ATP4B / Potassium-transporting ATPase subunit beta · IHC design guide

Design Immunohistochemistry for ATP4B

Plan ATP4B IHC in paraffin sections using stomach as a positive tissue control (HPA tissue IHC). Compare selective cytoplasmic staining in parietal cells with the apical canalicular membrane location annotated for the protein (HPA tissue IHC; UniProt).

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

Immunohistochemistry protocol sheet for ATP4B (IHC for ATP4B): expected localisation Parietal cytoplasm observed; apical membrane expected (HPA tissue IHC; UniProt), antibody A08719-2, validated IHC image, and IHC protocol steps — Printable **ATP4B** IHC protocol sheet — expected localisation Parietal cytoplasm observed; apical membrane expected (HPA tissue IHC; UniProt), antibody A08719-2, controls and protocol steps. Open the full ATP4B IHC guide →

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ATP4B 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	Parietal cytoplasm observed; apical membrane expected (HPA tissue IHC; UniProt)
	Staining pattern	Selective cytoplasmic staining in gastric parietal cells (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A08719-2)
	Positive control ⓘ	Stomach
	Negative control ⓘ	Adipose tissue+4 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). Selected-image fixative and duration unreported (datasheet A08719-2); verify before use.
	Caveat	Sections lacking parietal cells may appear negative (HPA tissue IHC)
	Regulation	Expression regulation not annotated (UniProt)
	Isoform / epitope	No isoforms annotated; cytoplasmic versus extracellular epitope matters (UniProt)

Section 1

Recommended ATP4B IHC & IF Protocols

The catalog antibody’s paraffin-section IHC protocol (datasheet A08719-2) is accompanied by four published ATP4B IHC protocols (PMC12697849; PMC8128498; PMC13395671; PMC11134753).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded mouse stomach tissue; fixative not specified (datasheet A08719-2)
Fixation	Image fixative and duration unreported (datasheet A08719-2); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: EDTA pH 8.0 (datasheet A08719-2); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A08719-2)
Primary antibody	Rabbit anti-ATP4B, 2-5 μg/ml (datasheet A08719-2)
Primary incubation	Overnight at 4 °C (datasheet A08719-2)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A08719-2)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	ATP4B-positive staining in glandular cells of stomach (HPA tissue IHC: High). HPA tissue profile: Selective cytoplasmic expression in parietal cells. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA pH 8.0 for the catalog antibody (datasheet A08719-2). Citric acid pH 6.0 is reported for esophageal tissue (PMC8128498).

Section 2

What Is the Expected ATP4B Staining Pattern?

ATP4B should stain gastric parietal cells: HPA reports selective cytoplasmic staining within stomach glandular cells, with high staining and Enhanced tissue IHC reliability (HPA tissue IHC). UniProt places the protein at the apical canalicular membrane, with one transmembrane segment at residues 37–57 and an extracellular region at 58–291 (UniProt P51164 topology). Interpret the apparent cytoplasmic pattern in paraffin sections alongside that membrane assignment.

What am I looking at on my slide?
Strong staining in a subset of stomach glandular cells, with an apparent cytoplasmic pattern (HPA tissue IHC).	This matches HPA’s high, selective parietal-cell result (HPA tissue IHC). Assess the pattern within recognizable gastric glands; UniProt assigns ATP4B to the parietal-cell apical canalicular membrane (UniProt P51164 subcellular location).
Staining is predominantly nuclear, rather than in the expected parietal-cell region (HPA tissue IHC; UniProt P51164 subcellular location).	Nuclear localization is unsupported by the supplied sources. Treat it as possible nonspecific staining or detection artefact (general IHC practice); check controls and morphology before counting cells as ATP4B positive.
Strong staining appears in nonparietal cells or in a listed tissue where HPA reports no detection (HPA tissue IHC).	Suspect cross-reactivity or endogenous detection activity (general IHC practice). For example, HPA reports no detection in appendix glandular cells and bronchial respiratory epithelial cells; their staining would need independent validation (HPA tissue IHC).
Diffuse chromogen covers cells and surrounding tissue without a recognizable selective pattern (HPA tissue IHC).	Background can obscure the expected parietal-cell staining (HPA tissue IHC). Review blocking, antibody concentration, washing and detection controls as general IHC variables (general IHC practice); diffuse color alone is insufficient to score ATP4B.
No signal is visible in stomach glandular cells expected to contain parietal cells (HPA tissue IHC).	First verify that the section contains identifiable parietal cells. HPA reports high stomach glandular-cell staining, but a negative slide alone cannot distinguish absent target from assay failure (HPA tissue IHC; general IHC practice).

💡Expected ATP4B appearanceCall a result positive when staining is strong and selective in gastric parietal cells, appearing cytoplasmic by HPA tissue IHC while consistent with an apical canalicular membrane protein (HPA tissue IHC; UniProt P51164 subcellular location); predominant nuclear color or widespread staining of unrelated cells is suspect (general IHC practice).

How each factor affects the staining
Compartment and epitope orientation (UniProt P51164 topology).	ATP4B has a short cytoplasmic segment at residues 1–36, one membrane span at 37–57, and an extracellular region at 58–291 (UniProt P51164 topology). Antibody epitope location is not supplied, so these features do not predict which retrieval condition will work.
Glycosylation (UniProt P51164 PTMs).	UniProt annotates seven glycosylation sites at residues 99, 103, 130, 146, 161, 193 and 222 (UniProt P51164 PTMs). Without the antibody’s epitope or comparative staining data, their effect on this IHC result is unknown.
Tissue evidence and validation (HPA tissue IHC; HPA antibodies).	HPA rates the tissue pattern Enhanced because antibody staining is highly consistent with RNA expression; stomach RNA is tissue enriched (HPA tissue IHC). Both listed antibodies, HPA045400 and HPA052649, have Enhanced IHC status (HPA antibodies).
Fixation and antigen retrieval (general IHC practice).	Retrieval conditions can be optimized using matched sections and controls (general IHC practice). The supplied HPA and UniProt records give no ATP4B-specific fixation sensitivity or retrieval comparison; no target-specific direction of effect can be assigned.
IF/ICC interpretation (HPA subcellular; HPA antibodies).	Q: Should IF/ICC show the same pattern as tissue IHC? A: HPA reports an approved plasma-membrane location in ICC-IF and Approved ICC status for HPA045400 (HPA subcellular; HPA antibodies). Interpret that evidence in its cell-imaging context, not as an IHC-P protocol.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Stomach section has no clear parietal-cell signal (HPA tissue IHC).	The section may lack identifiable parietal cells, or the IHC run may have failed (general IHC practice). The supplied sources do not identify an ATP4B-specific fixation cause.	Confirm gland morphology and include a stomach positive control; then review retrieval, antibody concentration and detection using the applicable validated IHC procedure (general IHC practice).
Signal appears mainly nuclear (HPA tissue IHC; UniProt P51164 subcellular location).	That compartment disagrees with both the reported tissue pattern and membrane localization; nonspecific detection is possible (HPA tissue IHC; UniProt P51164 subcellular location; general IHC practice).	Check a no-primary control and evaluate whether staining persists independently of the primary antibody; rescore only a selective, morphologically plausible parietal-cell pattern (general IHC practice).
Color is widespread across the section (HPA tissue IHC).	Diffuse background may reflect nonspecific binding, incomplete washing or detection background (general IHC practice); it does not match HPA’s selective parietal-cell pattern (HPA tissue IHC).	Review blocking and washes, assess a no-primary control, and adjust antibody concentration within the validated IHC workflow (general IHC practice).
Nonparietal cells stain strongly in stomach (HPA tissue IHC).	Cell-type assignment may be mistaken, or the signal may reflect cross-reactivity or endogenous activity (general IHC practice). HPA describes selective parietal-cell expression (HPA tissue IHC).	Recheck gland and cell morphology, compare an appropriate negative tissue, and inspect detection controls before assigning ATP4B positivity (HPA tissue IHC; general IHC practice).
A listed negative tissue stains, such as appendix glandular cells (HPA tissue IHC).	HPA reports ATP4B as not detected there; staining could arise from cross-reactivity or the detection system (HPA tissue IHC; general IHC practice).	Repeat alongside a stomach positive control and a no-primary control. Treat persistent unexpected staining as unvalidated until its specificity is independently checked (HPA tissue IHC; general IHC practice).
An IF/ICC plasma-membrane image seems inconsistent with cytoplasmic-looking tissue IHC (HPA subcellular; HPA tissue IHC).	HPA describes plasma-membrane localization in ICC-IF and selective cytoplasmic appearance in tissue sections; the records use different imaging contexts (HPA subcellular; HPA tissue IHC).	Score the paraffin IHC slide against its tissue and cell-type pattern, while retaining UniProt’s apical canalicular membrane assignment; use the separate IF/ICC guide for that application (HPA tissue IHC; UniProt P51164 subcellular location).

Source: UniProt P51164 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing ATP4B staining

IHC analysis of ATP4B using anti-ATP4B antibody (A08719-2). ATP4B was detected in a paraffin-embedded section of mouse stomach tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-ATP4B Antibody (A08719-2) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A08719-2 · IHCIHC analysis of ATP4B using anti-ATP4B antibody (A08719-2). ATP4B was detected in a paraffin-embedded section of mouse stomach tissue. Heat

IF analysis of ATP4B using anti-ATP4B antibody (A08719-2). ATP4B was detected in a paraffin-embedded section of mouse stomach tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 5 μg/mL rabbit anti-ATP4B Antibody (A08719-2) overnight at 4°C. DyLight®594 Conjugated Goat Anti-Rabbit IgG (BA1142) was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A08719-2 · IFIF analysis of ATP4B using anti-ATP4B antibody (A08719-2). ATP4B was detected in a paraffin-embedded section of mouse stomach tissue. Heat m

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000186009-ATP4B/tissue/cerebral+cortex#imid_16713819

HPA · StomachHuman Protein Atlas IHC, Stomach. Source: https://www.proteinatlas.org/ENSG00000186009-ATP4B/tissue/stomach+2#imid_16713711

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000186009-ATP4B/tissue/colon#imid_16713753

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000186009-ATP4B/tissue/liver#imid_16713762

HPA · ICC-IF SH-SY5YHuman Protein Atlas immunofluorescence, SH-SY5Y, antibody HPA045400; green = ATP4B, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for ATP4B IHC & IF

🧪Run stomach first and look for staining in glandular cells (High; HPA: stomach glandular cells). Run adrenal gland as a negative, where glandular cells are Not detected (HPA: adrenal gland glandular cells); on the stomach slide, compare neighboring non-parietal cells for absence of the characteristic apical canalicular pattern (UniProt P51164 localization; standard IHC practice).

Positive control tissue: Stomach (Glandular cells, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ATP4B in SH-SY5Y, THP-1, U2OS, Sperm, with annotated localisation: Plasma membrane (approved) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control and a rabbit IgG isotype control matched to the primary antibody’s clonality where applicable (selected A08719-2 caption: rabbit primary; standard IHC practice). Use ATP4B-deficient material, if available, as a biological negative, and quench endogenous peroxidase in stomach sections before HRP/DAB detection (standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence; the selected A08719-2 paraffin-section caption does not state a fixative (selected A08719-2 caption). That caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required (selected A08719-2 caption). The evidence does not establish that frozen sections or IF are easier for ATP4B; for chromogenic stomach IHC, monitor endogenous peroxidase background (selected A08719-2 caption: HRP/DAB; standard IHC practice).

HPA tissue IHC evidence for ATP4B

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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
Stomach	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 →
Appendix	Glandular cells	Not detected	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Not detected	Protein (IHC)	HPA →
Breast	Adipocytes	Not detected	Protein (IHC)	HPA →

Section 3

Advanced ATP4B IHC Tips

Troubleshoot ATP4B staining in paraffin sections by checking retrieval, cell identity, membrane localisation and the controls used to interpret chromogenic signal.

What retrieval should I try when ATP4B staining is weak in paraffin sections?

Use heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A08719-2). This is the retrieval used for the catalog antibody’s mouse stomach image, followed by 2 μg/ml primary antibody overnight at 4°C (caption A08719-2). Check that sections were fully immersed and heated evenly, then compare staining with a known positive stomach section in the same run (standard IHC practice; HPA: stomach glandular cells, High). If signal remains weak, vary heating duration on matched sections while keeping EDTA at pH 8.0 constant (standard IHC practice). Score the change in parietal-cell pattern alongside any increase in diffuse background (HPA: selective parietal-cell expression; standard IHC practice).

Could fixation explain inconsistent ATP4B staining between paraffin blocks?

The selected mouse stomach caption identifies a paraffin-embedded section but does not state its fixative, so target-specific fixation sensitivity is unknown (caption A08719-2). Record the fixative, fixation duration and tissue thickness for each block before attributing a weak result to ATP4B biology (standard IHC practice). Process matched sections with EDTA retrieval at pH 8.0 and the same 2 μg/ml primary incubation used in the catalog image (caption A08719-2; standard IHC practice). Compare the distribution and intensity of staining in morphologically intact glands against a stomach control processed in the same run (HPA: stomach glandular cells, High; standard IHC practice). A block-level difference then remains an observation requiring controlled follow-up, rather than evidence of a known ATP4B fixation effect (caption A08719-2; standard IHC practice).

Should ATP4B appear at the membrane or throughout the cytoplasm?

Prioritise staining in gastric parietal cells and examine its relationship to the apical canalicular membrane, where ATP4B is localised (HPA: selective parietal-cell expression; UniProt P51164 subcellular localisation). The protein has one membrane-spanning segment at residues 37–57, with residues 1–36 cytoplasmic and 58–291 extracellular (UniProt P51164 topology). HPA describes a selective cytoplasmic pattern in parietal cells, so a granular intracellular appearance in chromogenic sections should be assessed against cell morphology and membrane-associated staining (HPA: tissue IHC profile; standard IHC practice). Compare serial sections stained with the same retrieval and detection settings before interpreting a changed pattern (standard IHC practice). Broad staining of unrelated glandular cells requires a background and specificity check (HPA: selective parietal-cell expression; standard IHC practice).

How does ATP4B topology affect interpretation of an unknown antibody epitope?

The supplied record lists a single 291-aa chain and no annotated isoforms, so an isoform-specific explanation is unsupported here (UniProt P51164 processing and isoforms). ATP4B has a short cytoplasmic N-terminal region at residues 1–36 and an extracellular region at 58–291 (UniProt P51164 topology). Seven glycosylation sites are annotated in the extracellular region, but the selected caption does not identify the antibody’s epitope (UniProt P51164 glycosylation; caption A08719-2). If staining changes with retrieval, compare matched stomach sections before assigning that change to epitope exposure or glycosylation (HPA: stomach glandular cells, High; standard IHC practice). Interpret an unexpected compartment cautiously until the epitope and staining specificity are established (standard IHC practice).

How can IF help resolve an ambiguous ATP4B IHC pattern?

Use IF on a matched section to check whether the chromogenic pattern follows parietal cells, the selectively stained population in tissue IHC (HPA: tissue IHC profile; standard IHC practice). Multiplex ATP4B with a separately validated parietal-cell marker, and choose fluorophores after checking stomach-section autofluorescence in unstained and secondary-only controls (HPA: selective parietal-cell expression; standard IF practice). ATP4B spans the membrane at residues 37–57, leaving residues 1–36 cytoplasmic and 58–291 extracellular (UniProt P51164 topology). Set permeabilisation according to the antibody’s known epitope side, or compare permeabilised and nonpermeabilised sections if that side is unknown (standard IF practice). Treat the catalog’s 2 μg/ml incubation as IHC image evidence, not an established IF condition (caption A08719-2).

How should I investigate diffuse DAB staining outside gastric parietal cells?

First compare the distribution with ATP4B’s selective parietal-cell tissue pattern and expected apical canalicular localisation (HPA: tissue IHC profile; UniProt P51164 subcellular localisation). The catalog image used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, a peroxidase-conjugated secondary and DAB development (caption A08719-2). Include a no-primary section and an appropriate peroxidase block to assess detection-related signal before changing antibody concentration (standard IHC practice). If background persists, compare shorter chromogen development and a primary-antibody titration on adjacent sections while preserving the same retrieval conditions (standard IHC practice). Judge improvement by retained staining in intact parietal cells and reduced signal elsewhere (HPA: selective parietal-cell expression; standard IHC practice).

What should I measure when comparing ATP4B IHC across stomach sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define gastric glands and morphologically intact parietal cells before scoring, because tissue IHC reports selective expression in parietal cells (HPA: tissue IHC profile; standard IHC practice). Report the percentage of positive parietal cells and an H-score if staining intensity can be scored consistently; density per mm² is useful when cell abundance differs (standard IHC practice). Normalise positive-cell counts to the total parietal cells assessed, or density to the measured viable gland area (standard IHC practice). Keep section thickness, EDTA retrieval at pH 8.0, detection and imaging settings consistent across the comparison (datasheet A08719-2; standard IHC practice). Record staining intensity and cell abundance separately so a loss of parietal cells is not mistaken for weaker staining per cell (standard IHC practice).

How can I distinguish true ATP4B staining from an IHC artefact?

A convincing positive pattern follows gastric parietal cells and is compatible with ATP4B’s apical canalicular membrane localisation (HPA: selective parietal-cell expression; UniProt P51164 subcellular localisation). Interpret predominantly nuclear signal, widespread staining of unrelated cells, or staining confined to section edges cautiously because these do not match that expected distribution (HPA: tissue IHC profile; UniProt P51164 subcellular localisation; standard IHC practice). Exclude necrotic areas and compare with a no-primary control to identify tissue damage or detection-system background (standard IHC practice). Check endogenous peroxidase control when DAB signal is unexpected, since the catalog image used peroxidase detection and DAB (caption A08719-2; standard IHC practice). Compare with a concurrently stained stomach positive control before calling a negative specimen ATP4B-deficient (HPA: stomach glandular cells, High; standard IHC practice).

Boster reagents

Best ATP4B / Potassium-transporting ATPase subunit beta IHC Antibodies

A08719-2 has IHC images from mouse and rat paraffin stomach sections and IF images from mouse and rat paraffin stomach sections (catalog image captions); human reactivity is listed (catalog applications/reactivity).

Real IHC data

Anti-ATP4B Antibody ®

Cat # A08719-2

View product →

A08719-2 is listed for IHC and IF in human, mouse and rat (catalog applications/reactivity). Its images document IHC and IF on mouse and rat paraffin stomach sections (catalog image captions).

Which to pick: For tissue IHC, choose A08719-2: its IHC captions show mouse and rat paraffin stomach sections with EDTA pH 8.0 retrieval and 2 μg/ml primary antibody (A08719-2 IHC image captions); the fixative is unreported (A08719-2 IHC image captions). For IF, A08719-2 has mouse and rat paraffin stomach images at 5 μg/ml (A08719-2 IF image captions); ICC validation is unreported (catalog applications and image captions). For cross-species work, A08719-2 lists human, mouse and rat reactivity, while the supplied IHC and IF images document mouse and rat samples (catalog reactivity; A08719-2 image captions).

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

References

UniProt Consortium. UniProt entry P51164 (ATP4B_HUMAN, Potassium-transporting ATPase subunit beta).
Human Protein Atlas. ATP4B tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. ATP4B subcellular location (ICC-IF): Localized to the plasma membrane..
Human Protein Atlas. ATP4B antibody validation summary (2 antibodies).
AI-Assisted Detection of Early Gastric Cancer via Visualization of Mucosal Acidity Compromise During Endoscopy. Advanced science (Weinheim, Baden-Wurttemberg, Germany) 2025 — PMC12697849.
Epigenetic Study of Esophageal Carcinoma Based on Methylation, Gene Integration and Weighted Correlation Network Analysis. OncoTargets and therapy 2021 — PMC8128498.
Huangjin Shuangshen decoction alleviates chronic atrophic gastritis by suppressing TNF/NF-κB signaling and promoting CFTR-associated gastric mucosal barrier repair. Frontiers in immunology 2026 — PMC13395671.
Omeprazole taken once every other day can effectively prevent aspirin-induced gastrointestinal mucosal damage in rats. BMC gastroenterology 2024 — PMC11134753.
PubMed PMID:1656976 — UniProt-cited evidence.
PubMed PMID:15057823 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.