FZD3 / Frizzled-3 · IHC design guide

Design Immunohistochemistry for FZD3

Plan FZD3 paraffin-section IHC around the observed general cytoplasmic tissue pattern (HPA tissue IHC) and its annotated cell membrane localisation (UniProt). Use the catalog antibody’s IHC protocol, and interpret staining with the reported low agreement between antibody staining and RNA expression in mind (HPA tissue IHC).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for FZD3 (IHC for FZD3): expected localisation General cytoplasmic tissue staining (HPA tissue IHC); cell membrane localisation (UniProt), antibody A04680-1, validated IHC image, and IHC protocol steps — Printable **FZD3** IHC protocol sheet — expected localisation General cytoplasmic tissue staining (HPA tissue IHC); cell membrane localisation (UniProt), antibody A04680-1, controls and protocol steps. Open the full FZD3 IHC guide →

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FZD3 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	General cytoplasmic tissue staining (HPA tissue IHC); cell membrane localisation (UniProt)
	Staining pattern	General cytoplasmic staining across cell types (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6 HIER, heat-mediated (datasheet A04680-1)
	Positive control ⓘ	Adipose tissue+4 more · see all
	Negative control ⓘ	None in HPA (detected in all 45 tissues); use no-primary + isotype controls

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	Abundance regulation is unspecified (UniProt)
	Isoform / epitope	2 isoforms; map extracellular vs cytoplasmic epitopes (UniProt)

Section 1

Recommended FZD3 IHC & IF Protocols

The catalog antibody protocol uses citrate pH 6 retrieval (datasheet A04680-1). The published IHC protocols below report methods for human tissue and mouse hippocampal sections (PMC2453022; PMC7746375; PMC13617583).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A04680-1)
Fixation	Image fixative and duration unreported (datasheet A04680-1); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: Citrate pH 6, 20 min (datasheet A04680-1)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A04680-1)
Primary antibody	Rabbit anti-FZD3, 1μg/ml (datasheet A04680-1)
Primary incubation	Overnight at 4 °C (datasheet A04680-1)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A04680-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	FZD3-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control.

💡Decision noteStart with heat-mediated citrate pH 6 retrieval (datasheet A04680-1); one published FZD3 IHC protocol also used microwave heating in citrate pH 6 (PMC2453022).

Section 2

What Is the Expected FZD3 Staining Pattern?

FZD3 is a 7-pass cell-surface receptor, with an apical membrane location also reported (UniProt Q9NPG1 topology and subcellular location). In paraffin-section IHC, HPA instead describes a general cytoplasmic staining pattern, including high staining in adipocytes, glandular cells, hematopoietic cells, neurons, and cervical squamous epithelium (HPA tissue IHC). Treat that pattern as a reference, not proof of specificity: HPA rates the tissue IHC evidence Approved but reports low consistency with RNA expression (HPA tissue IHC).

What am I looking at on my slide?
Cytoplasmic stain in adipocytes, colon or adrenal glandular cells, bone marrow hematopoietic cells, or caudate neurons.	This matches HPA's reported High staining in those cell populations and its general cytoplasmic profile (HPA tissue IHC). Compare staining within the identified cell population; HPA's low RNA–staining consistency limits how confidently intensity alone identifies FZD3 (HPA tissue IHC).
A cell-edge accent accompanies cytoplasmic staining in a cell population reported positive by HPA.	Membrane staining is biologically plausible for a cell-surface receptor with 7 transmembrane segments (UniProt Q9NPG1 topology and subcellular location). HPA's tissue IHC profile is generally cytoplasmic, so a sharp edge alone should not be made a required pass criterion (HPA tissue IHC).
Predominantly nuclear chromogen in a paraffin section.	This departs from HPA's general cytoplasmic tissue IHC profile and UniProt's membrane assignment (HPA tissue IHC; UniProt Q9NPG1 subcellular location). Investigate localisation and controls before calling it FZD3; HPA's nucleoplasmic finding comes from ICC-IF and does not validate nuclear IHC staining (HPA subcellular ICC-IF).
Strong staining in an unexpected cell population, or a similar deposit across unrelated cell types.	Cross-reactivity or endogenous detection activity are possibilities to check, not established diagnoses (general IHC practice). FZD3 is widely expressed, so a cell type absent from HPA's short positive list is not automatically negative (UniProt Q9NPG1 tissue specificity; HPA tissue IHC).
No stain in a section expected to contain HPA high-staining cells.	First check that the relevant cells are present and assess the run controls (general IHC practice). Absence in one section does not by itself overturn HPA's reported high staining, especially given the Approved rating and low RNA–staining consistency (HPA tissue IHC).

💡Expected FZD3 appearanceA convincing IHC-positive result shows interpretable cytoplasmic chromogen in an identified HPA High cell population, with possible cell-edge accent; predominant nuclear stain or uniform deposit across unrelated cells warrants control checks (HPA tissue IHC; UniProt Q9NPG1 topology; general IHC practice).

How each factor affects the staining
Tissue and cell population	HPA reports High staining in selected adipocytes, glandular cells, hematopoietic cells, caudate neurons, and cervical squamous epithelial cells, but Low staining in oral-mucosa squamous epithelial cells (HPA tissue IHC). Identify the cells before comparing sections (general IHC practice).
Topology and epitope location	FZD3 has extracellular, transmembrane, and cytoplasmic regions; its FZ domain spans residues 23–136 (UniProt Q9NPG1 topology and domains). The antibody epitope is unspecified here, so topology alone cannot predict which staining compartment that reagent will show.
Protein processing and variants	The signal peptide spans residues 1–22, and UniProt lists Long and Short isoforms (UniProt Q9NPG1 processing and isoforms). No epitope or isoform coverage is supplied, so neither feature supports an isoform-specific reading of the chromogen.
Evidence strength and antibody validation	HPA calls tissue IHC Approved while noting low consistency with RNA expression; CAB024995 is IHC Approved, with no IHC Enhanced designation supplied (HPA tissue IHC; HPA antibodies). Treat distribution as a comparison point, then interpret it with assay controls (general IHC practice).
IF/ICC: should nuclear signal be expected?	HPA reports mainly nucleoplasmic ICC-IF signal, with nuclear membrane and vesicle signal also approved (HPA subcellular ICC-IF). That is an IF observation; the paraffin IHC reference remains HPA's general cytoplasmic profile (HPA tissue IHC).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
The expected high-staining cells are absent or hard to identify.	The chosen field or section may lack the relevant cell population (general IHC practice).	Inspect tissue architecture and select a field containing a documented HPA High population before judging the stain (HPA tissue IHC; general IHC practice).
Relevant cells are present, but chromogen is absent.	An assay step or reagent may have failed; this section alone does not identify which one (general IHC practice).	Review run controls, antibody dilution, retrieval, and detection setup as general IHC checks. No FZD3-specific retrieval condition or dilution is supplied (general IHC practice).
Most nuclei are strongly stained.	The pattern conflicts with the HPA tissue IHC profile; the separate ICC-IF nucleoplasmic observation cannot establish an IHC cause (HPA tissue IHC; HPA subcellular ICC-IF).	Compare the primary-omission control and cellular localisation, then reassess whether the signal is specific (general IHC practice).
Diffuse chromogen obscures cell boundaries.	Non-specific binding or excess detection background may reduce interpretability (general IHC practice).	Check blocking, antibody dilution, washes, and the primary-omission control; reassess staining only where cells remain identifiable (general IHC practice).
Chromogen appears broadly in unrelated cell types.	Endogenous detection activity or cross-reactivity is possible, but broad expression also limits tissue exclusion as a test (general IHC practice; UniProt Q9NPG1 tissue specificity).	Check the appropriate detection-background control and compare localisation with HPA's reported cytoplasmic pattern in identified cells (general IHC practice; HPA tissue IHC).
Only oral-mucosa squamous epithelium is weak or unstained.	HPA lists this cell population as Low, so a weak result there alone is a poor test of assay sensitivity (HPA tissue IHC).	Assess a section containing a documented HPA High population and its run controls before changing assay settings (HPA tissue IHC; general IHC practice).

Source: UniProt Q9NPG1 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing FZD3 staining

IHC analysis of FZD3 using anti-FZD3 antibody (A04680-1). FZD3 was detected in paraffin-embedded section of human colon cancer tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-FZD3 Antibody (A04680-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

A04680-1 · IHCIHC analysis of FZD3 using anti-FZD3 antibody (A04680-1). FZD3 was detected in paraffin-embedded section of human colon cancer tissue. Heat

IHC analysis of FZD3 using anti-FZD3 antibody (A04680-1). FZD3 was detected in paraffin-embedded section of human mammary cancer tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-FZD3 Antibody (A04680-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

A04680-1 · IHCIHC analysis of FZD3 using anti-FZD3 antibody (A04680-1). FZD3 was detected in paraffin-embedded section of human mammary cancer tissue. Hea

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000104290-FZD3/tissue/cerebral+cortex#imid_6760504

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000104290-FZD3/tissue/colon#imid_6760430

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000104290-FZD3/tissue/liver#imid_6760423

HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000104290-FZD3/tissue/pancreas#imid_6760382

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA042320; green = FZD3, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for FZD3 IHC & IF

🧪Run caudate first and score its neuronal cells for FZD3 staining (HPA: High in caudate neuronal cells). HPA detects FZD3 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and treat unstained neighboring cells on the positive slide as background comparators rather than proven FZD3-negative cells (HPA: no negative tissue rows; FZD3 detected in all 45 scored tissues).

Positive control tissue: Adipose tissue (Adipocytes, HPA High)

Negative control tissue: None in HPA: FZD3 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show FZD3 in A-431, MCF-7, U2OS, RPTEC/TERT1, hTERT-RPE1 (serum starved), NIH 3T3, with annotated localisation: Nucleoplasm (approved) (HPA subcellular).

Technical controls: Run a secondary-only slide with no primary antibody and a host- and immunoglobulin-class-matched isotype control; the caption identifies the primary as rabbit IgG, but does not state its clonality (A04680-1 tissue-IHC caption; standard IHC control practice). Use FZD3-knockout material as a biological specificity control if available, and quench endogenous peroxidase before chromogenic detection; neuronal lipofuscin can produce pigment that must be distinguished from DAB signal (standard IHC practice).

⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the selected A04680-1 paraffin-section caption leaves the fixative unreported (A04680-1 tissue-IHC caption). That caption uses heat retrieval in citrate buffer at pH 6 for 20 minutes, but does not establish that retrieval is required for every specimen (A04680-1 tissue-IHC caption). The evidence does not establish frozen sections or IF/ICC as easier than paraffin IHC; for caudate, assess neuronal pigment separately from chromogenic staining (A04680-1 tissue-IHC caption; HPA: ICC-IF images; standard IHC practice).

HPA tissue IHC evidence for FZD3

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.

Positive expression · recommended positive controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	High	Protein (IHC)	HPA →
Adrenal gland	Glandular cells	High	Protein (IHC)	HPA →
Appendix	Glandular cells	High	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	High	Protein (IHC)	HPA →
Caudate	Neuronal cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
None in HPA: FZD3 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

Section 3

Advanced FZD3 IHC Tips

Troubleshoot FZD3 chromogenic IHC by checking retrieval, controls, cellular localisation, and scoring before interpreting staining differences.

How should I optimise antigen retrieval for FZD3 in paraffin sections?

Start with heat-mediated citrate buffer at pH 6 for 20 minutes before chromogenic FZD3 IHC (datasheet A04680-1). The demonstrated colon cancer section used this retrieval, followed by a 10% goat serum block and 1 μg/ml primary antibody overnight at 4°C (caption A04680-1). If staining is weak, compare shorter and longer heating intervals on adjacent sections while holding antibody concentration and detection constant (general IHC practice). Score membrane-associated and cytoplasmic staining separately: FZD3 has 7 transmembrane segments, whereas reported tissue IHC staining is generally cytoplasmic (UniProt Q9NPG1 topology; HPA tissue IHC).

What fixation variables matter when FZD3 staining is weak or uneven?

Target-specific fixation sensitivity is unknown because the selected paraffin-section caption does not state a fixative (caption A04680-1). Record fixative identity, exposure time, processing delay, and section age, then compare matched sections using citrate pH 6 retrieval and identical detection conditions (general IHC practice; datasheet A04680-1). Include a positive tissue control in each batch; colon glandular cells are reported to stain at a high level, but morphology must remain interpretable (HPA tissue IHC; general IHC practice). Use no-primary controls and a consistent counterstain to identify processing-related haze, and establish any FZD3-specific fixation effect by direct comparison (general IHC practice).

Where should FZD3 staining appear, and how should nuclear signal be handled?

FZD3 is annotated at the cell surface and apical membrane, with 7 transmembrane segments and a cytoplasmic C terminus (UniProt Q9NPG1 topology/localisation). Tissue IHC is reported as generally cytoplasmic, while cell imaging reports nucleoplasm, nuclear membrane, and vesicles; record these compartments separately (HPA tissue IHC; HPA subcellular). Check whether membrane outlines persist across intact cells and whether cytoplasmic staining follows cell boundaries rather than folds or necrotic regions (general IHC practice). Interpret isolated nuclear chromogen cautiously until it is reproduced with an independent epitope reagent or orthogonal expression evidence, given the reported low consistency between tissue staining and RNA data (HPA tissue IHC; general IHC practice).

Can this antibody distinguish the Long and Short FZD3 isoforms?

FZD3 has Long and Short isoforms, but the supplied antibody evidence does not map the catalog antibody’s immunogen or epitope (UniProt Q9NPG1 isoforms; caption A04680-1). Chromogenic staining with A04680-1 therefore cannot be assigned to either isoform without epitope mapping or isoform-specific controls (UniProt Q9NPG1 isoforms; general IHC practice). The mature chain spans residues 23–666; its FZ domain lies at 23–136, and glycosylation sites are annotated at 42, 265, and 356 (UniProt Q9NPG1 processing/domains/glycosylation). If epitope coordinates become available, compare them with both isoform sequences and membrane topology before interpreting retrieval effects (UniProt Q9NPG1 topology; general IHC practice).

How can IF help identify the cells producing chromogenic FZD3 signal?

Compare FZD3 chromogenic IHC with multiplex IF on adjacent sections, pairing FZD3 with a marker for the expected cell type; colon glandular cells are reported high (HPA tissue IHC; general IF practice). Choose separated fluorophores and put the weaker signal in a channel with low tissue autofluorescence; include single-stain and no-primary controls (general IF practice). Use mild permeabilisation for an intracellular epitope and minimise it for a surface epitope; FZD3 has 7 membrane-spanning segments, but this antibody’s epitope is unspecified (UniProt Q9NPG1 topology; caption A04680-1; general IF practice). Compare staining within individual cells and compartments, using the documented paraffin-section IHC workflow as the chromogenic reference (caption A04680-1; general IF practice).

How do I reduce diffuse or edge-heavy FZD3 DAB background?

Compare no-primary and secondary-only sections with the test slide to identify secondary-antibody binding or endogenous enzyme staining (general IHC practice). The demonstrated workflow used 10% goat serum, a biotinylated secondary antibody for 30 minutes at 37°C, and SABC with DAB (caption A04680-1). Apply an appropriate peroxidase block and assess endogenous biotin with this avidin-biotin detection method; optimise blocking and wash stringency on matched sections (general IHC practice; caption A04680-1). Flag dark tissue edges, folds, and necrosis before scoring, and reassess primary concentration if diffuse staining persists outside intact cells (general IHC practice).

How should FZD3 staining be scored across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the analysis region and cell type before scoring: FZD3 is widely expressed, and colon glandular cells show high tissue IHC staining (UniProt Q9NPG1 tissue specificity; HPA tissue IHC). For comparable sections, report percent positive cells and an H-score using predefined intensity categories and a consistent chromogen threshold (general IHC practice). Normalise positive cells to evaluable cells of the same type, or stained area to viable tissue area in mm²; exclude folds, edges, and necrosis (general IHC practice). Record membrane-associated and cytoplasmic scores separately because FZD3 topology and reported tissue staining indicate distinct compartments (UniProt Q9NPG1 topology; HPA tissue IHC).

How can I distinguish genuine FZD3 staining from artefact?

Look for reproducible cellular staining in intact, anatomically matched regions; colon glandular cells provide a documented high-staining comparison (HPA tissue IHC; general IHC practice). Membrane or apical staining fits FZD3 localisation, and cytoplasmic tissue staining is reported; isolated nuclear DAB needs independent validation despite nuclear localisation in cell imaging (UniProt Q9NPG1 localisation; HPA tissue IHC; HPA subcellular). Exclude edge-only, fold-associated, necrotic, or no-primary-positive deposits, and control endogenous peroxidase during DAB development (general IHC practice). Interpret staining alongside morphology and independent expression evidence: HPA rates tissue IHC approved but reports low consistency with RNA expression (HPA tissue IHC; general IHC practice).

Boster reagents

Best FZD3 / Frizzled-3 IHC Antibodies

The IHC-validated anti-FZD3 antibody has paraffin-section images from human colon and mammary cancer and mouse and rat kidney (catalog IHC captions).

Real IHC data

Anti-FZD3 Antibody ®

Cat # A04680-1

View product →

A04680-1 is listed for IHC in human, mouse and rat (catalog applications and reactivity). Its IHC images show paraffin sections of human colon and mammary cancer and mouse and rat kidney (catalog IHC captions).

Which to pick: For tissue IHC, choose A04680-1, a rabbit polyclonal antibody with paraffin-section images; the fixative is unreported (catalog dilution_raw; catalog IHC captions). For cross-species IHC, A04680-1 has images from human, mouse and rat tissue (catalog IHC captions). For IF/ICC, seek a separately validated reagent: A04680-1 has no listed IF/ICC application or IF image (catalog applications; catalog IF image alts).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry Q9NPG1 (FZD3_HUMAN, Frizzled-3).
Human Protein Atlas. FZD3 tissue IHC expression (reliability: Approved).
Human Protein Atlas. FZD3 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the nuclear membrane and vesicles..
Human Protein Atlas. FZD3 antibody validation summary (2 antibodies).
Common dysregulation of Wnt/Frizzled receptor elements in human hepatocellular carcinoma. British journal of cancer 2008 — PMC2453022.
Requirement of DLG1 for cardiovascular development and tissue elongation during cochlear, enteric, and skeletal development: possible role in convergent extension. PloS one 2015 — PMC4393223.
NEAT1 regulates microtubule stabilization via FZD3/GSK3β/P-tau pathway in SH-SY5Y cells and APP/PS1 mice. Aging 2020 — PMC7746375.
Identification and Experimental Validation of PANoptosis Key Genes for Constructing a PANoptosis Risk Diagnostic Model in Intervertebral Disc Degeneration. The journal of gene medicine 2026 — PMC13617583.
PubMed PMID:10777673 — UniProt-cited evidence.
PubMed PMID:10873558 — UniProt-cited evidence.
PubMed PMID:11407985 — UniProt-cited evidence.