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

Design Immunohistochemistry for ATP4B

Expect selective cytoplasmic staining in stomach parietal cells (HPA tissue IHC). This paraffin IHC guide pairs that pattern with the apical canalicular membrane location (UniProt) and the catalog antibody’s 2–5 μg/ml IHC range (datasheet A08719-2).

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-cell cytoplasm (HPA tissue IHC), antibody A08719-2, validated IHC image, and IHC protocol steps — Printable **ATP4B** IHC protocol sheet — expected localisation Parietal-cell cytoplasm (HPA tissue IHC), 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-cell cytoplasm (HPA tissue IHC)
	Staining pattern	Selective cytoplasmic staining in 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	Cytoplasmic IHC may not resolve apical canaliculi (HPA tissue IHC; UniProt)
	Regulation	Stomach-enriched RNA expression (HPA tissue IHC)
	Isoform / epitope	No isoforms annotated; epitope side matters across TM 37–57 (UniProt)

Section 1

Recommended ATP4B IHC & IF Protocols

The catalog antibody’s IHC-P protocol (datasheet A08719-2) is followed by two published ATP4B paraffin-section protocols (PMC12697849; PMC8128498).

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 EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A08719-2); the esophageal tissue protocol used pH 6.0 citrate (PMC8128498).

Section 2

What Is the Expected ATP4B Staining Pattern?

ATP4B should stain stomach parietal cells selectively: HPA describes a cytoplasmic-appearing pattern with Enhanced tissue IHC reliability and high staining in stomach glandular cells (HPA tissue IHC). UniProt places ATP4B at the apical canalicular membrane of parietal cells and identifies one membrane-spanning segment at residues 37–57 (UniProt P51164 topology). Interpret the slide using both the observed HPA pattern and the membrane location.

What am I looking at on my slide?
Strong, selective staining in stomach parietal cells, cytoplasmic-appearing with canalicular emphasis where visible.	This fits the observed parietal-cell pattern (HPA tissue IHC). Apical canalicular emphasis fits ATP4B localisation (UniProt P51164 subcellular location).
Predominantly nuclear staining, with little convincing parietal-cell staining.	Nuclear-only signal conflicts with the observed cytoplasmic-appearing pattern (HPA tissue IHC) and membrane location (UniProt P51164). Treat it as suspect; check controls before scoring.
Strong staining in other cell types or in a tissue listed as not detected.	This conflicts with selective parietal-cell expression (HPA tissue IHC). Cross-reactivity or endogenous detection activity is possible (standard IHC-P practice); examine the no-primary control.
Diffuse chromogen across cells and stroma obscures the glands.	A broad haze cannot establish the selective parietal-cell pattern (HPA tissue IHC). Assess background with a no-primary control and review blocking, antibody concentration and detection (standard IHC-P practice).
No staining in an adequately sampled stomach section.	Stomach is the expected high-staining tissue (HPA tissue IHC). Check that parietal cells are present, then review the IHC-P controls and reagent performance (standard IHC-P practice).

💡Expected ATP4B appearanceCall a positive result when stomach parietal cells stain strongly and selectively, with cytoplasmic-appearing and, where resolvable, apical canalicular signal (HPA tissue IHC; UniProt P51164 subcellular location); nuclear-only or widespread unrelated-cell staining is suspect against that pattern.

How each factor affects the staining
Cell identity and tissue	Use stomach sections containing identifiable parietal cells for the positive reference: HPA reports high glandular-cell staining and selective parietal-cell expression (HPA tissue IHC).
Membrane topology	ATP4B has one transmembrane segment at 37–57, a cytoplasmic N-terminus and an extracellular C-terminal region (UniProt P51164 topology). These facts do not identify the antibody epitope.
Glycosylation	UniProt annotates 7 glycosylation sites (UniProt P51164). Their effect on this antibody's IHC-P staining is unreported in the supplied sources; do not infer a retrieval requirement.
HPA validation	The tissue profile has Enhanced reliability through staining/RNA consistency (HPA tissue IHC). HPA045400 and HPA052649 each have Enhanced IHC status (HPA antibodies); that does not validate an unspecified catalog antibody.
Processing and isoforms	UniProt lists a 1–291 chain, no annotated propeptide and no isoforms (UniProt P51164). The supplied record gives no basis for predicting a processed-form-specific staining pattern.
Negative reference tissues	HPA lists adipocytes in adipose tissue and glandular cells in adrenal gland as not detected (HPA tissue IHC). Use such results as context; they do not replace controls for nonspecific staining.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Stomach section has no convincing positive cells.	Parietal cells may be absent from the sampled area, or the IHC-P run may have failed (HPA tissue IHC; standard IHC-P practice).	Confirm parietal-cell morphology on the counterstained section; review positive control, detection reagents and the antibody's stated IHC-P conditions (standard IHC-P practice).
Staining is weak despite visible parietal cells.	A weak run may obscure the high stomach staining reported by HPA (HPA tissue IHC). The supplied sources do not establish ATP4B-specific fixation or retrieval sensitivity.	Compare with a successful control and review the antibody's IHC-P retrieval and dilution instructions before adjusting conditions (standard IHC-P practice).
Chromogen is widespread or obscures cell boundaries.	Diffuse background prevents assessment of the selective pattern (HPA tissue IHC); excessive antibody or detection background are possible general causes (standard IHC-P practice).	Inspect the no-primary control, then review blocking, wash steps, antibody concentration and detection timing (standard IHC-P practice).
Signal is predominantly nuclear.	Nuclear-only staining conflicts with HPA's cytoplasmic-appearing observation and UniProt's membrane assignment (HPA tissue IHC; UniProt P51164).	Treat nuclear signal as unconfirmed; compare cell morphology, the no-primary control and a known-positive stomach section before scoring (standard IHC-P practice).
Nonparietal cells stain strongly, including cells in a listed negative tissue.	The distribution conflicts with selective parietal-cell expression and HPA not-detected results (HPA tissue IHC). Cross-reactivity or endogenous detection activity is possible (standard IHC-P practice).	Check the no-primary control and detection system; require a selective parietal-cell pattern in stomach before interpreting the signal as ATP4B (HPA tissue IHC; standard IHC-P practice).
What pattern should an IF/ICC image show?	HPA reports plasma-membrane localisation by ICC-IF, while UniProt specifies the parietal-cell apical canalicular membrane (HPA subcellular; UniProt P51164).	Assess membrane localisation in its cell context; use the separate IF/ICC guide for assay setup. An ICC-IF image alone does not establish the stomach IHC-P pattern (HPA subcellular; HPA tissue IHC).

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: glandular cells should stain, with apical canalicular membrane signal expected in parietal cells (HPA: High in stomach glandular cells; UniProt P51164: parietal-cell localization). Run adrenal gland as the negative tissue (HPA: Not detected in adrenal gland glandular cells); on the stomach slide, surrounding stroma should show only background rather than the parietal-cell membrane pattern (UniProt P51164: parietal-cell 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; a nonimmune rabbit IgG control matched to the primary’s clonality and isotype when known; and ATP4B knockout tissue or a validated peptide-block control if available (selected A08719-2 tissue-IHC caption: rabbit primary; standard IHC practice). For stomach DAB IHC, quench endogenous peroxidase; for IF, check an unstained section for tissue autofluorescence (selected A08719-2 tissue-IHC caption: stomach HRP/DAB staining; standard IHC/IF practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state the fixative (selected A08719-2 tissue-IHC caption: fixative not stated). Heat-mediated EDTA retrieval at pH 8.0 was used for the pictured mouse stomach section, but its necessity across specimens is unreported (selected A08719-2 tissue-IHC caption). The supplied evidence does not establish whether frozen sections or IF/ICC are easier; HPA reports plasma membrane localization by ICC-IF, and stomach autofluorescence warrants checking for IF (HPA subcellular: plasma membrane approved; standard IF 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 using the catalog antibody’s IHC conditions and the expected parietal-cell pattern.

What retrieval should I try first when ATP4B staining is weak?

Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A08719-2). The catalog antibody produced a mouse stomach IHC image after this retrieval, followed by 2 μg/ml primary antibody overnight at 4°C (datasheet A08719-2). If staining remains weak, first check that the section contains intact gastric glands and that parietal cells are represented (HPA: selective parietal-cell expression). Then compare retrieval duration on adjacent sections while keeping primary concentration and DAB development consistent (standard IHC practice). Excessive retrieval can damage morphology and make an apparent gain in signal difficult to interpret (standard IHC practice).

Could fixation explain a weak or uneven ATP4B stain?

Target-specific fixation sensitivity is unknown: the catalog image identifies a paraffin-embedded mouse stomach section but does not state its fixative (datasheet A08719-2). Record the fixative, fixation duration, section age and processing history before changing staining conditions (standard IHC practice). Compare sections processed together, using gastric glands with identifiable parietal cells as the biological reference (HPA: selective parietal-cell expression). If preservation differs between samples, assess morphology alongside ATP4B staining before attributing a signal difference to expression (standard IHC practice). Keep EDTA retrieval at pH 8.0 and the 2 μg/ml primary condition fixed during that comparison (datasheet A08719-2).

Where should ATP4B signal appear within stained gastric cells?

Look for staining in parietal cells, with an apical canalicular membrane component expected from the annotated location (UniProt P51164 localisation). The extracellular portion spans residues 58–291 after a single transmembrane segment at 37–57 (UniProt P51164 topology). HPA describes its tissue IHC pattern as selective cytoplasmic expression in parietal cells, so a resolved membrane rim is not required in every chromogenic section (HPA: tissue IHC profile). Interpret the pattern with gland architecture and cellular morphology visible on the counterstain (standard IHC practice). Diffuse staining of unrelated cells warrants review of background and detection controls before it is scored as ATP4B (HPA: selective parietal-cell expression; standard IHC practice).

How do topology and glycosylation affect ATP4B epitope troubleshooting?

ATP4B has one annotated transmembrane segment at residues 37–57, a cytoplasmic N terminus at 1–36 and an extracellular region at 58–291 (UniProt P51164 topology). UniProt lists 7 glycosylation sites in the extracellular region and no annotated isoforms (UniProt P51164 glycosylation and isoforms). If retrieval changes signal, review the antibody’s stated immunogen or epitope before assigning the change to a particular side of the membrane (standard IHC practice). The supplied catalog caption does not map the antibody’s epitope, so staining alone cannot establish which region it recognizes (datasheet A08719-2). Compare serial sections under the same EDTA pH 8.0 condition when assessing pattern reproducibility (datasheet A08719-2; standard IHC practice).

How should I assess ATP4B when adding an IF multiplex panel?

Pair ATP4B with a separately validated parietal-cell marker and check whether the signals identify the same cells in gastric glands (HPA: selective parietal-cell expression; standard IF practice). Select fluorophores and imaging channels after examining an unstained section for tissue autofluorescence, and include single-stain controls when assessing bleed-through (standard IF practice). If the antibody recognizes the cytoplasmic 1–36 region, permeabilisation may be needed; accessibility of an extracellular 58–291 epitope depends on specimen preparation (UniProt P51164 topology; standard IF practice). The antibody epitope is not specified in the supplied IHC caption, so test accessibility empirically (datasheet A08719-2). This IF comparison is a separate assay and does not inherit the catalog antibody’s chromogenic IHC performance (datasheet A08719-2; standard IF practice).

How can I distinguish ATP4B staining from chromogenic background?

Use the catalog image conditions as a reference: 10% goat serum block, 2 μg/ml primary overnight at 4°C, and DAB detection (datasheet A08719-2). Include a no-primary section to assess secondary and detection-system signal, and apply an endogenous peroxidase block when using HRP–DAB (standard IHC practice). Compare any residual color with the expected enrichment in gastric parietal cells and with tissue morphology (HPA: selective parietal-cell expression; standard IHC practice). Review staining concentrated at section edges, folds or damaged areas separately from intact glands (standard IHC practice). If background persists, adjust blocking, washing or DAB development one variable at a time while retaining EDTA at pH 8.0 as the retrieval reference (datasheet A08719-2; standard IHC practice).

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

Define gastric glands and morphologically identified parietal cells as the analysis region before scoring (HPA: selective parietal-cell expression; standard IHC practice). Report the percentage of positive parietal cells and, when intensity is reproducible, an H-score based on the proportions at each intensity level (standard IHC practice). Normalize positive-cell counts to the number of evaluable parietal cells; if measuring stained area, normalize to evaluable gland area in mm² (standard IHC practice). Use the same imaging, thresholding and DAB development settings for sections being compared (standard IHC practice). Document whether scoring includes cytoplasmic staining, canalicular staining or both, since HPA reports a cytoplasmic tissue pattern and UniProt annotates an apical canalicular location (HPA: tissue IHC profile; UniProt P51164 localisation).

When is an apparent ATP4B-positive result biologically convincing?

A convincing result enriches identifiable gastric parietal cells and agrees with the annotated apical canalicular location when that detail is resolved (HPA: selective parietal-cell expression; UniProt P51164 localisation). HPA reports high staining in stomach glandular cells and no detected staining in several listed non-gastric tissues, providing context for an unexpected positive result (HPA: tissue IHC). Inspect the counterstain before interpreting color in necrotic areas, section edges or poorly preserved glands (standard IHC practice). Check a no-primary control and the endogenous peroxidase block if DAB appears broadly in cells outside the expected population (standard IHC practice). The ATP4B beta subunit supports pump assembly and targeting, but chromogenic staining alone does not measure H+/K+ transport activity (UniProt P51164 function; standard IHC practice).

Boster reagents

Best ATP4B / Potassium-transporting ATPase subunit beta IHC Antibodies

The IHC-validated antibody has paraffin-section IHC and IF images from mouse and rat stomach (catalog IHC/IF captions).

Real IHC data

Anti-ATP4B Antibody ®

Cat # A08719-2

View product →

Only A08719-2 will render; its listed applications include IHC and IF, with human, mouse and rat reactivity (catalog applications/reactivity). Its images show paraffin-section IHC and IF in mouse and rat stomach (catalog IHC/IF captions).

Which to pick: For chromogenic tissue IHC, choose A08719-2: its paraffin-section stomach captions document EDTA pH 8.0 retrieval and DAB detection in mouse and rat; the fixative is unreported (catalog IHC captions). For fluorescence, the same SKU has paraffin-section IF images in mouse and rat stomach, while ICC validation is unreported (catalog IF captions; catalog applications). For work across species, the catalog lists human, mouse and rat reactivity, but the pictured IHC and IF results cover mouse and rat only; it lists a rabbit host and does not report clonality (catalog reactivity/host/clone; catalog IHC/IF 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.