IHC; general IHC practice). |
| All compartments have diffuse chromogen. | Background from detection chemistry, endogenous activity or inadequate blocking or washing can obscure cellular staining (general chromogenic IHC practice). | Inspect an antibody-omission control, verify endogenous-activity blocking appropriate to the detection system, and review washing and counterstain before scoring FZD6 (general chromogenic IHC practice). |
| Unexpected nuclear staining dominates the section. | Nuclear localization is absent from the supplied FZD6 annotations and HPA tissue-IHC pattern (UniProt O60353 subcellular location; HPA tissue IHC). | Check control sections and morphology, then compare cytoplasmic staining in a named HPA high-staining population; do not count nuclear color alone as a confirmed positive (HPA tissue IHC; general IHC practice). |
| Strong staining appears in lymph-node non-germinal-center cells. | That population is reported as not detected; cross-reactivity or endogenous detection activity is possible (HPA tissue IHC; general chromogenic IHC practice). | Compare the same population with an antibody-omission control and inspect the positive control before interpreting the unexpected signal (general IHC practice). |
| Results differ between IHC and IF/ICC. | HPA describes cytoplasmic tissue-IHC expression but supported plasma-membrane localization by ICC-IF; the observations come from different applications (HPA tissue IHC; HPA subcellular ICC-IF). | Score each application against its own reported pattern and controls. For IF/ICC interpretation, use its separate guide; do not infer an IHC-P protocol or reject cytoplasmic IHC solely from ICC-IF images (HPA tissue IHC; HPA subcellular ICC-IF; general IHC practice). |
Experiment evidence
IHC & IF images showing FZD6 staining
A04241-1 · IHCImmunohistochemistry analysis of paraffin-embedded human brain tissue, using FZD6 Antibody. The picture on the right is blocked with the syn
A04241-1 · IFImmunofluorescence analysis of MCF7 cells, using FZD6 Antibody. The picture on the right is blocked with the synthesized peptide.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000164930-FZD6/tissue/cerebral+cortex#imid_4765660
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000164930-FZD6/tissue/colon#imid_4765586
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000164930-FZD6/tissue/liver#imid_4765571
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000164930-FZD6/tissue/kidney#imid_4765572
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA017991; green = FZD6, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA017991; green = FZD6, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for FZD6 IHC & IF
🧪Run bronchus first: respiratory epithelial cells should stain (HPA: High in bronchus respiratory epithelial cells). Use lymph node non-germinal center cells as the biological tissue negative (HPA: Not detected); cells without specific staining on the bronchus slide should show counterstain alone, without a defined membrane signal (UniProt O60353: cell membrane localization).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Lymph node (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show FZD6 in A-431, U-251MG, U2OS, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Plasma membrane (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype-matched control from the primary antibody’s host species at the same concentration (standard IHC practice). Use FZD6 knockout tissue if available or a peptide-block control, as shown for the catalog antibody A04241-1 (selected tissue-IHC caption); quench endogenous peroxidase for chromogenic detection and check bronchial luminal material for trapped stain (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or antigen-retrieval dependency is reported in the supplied evidence; optimize retrieval empirically (supplied target/application evidence). The A04241-1 paraffin-section caption does not state its fixative (selected tissue-IHC caption). HPA supports plasma-membrane localization in ICC-IF, but the supplied evidence does not establish that IF or frozen sections are easier than paraffin IHC (HPA: plasma membrane, supported); inspect bronchial mucus for nonspecific chromogen retention (standard IHC practice).
HPA tissue IHC evidence for FZD6
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Lymph node | Non-germinal center cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced FZD6 IHC Tips
Troubleshoot FZD6 chromogenic IHC in paraffin sections by checking retrieval, staining compartment, controls and scoring before interpreting biological differences.
How should I retrieve FZD6 in paraffin sections when staining is weak?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval rule). Let sections cool in the retrieval buffer, then compare staining with an identically processed control section to assess whether weak signal persists (standard IHC practice). The catalog antibody has a paraffin-section brain image with peptide-blocked staining, but its caption reports no retrieval conditions (catalog antibody A04241-1 caption). If staining remains weak, test an alternative retrieval buffer on adjacent sections while holding antibody incubation and detection constant (standard IHC practice). Score membrane and cytoplasmic staining separately, since both patterns have supporting localization evidence (HPA subcellular; HPA tissue IHC).
Can fixation explain variable FZD6 staining between paraffin samples?
Target-specific fixation sensitivity is unknown: the catalog antibody’s paraffin-section brain caption does not state the fixative or fixation duration (catalog antibody A04241-1 caption). Record each specimen’s fixative and processing history before comparing stain intensity, since variable preparation can affect antigen accessibility in IHC (standard IHC practice). For a prospective series, use a consistent routine fixation and processing schedule, then stain comparison sections in the same run (standard IHC practice). If one batch stains weakly, compare retrieval and detection on adjacent sections before assigning the difference to FZD6 abundance (standard IHC practice). Neither tissue staining patterns nor receptor topology establish a FZD6-specific fixation effect (HPA tissue IHC; UniProt O60353 topology).
Should FZD6 staining appear at the membrane or in the cytoplasm?
Expect a membrane-associated pattern because FZD6 is a 7-pass receptor with cell-surface and other membrane localizations (UniProt O60353 topology and subcellular location). Plasma membrane localization is supported in cell images, whereas cilium and basal-body assignments are uncertain (HPA subcellular). Cytoplasmic staining also occurs across most tissues in the tissue IHC profile, so cytoplasmic signal alone should not be dismissed (HPA tissue IHC). Compare the membrane and cytoplasmic compartments on the same section, using a matched negative control to judge diffuse deposit (standard IHC practice). Interpret apical enrichment cautiously: apical localization with FZD3 is reported by similarity, not as direct evidence for this specimen (UniProt O60353 subcellular location).
How does the antibody epitope affect FZD6 staining and isoform claims?
FZD6 has 2 reported isoforms, but the supplied catalog caption does not identify this antibody’s epitope or establish isoform selectivity (UniProt O60353 isoforms; catalog antibody A04241-1 caption). Check the antibody’s documented immunogen before claiming that staining represents either isoform specifically (standard IHC practice). The mature chain spans residues 19–706, with extracellular, transmembrane and cytoplasmic regions that expose different epitopes to section processing (UniProt O60353 processing and topology). Glycosylation is annotated at residues 38 and 352; consider epitope accessibility if an antibody targets either nearby extracellular region (UniProt O60353 glycosylation and topology). Treat the peptide-blocked brain image as evidence of competition for this antibody, rather than proof of isoform specificity (catalog antibody A04241-1 caption).
How can I check FZD6 localization with multiplex IF?
Use IF as a separate validation experiment and pair FZD6 with a marker for the cell population identified in the IHC section (standard IF practice). Choose a fluorophore channel that separates antibody signal from the specimen’s autofluorescence, and include single-color and no-primary controls when setting acquisition (standard IF practice). If the antibody recognizes an extracellular epitope, first assess staining without detergent; a cytoplasmic-tail epitope generally requires permeabilisation after fixation (UniProt O60353 topology; standard IF practice). The antibody epitope is unspecified in the supplied caption, so determine which access condition is appropriate before interpreting a negative result (catalog antibody A04241-1 caption). Plasma membrane is the supported subcellular reference pattern for comparison (HPA subcellular).
How do I distinguish FZD6 staining from chromogenic background?
Inspect a no-primary section for detection-system background and include a peroxidase block before chromogenic detection when using an HRP and DAB workflow (standard IHC practice). Check whether brown deposit follows cell boundaries or fills recognizable cells, rather than appearing over folds, cut edges or damaged regions (standard IHC practice). The catalog antibody’s paraffin-section brain image includes a synthesized-peptide block, which provides a useful comparison for antibody-dependent staining with that exact reagent (catalog antibody A04241-1 caption). Peptide competition alone cannot establish the compartment or cell identity of every stained structure (standard IHC interpretation). Compare candidate signal with the supported plasma membrane localization and the reported cytoplasmic tissue pattern (HPA subcellular; HPA tissue IHC).
How should I score FZD6 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the sampled cell population and score membrane and cytoplasmic staining separately before combining results across sections (HPA subcellular; HPA tissue IHC; standard IHC practice). For each compartment, record the percentage of cells at intensity scores 0–3 and calculate an H-score from 0–300 (standard IHC practice). Alternatively, report the percentage of positive cells using one prespecified threshold across the series (standard IHC practice). Normalize counts to the number of eligible cells, or report positive-cell density per mm² of viable tissue when area is the denominator (standard IHC practice). Keep retrieval, detection, exposure to chromogen and scoring rules consistent between samples (standard IHC practice).
When does FZD6 staining support a biological conclusion?
Treat staining in the expected cell population and a plausible membrane or cytoplasmic compartment as stronger evidence than isolated brown deposits (HPA subcellular; HPA tissue IHC). High tissue IHC staining is reported in colon glandular cells and bronchial respiratory epithelial cells, while non-germinal-center lymph-node cells are reported as not detected (HPA tissue IHC). Use those cell-level references cautiously because the tissue IHC profile has medium agreement with RNA data and awaits external verification (HPA tissue IHC reliability). Exclude section edges, necrotic areas and endogenous-enzyme signal using morphology and a no-primary control before scoring (standard IHC practice). A peptide-blocked signal in the catalog brain image supports antibody-dependent staining there, but does not by itself establish function or pathway activity (catalog antibody A04241-1 caption; standard IHC interpretation).
Boster reagents
Best FZD6 / Frizzled-6 IHC Antibodies
A04241-1 has IHC data from paraffin-embedded human brain tissue and IF data from MCF7 cells (catalog image captions). Its listed reactivity covers human and mouse (catalog: A04241-1).
Real IHC data
Anti-Frizzled-6 FZD6 Antibody
Cat # A04241-1
A04241-1 is the only SKU with a card here; its IHC image shows paraffin-embedded human brain tissue with a peptide-blocked comparison (catalog IHC image caption). Its IF image shows MCF7 cells with a peptide-blocked comparison (catalog IF image caption).
Which to pick: For tissue IHC, choose A04241-1: IHC is listed at 1:100–1:300, and its own image documents paraffin-embedded human brain tissue; the fixative is unreported (catalog applications and dilution; catalog IHC image caption). For IF, A04241-1 has an MCF7 image, while A04241 lists IF and A04241T167 lists ICC; neither of the latter has an image in this payload (catalog applications and image captions). For human, mouse and rat IF or ICC work, consider A04241 or A04241T167 according to the listed application; their clonality is unreported, and neither lists IHC (catalog applications, reactivity and clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O60353 (FZD6_HUMAN, Frizzled-6).
- Human Protein Atlas. FZD6 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. FZD6 subcellular location (ICC-IF): Mainly localized to the plasma membrane. In addition localized to the primary cilium and basal body..
- Human Protein Atlas. FZD6 antibody validation summary (1 antibodies).
- Functional and prognostic significance of the genomic amplification of frizzled 6 (FZD6) in breast cancer. The Journal of pathology 2017 — PMC5248601.
- FZD6 expression is negatively regulated by miR-199a-5p in human colorectal cancer. BMB reports 2015 — PMC4578624.
- The Prognostic Value of the Developmental Gene FZD6 in Young Saudi Breast Cancer Patients: A Biomarkers Discovery and Cancer Inducers OncoScreen Approach. Frontiers in molecular biosciences 2022 — PMC8883113.
- Association of Hsa_circ_0059511 with the sensitivity of the temozolomide chemotherapeutic treatment in human glioma cells. Scientific reports 2025 — PMC12311012.
- PubMed PMID:9480858 — UniProt-cited evidence.
- PubMed PMID:11707601 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for FZD6 (Frizzled-6). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC); cell membrane localization (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A04241-1 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Lymph node. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human brain tissue; fixative not specified (datasheet A04241-1); 2. Fixation: Image fixative and duration unreported (datasheet A04241-1); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 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-FZD6, 1:100-1:300 (datasheet A04241-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep paraffin-section fixation consistent. (standard IHC practice; not target-specific); Caveat: Cytoplasmic IHC differs from membrane localization (HPA tissue IHC; UniProt); Isoform / epitope: 2 isoforms; check epitope coverage and membrane side (UniProt); Regulation: No staining-intensity regulator specified (UniProt). Evidence: UniProt O60353, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the plasma membrane. In addition localized to…, Datasheet A04241-1, PMC5248601, PMC4578624. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/FZD6/immunohistochemistry-ihc-guide.