FXYD6 / FXYD domain-containing ion transport regulator 6 · IHC design guide

Design Immunohistochemistry for FXYD6

Plan chromogenic paraffin IHC for FXYD6 using kidney tubules or hepatocytes as high-staining controls (HPA tissue IHC). Start the IHC-validated antibody at 1:25 (datasheet: IHC-P) and assess the cytoplasmic pattern against a not-detected control such as adipocytes (HPA tissue IHC).

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

Immunohistochemistry protocol sheet for FXYD6 (IHC for FXYD6): expected localisation Cytoplasmic tissue staining (HPA tissue IHC); cell membrane location (UniProt), antibody A09541, validated IHC image, and IHC protocol steps — Printable **FXYD6** IHC protocol sheet — expected localisation Cytoplasmic tissue staining (HPA tissue IHC); cell membrane location (UniProt), antibody A09541, controls and protocol steps. Open the full FXYD6 IHC guide →

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FXYD6 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 location (UniProt)
	Staining pattern	Cytoplasmic staining in most tissues (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen)
	Positive control ⓘ	Cerebellum+4 more · see all
	Negative control ⓘ	Adipose tissue+4 more · see all

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific)
	Caveat	Endogenous liver peroxidase may raise DAB background (standard IHC practice; HPA tissue IHC: hepatocytes high)
	Regulation	Expression regulation unreported (UniProt)
	Isoform / epitope	2 isoforms; check cytoplasmic C-terminal epitope coverage (UniProt; datasheet: C-term)

Section 1

Recommended FXYD6 IHC & IF Protocols

The catalog antibody protocol is accompanied by three published chromogenic IHC protocols for FXYD6 (PMC3881923; PMC7874599; PMC11958163).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded H. liver tissue; fixative not specified (datasheet A09541)
Fixation	Image fixative and duration unreported (datasheet A09541); 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-FXYD6, 1:25 (datasheet A09541)
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	FXYD6-positive staining in cells in granular layer of cerebellum (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 heat retrieval at 95–98 °C for 20 min (page antigen-retrieval setting); adjust using the cited microwave protocols.

Section 2

What Is the Expected FXYD6 Staining Pattern?

FXYD6 is a cell-membrane protein with one transmembrane segment and an extracellular region spanning residues 19–35 (UniProt Q9H0Q3 topology). In paraffin IHC, expect chiefly cytoplasmic staining in cells of the cerebellar granular layer, kidney tubules, hepatocytes, and cerebral-cortex neuropil (HPA tissue IHC). HPA rates its tissue-IHC profile Enhanced for consistency with RNA expression; external verification remains pending (HPA tissue IHC).

What am I looking at on my slide?
Strong staining in kidney tubule cells or hepatocytes, with a predominantly cytoplasmic appearance.	This matches two HPA High tissue-IHC observations and its broader cytoplasmic profile (HPA tissue IHC). Record the stained cell population and compartment; the cell-membrane assignment permits a peripheral component, but does not require a crisp membrane rim in every paraffin section (UniProt Q9H0Q3 subcellular location; HPA tissue IHC).
Predominantly nuclear staining on a paraffin IHC slide, especially when expected cytoplasmic staining is absent.	Treat this as discordant with the reported tissue-IHC profile and investigate an artefact before calling it specific (HPA tissue IHC; general IHC practice). HPA separately approves nucleoplasm and cytosol localisation in ICC-IF, so a nuclear signal alone cannot settle the interpretation across assays (HPA ICC-IF).
Strong staining of adipocytes or bone-marrow hematopoietic cells while a positive control stains as expected.	Both populations are listed as Not detected in HPA tissue IHC. Check morphology and detection controls for cross-reactivity or endogenous detection activity before assigning FXYD6 expression (HPA tissue IHC; general IHC practice). A discrepant result calls for review, not automatic exclusion of a biological difference.
Broad haze across cells, extracellular spaces, or the entire section, with little distinction between cell types.	This obscures the cell-specific pattern needed for interpretation. Review blocking, washes, primary-antibody concentration, and chromogen development; compare a no-primary control for detection background (general IHC practice). HPA's reported cytoplasmic expression in most tissues does not make uniform slide-wide haze a specific result (HPA tissue IHC).
Little or no staining in kidney tubules, hepatocytes, or cerebellar granular-layer cells.	These are High HPA tissue-IHC populations, so first examine control-tissue integrity, antibody conditions, retrieval, and detection performance (HPA tissue IHC; general IHC practice). A negative run cannot establish biological absence until the assay produces its expected positive-control signal (general IHC practice).

💡Expected FXYD6 appearanceCall a positive paraffin-IHC result when identifiable kidney tubule cells, hepatocytes, or cerebellar granular-layer cells show clear, predominantly cytoplasmic staining at the HPA-reported High level; isolated nuclear staining or uniform slide-wide haze warrants control-based review (HPA tissue IHC; general IHC practice).

How each factor affects the staining
Compartment and topology	UniProt places FXYD6 at the cell membrane, with one transmembrane segment at residues 36–57; HPA describes tissue-IHC staining as cytoplasmic in most tissues (UniProt Q9H0Q3 topology; HPA tissue IHC). Interpret fine peripheral staining alongside the observed paraffin pattern rather than imposing a membrane-only scoring rule.
Choice of comparison tissue	Kidney tubule cells, hepatocytes, cerebellar granular-layer cells, cerebral-cortex neuropil, and seminal-vesicle glandular cells are High in HPA tissue IHC. Appendix, colon, and duodenal glandular cells are Medium; several listed populations are Not detected (HPA tissue IHC). Compare like cell types when assessing intensity.
Validation and interpretation limits	HPA reports an Enhanced tissue-IHC profile with external verification pending; antibody HPA041334 also has Enhanced IHC status (HPA tissue IHC; HPA antibodies). These support the reported pattern but do not establish the cause of every discordant stain or a target-specific fixation effect.
Processing and isoforms	UniProt lists a signal peptide at residues 1–18, a mature chain at 19–95, and 2 isoforms (UniProt Q9H0Q3). Antibody epitope and isoform coverage are unspecified here, so neither differential isoform staining nor processing-related signal loss can be predicted from this record.
IF/ICC Q: Should its localisation decide paraffin-IHC scoring?	A: HPA approves nucleoplasm and cytosol localisation in ICC-IF and lists Rh30 images (HPA ICC-IF). For this paraffin-IHC guide, score against the tissue-IHC cell and compartment pattern; the ICC-IF result is a separate assay observation (HPA tissue IHC; HPA ICC-IF).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
A known-positive tissue is blank, including kidney tubules or hepatocytes.	The run may lack usable primary-antibody, retrieval, or detection signal; these cell types are High in HPA tissue IHC (HPA tissue IHC; general IHC practice).	Confirm tissue and section quality, then review the validated IHC antibody conditions, retrieval, reagent sequence, and a working positive-control slide (general IHC practice). Do not assign FXYD6-negative status from a failed positive control.
The section shows only nuclear staining in a proposed IHC-positive population.	This differs from HPA's chiefly cytoplasmic tissue-IHC profile, although nucleoplasm is an approved ICC-IF location (HPA tissue IHC; HPA ICC-IF).	Inspect morphology and compare the positive control, no-primary control, and antibody-validation information (general IHC practice). Report the assay-specific discrepancy rather than transferring an ICC-IF compartment call directly to paraffin IHC.
A nominally negative population stains strongly, such as adipocytes or bone-marrow hematopoietic cells.	HPA records these populations as Not detected; nonspecific binding or endogenous detection activity is a possible IHC explanation (HPA tissue IHC; general IHC practice).	Verify the cell identity, examine a no-primary control, and review blocking and detection steps (general IHC practice). Treat a persistent discrepancy as requiring independent confirmation before calling it FXYD6.
Diffuse brown haze makes cytoplasmic staining difficult to locate.	Background from binding, inadequate washing, or detection development can obscure cell boundaries (general IHC practice).	Compare the no-primary control; review antibody concentration, blocking, washes, and development time within the validated workflow (general IHC practice). Score only staining that remains distinct from background in identifiable cells.
A weak glandular signal appears in appendix, colon, or duodenum.	HPA rates these glandular populations Medium, whereas several suggested positive-control populations are High (HPA tissue IHC).	Compare cells of the same type and include a High HPA tissue-IHC control before calling the run insensitive (HPA tissue IHC; general IHC practice). Preserve the observed intensity in the record rather than forcing a binary call.
An IF/ICC image and a paraffin-IHC slide appear to disagree on compartment.	HPA approves nucleoplasm and cytosol in ICC-IF, while its tissue-IHC summary describes cytoplasmic expression in most tissues (HPA ICC-IF; HPA tissue IHC).	Document assay, cell type, and compartment separately; interpret this IHC slide against HPA tissue-IHC observations and its own controls (HPA tissue IHC; general IHC practice). Use the dedicated IF/ICC guide for IF assay decisions.

Source: UniProt Q9H0Q3 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing FXYD6 staining

Immunohistochemical analysis of paraffin-embedded H. liver section using FXYD6 Antibody (C-term). A09541 was diluted at 1:100 dilution. A peroxidase-conjugated goat anti-rabbit IgG at 1:400 dilution was used as the secondary antibody, followed by DAB staining.

A09541 · IHCImmunohistochemical analysis of paraffin-embedded H. liver section using FXYD6 Antibody (C-term). A09541 was diluted at 1:100 dilution. A pe

Immunohistochemical analysis of paraffin-embedded M. kidney section using FXYD6 Antibody (C-term). A09541 was diluted at 1:100 dilution. A peroxidase-conjugated goat anti-rabbit IgG at 1:400 dilution was used as the secondary antibody, followed by DAB staining.

A09541 · IHCImmunohistochemical analysis of paraffin-embedded M. kidney section using FXYD6 Antibody (C-term). A09541 was diluted at 1:100 dilution. A p

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000137726-FXYD6/tissue/cerebral+cortex#imid_10630805

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000137726-FXYD6/tissue/colon#imid_10630739

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000137726-FXYD6/tissue/liver#imid_10630724

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000137726-FXYD6/tissue/kidney#imid_10630733

HPA · ICC-IF Rh30Human Protein Atlas immunofluorescence, Rh30, antibody HPA042284; green = FXYD6, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for FXYD6 IHC & IF

🧪Run liver first: hepatocytes should stain (HPA: High in hepatocytes; A09541 tissue-IHC caption: human liver paraffin section). Run adrenal gland as a negative tissue: its glandular cells are listed as not detected (HPA: Not detected in adrenal gland glandular cells). On the liver slide, use morphologically identifiable unstained cells as internal background comparators, without assuming every non-hepatocyte is FXYD6-negative.

Positive control tissue: Cerebellum (Cells in granular layer, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show FXYD6 in Rh30, with annotated localisation: Nucleoplasm (approved), Cytosol (approved) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control, a host- and isotype-matched control IgG, and FXYD6 knockout material or competition with the matching immunizing peptide if available. Quench endogenous peroxidase and assess native pigment before scoring liver DAB signal (A09541 tissue-IHC caption: peroxidase secondary and DAB).

⚠️Feasibility: Paraffin-section IHC is documented at 1:100 for the selected antibody, but its caption does not report the fixative (A09541 tissue-IHC caption: paraffin-embedded human liver; fixative unreported). A target-specific fixation window and retrieval dependency are unreported in the supplied evidence, so retrieval conditions need empirical optimization; the evidence also does not establish that frozen sections or IF are easier. Interpret localization cautiously: UniProt places FXYD6 at the cell membrane, while HPA ICC-IF reports nucleoplasm and cytosol in Rh30 (UniProt Q9H0Q3 subcellular location; HPA subcellular: Rh30).

HPA tissue IHC evidence for FXYD6

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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
Cerebellum	Cells in granular layer	High	Protein (IHC)	HPA →
Cerebral cortex	Neuropil	High	Protein (IHC)	HPA →
Kidney	Cells in tubules	High	Protein (IHC)	HPA →
Liver	Hepatocytes	High	Protein (IHC)	HPA →
Seminal vesicle	Glandular cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Adrenal gland	Glandular cells	Not detected	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Not detected	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	Not detected	Protein (IHC)	HPA →
Epididymis	Glandular cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced FXYD6 IHC Tips

Troubleshoot FXYD6 staining in paraffin sections by checking retrieval, cellular distribution, controls, and scoring before interpreting chromogenic signal.

How should I retrieve FXYD6 in paraffin sections when staining is weak?

Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 minutes (page retrieval setting). After cooling, compare the retrieved section with a matched section processed without retrieval to judge whether the treatment improves specific staining (standard IHC practice). Use liver hepatocytes as a positive reference because they show high FXYD6 staining in tissue IHC (HPA: High in hepatocytes). If signal remains weak, adjust retrieval time in small steps while checking whether tissue edges or background also darken (standard IHC practice). The A09541 tissue caption reports paraffin sections and a 1:100 primary dilution, but gives no retrieval method (A09541 caption).

Could fixation explain weak FXYD6 staining in my sections?

Target-specific sensitivity of FXYD6 to fixation is unknown from the supplied evidence; the paraffin-section caption does not state a fixative (A09541 caption). Record the fixative, fixation duration, and processing history for every specimen so differences can be investigated (standard IHC practice). Compare sections processed together, using hepatocytes as a positive reference because their tissue staining is high (HPA: High in hepatocytes). If a batch stains poorly, first confirm retrieval at pH 6.0 and 95–98 °C for 20 minutes before comparing separately processed material (page retrieval setting). Do not assign a fixation effect from the reported tissue staining pattern alone (HPA: tissue IHC profile).

Should FXYD6 staining be membranous or cytoplasmic in tissue IHC?

Review both membrane-associated and cytoplasmic staining: FXYD6 is annotated at the cell membrane, while tissue IHC reports cytoplasmic expression in most tissues (UniProt Q9H0Q3 subcellular location; HPA: tissue IHC profile). Its transmembrane segment spans residues 36–57, with residues 58–95 on the cytoplasmic side (UniProt Q9H0Q3 topology). In liver sections, assess signal within hepatocytes because they are reported as strongly positive (HPA: High in hepatocytes). Compare staining with a no-primary control and examine whether the apparent compartment persists away from section edges (standard IHC practice). Record membrane and cytoplasmic patterns separately rather than merging them into one intensity score (standard IHC practice).

How can epitope position affect interpretation across FXYD6 isoforms?

FXYD6 has 2 annotated isoforms, but the supplied record does not map their sequence differences to antibody binding (UniProt Q9H0Q3 isoforms). The A09541 tissue caption identifies a C-terminal antibody; it does not establish which isoforms that antibody detects in sections (A09541 caption). For the annotated topology, residues 58–95 face the cytoplasm, whereas residues 19–35 face outside the cell (UniProt Q9H0Q3 topology). Check the antibody's epitope documentation against each isoform sequence before treating a negative result as isoform absence (standard IHC practice). Report staining as FXYD6 immunoreactivity unless isoform-specific binding has been demonstrated (standard IHC practice).

How should I assess FXYD6 by multiplex immunofluorescence?

Pair FXYD6 with a marker for the expected cell population, such as a hepatocyte marker in liver, and inspect the channels separately before assessing overlap (HPA: High in hepatocytes; standard IF practice). Select a fluorophore in a spectral range with low background in the specimen, and acquire an unstained section to assess tissue autofluorescence (standard IF practice). For a C-terminal epitope, test gentle permeabilisation because the annotated C-terminal region faces the cytoplasm (A09541 caption: C-term; UniProt Q9H0Q3 topology). Interpret IF compartment patterns cautiously: cell imaging reports nucleoplasm and cytosol, while UniProt annotates the cell membrane (HPA: subcellular; UniProt Q9H0Q3 subcellular location).

What should I check when FXYD6 DAB staining is diffuse?

Run a no-primary control through the same secondary antibody and DAB steps to locate background from detection chemistry (standard IHC practice). Include a peroxidase-blocking step and check whether persistent signal follows endogenous enzyme activity rather than primary-antibody binding (standard chromogenic IHC practice). The A09541 caption used a 1:100 primary dilution, a 1:400 peroxidase-conjugated secondary dilution, and DAB; these values describe that image, not a background threshold (A09541 caption). If background rises across the section, compare stronger blocking and a less concentrated primary while retaining a positive reference (standard IHC practice). Assess specific signal in hepatocytes separately from background outside them (HPA: High in hepatocytes; standard IHC practice).

How can I quantify FXYD6 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the compartment and cell population before scoring, since tissue IHC reports predominantly cytoplasmic expression and high staining in hepatocytes (HPA: tissue IHC profile; HPA: High in hepatocytes). For hepatocyte staining, record the percentage of positive viable cells and an H-score from 0–300 based on the percentages at intensity grades 0–3 (standard IHC scoring practice). Apply the same threshold, counterstain assessment, and image settings to every section (standard IHC practice). Normalise positive-cell counts to the number of viable hepatocytes assessed, or area-based counts to examined viable tissue area in mm² (standard IHC quantification practice). Report cytoplasmic and membrane scores separately if both are present (standard IHC practice).

How do I distinguish true FXYD6 staining from artefact?

Start with the expected cells: hepatocytes show high staining, whereas adipocytes are reported as not detected in tissue IHC (HPA: High in hepatocytes; HPA: Not detected in adipocytes). Evaluate cytoplasmic signal alongside any membrane pattern, because tissue IHC describes cytoplasmic expression while UniProt annotates FXYD6 at the cell membrane (HPA: tissue IHC profile; UniProt Q9H0Q3 subcellular location). Treat staining confined to section edges or necrotic areas as suspect, and compare it with intact tissue (standard IHC practice). If DAB persists in a no-primary control, investigate endogenous peroxidase or secondary-antibody background before assigning it to FXYD6 (standard chromogenic IHC practice). A single unexpected compartment does not establish a new localisation without further validation (standard IHC practice).

Boster reagents

Best FXYD6 / FXYD domain-containing ion transport regulator 6 IHC Antibodies

A09541 has paraffin-section IHC images from human liver and mouse kidney (A09541 image captions); its listed reactivity also includes rat (catalog: A09541). No IF image is supplied (catalog: A09541).

Real IHC data Immunohistochemical analysis of paraffin-embedded H. liver section using FXYD6 Antibody (C-term). A09541 was diluted at 1:100 dilution. A peroxidase-conjugated goat anti-rabbit IgG at 1:400 dilution was used as the secondary antibody, followed by DAB staining.

Anti-FXYD6 Antibody (C-term)

Cat # A09541

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A09541 will render with its own IHC figure from a paraffin-embedded human liver section (A09541 image caption). Its second IHC caption shows paraffin-embedded mouse kidney; the application list includes IHC-P, and reactivity lists human, mouse and rat (A09541 image caption; catalog: A09541).

Which to pick: Choose A09541 for paraffin-section tissue IHC: it is a rabbit polyclonal antibody listed for IHC-P, with human liver and mouse kidney images (catalog: A09541; A09541 image captions). Its listed reactivity covers human, mouse and rat, although the supplied IHC captions show only human and mouse tissue (catalog: A09541; A09541 image captions). No IF/ICC application or image is supplied for A09541, so it is not an IF/ICC-validated choice here; the captions report paraffin embedding but do not report the fixative (catalog: A09541; A09541 image captions).

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

References

UniProt Consortium. UniProt entry Q9H0Q3 (FXYD6_HUMAN, FXYD domain-containing ion transport regulator 6).
Human Protein Atlas. FXYD6 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. FXYD6 subcellular location (ICC-IF): Localized to the nucleoplasm and cytosol..
Human Protein Atlas. FXYD6 antibody validation summary (2 antibodies).
FXYD6 is a new biomarker of cholangiocarcinoma. Oncology letters 2014 — PMC3881923.
FXYD6: a novel therapeutic target toward hepatocellular carcinoma. Protein & cell 2014 — PMC4085285.
FXYD6 overexpression in HBV-related hepatocellular carcinoma with cirrhosis. Open life sciences 2020 — PMC7874599.
Identification of prognostic subtypes and the role of FXYD6 in ovarian cancer through multi-omics clustering. Frontiers in immunology 2025 — PMC11958163.
PubMed PMID:11230166 — UniProt-cited evidence.
PubMed PMID:12975309 — UniProt-cited evidence.
PubMed PMID:15146197 — UniProt-cited evidence.