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ATP1A1 / Sodium/potassium-transporting ATPase subunit alpha-1 · IHC design guide
Design Immunohistochemistry for ATP1A1
Plan ATP1A1 chromogenic IHC on paraffin sections using the membranous tissue pattern as a reference (HPA tissue IHC). Compare staining by cell type, including high staining in kidney collecting ducts (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ATP1A1 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 | Membranous in tissue (HPA tissue IHC); basolateral location annotated (UniProt) | |
| Staining pattern | Membranous staining in most tissues (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB9482) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image PB9482) | |
| Caveat | Cell-type variation: cardiomyocytes not detected (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | 4 isoforms; cytoplasmic and extracellular regions are annotated, but variant epitope coverage is unknown (UniProt) |
Section 1
Recommended ATP1A1 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: PB9482) is followed by three published ATP1A1 IHC protocols (PMC12442677; PMC11083557; PMC9204326).
| Sample | Paraffin-embedded human ovarian cancer tissue; fixative not specified (datasheet PB9482) |
| Fixation | Image fixative and duration unreported (datasheet PB9482); 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 PB9482); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9482) |
| Primary antibody | Rabbit anti-ATP1A1, 2-5 μg/ml (datasheet PB9482) |
| Primary incubation | Overnight at 4 °C (datasheet PB9482) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet PB9482) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ATP1A1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Membranous expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA, pH 8.0, retrieval for the catalog antibody (datasheet: PB9482); use a paper’s stated conditions when reproducing its protocol.
Section 2
What Is the Expected ATP1A1 Staining Pattern?
ATP1A1 is a membrane protein with 10 transmembrane segments; UniProt lists cell membrane and basolateral membrane localisation (UniProt P05023 topology and subcellular location). In paraffin IHC, expect membranous staining in many tissues, with strong staining in specified glandular, endocrine and kidney collecting duct cells (HPA tissue IHC: Enhanced reliability; membranous expression in most tissues; High in those cell types).
| Clear membrane staining in kidney collecting ducts or adrenal glandular cells. | This fits the expected compartment and a documented High staining cell population (UniProt P05023 subcellular location; HPA tissue IHC: High in collecting ducts and adrenal glandular cells). Assess the named cells rather than calling the whole tissue uniformly positive (HPA tissue IHC). |
| Predominantly nuclear staining, with little membrane staining. | Nuclear localisation is outside the supplied ATP1A1 localisation evidence (UniProt P05023 subcellular location; HPA tissue IHC: membranous expression). Treat this pattern as suspect and review controls and staining conditions (general IHC practice). |
| Strong staining in adipocytes or bone marrow hematopoietic cells. | HPA reports ATP1A1 as Not detected in these specific cell populations (HPA tissue IHC). Consider cross-reactivity or endogenous detection activity; compare with a no-primary control before assigning the signal to ATP1A1 (general IHC practice). |
| Diffuse stain across tissue, including extracellular spaces. | A widespread haze cannot establish the expected cell membrane pattern (UniProt P05023 subcellular location; HPA tissue IHC: membranous expression). Review the no-primary control, background blocking and chromogen development (general IHC practice). |
| No signal in kidney collecting ducts despite visible tissue. | That disagrees with the reported High staining in collecting ducts (HPA tissue IHC). Check whether the assay controls worked, then review retrieval, antibody incubation and detection steps (general IHC practice); the supplied sources do not establish an ATP1A1-specific retrieval requirement. |
💡Expected ATP1A1 appearanceCall a result positive when distinct cell membrane staining is strong in kidney collecting ducts or documented glandular or endocrine cells (HPA tissue IHC: High; UniProt P05023 subcellular location); isolated nuclear staining or diffuse haze is suspect (HPA tissue IHC: membranous expression).
| Cell selection for interpretation | HPA reports High staining in kidney collecting ducts, adrenal and breast glandular cells, and appendix, colon and duodenum endocrine cells (HPA tissue IHC). Its Not detected calls apply to named cell populations, such as adipocytes and cardiomyocytes, rather than every cell in those tissues (HPA tissue IHC). |
| Membrane topology and epitope location | ATP1A1 has 10 transmembrane segments and both cytoplasmic and extracellular regions (UniProt P05023 topology). The supplied record gives no antibody epitope position or ATP1A1-specific antigen retrieval condition; choose and assess retrieval using the antibody's IHC instructions and assay controls (general IHC practice). |
| Strength of the tissue evidence | HPA grades its tissue IHC profile Enhanced, while describing medium consistency between antibody staining and RNA expression (HPA tissue IHC: reliability and description). CAB018702 and CAB069993 each have Enhanced IHC validation (HPA antibodies); interpret unexpected cells against the observed pattern and controls rather than treating the grade as proof of every stained cell. |
| Isoforms and antibody scope | UniProt lists 4 ATP1A1 isoforms (UniProt P05023 isoforms). No supplied antibody epitope or isoform recognition data establish which isoforms an IHC reagent detects, so a negative stain alone cannot resolve isoform expression (UniProt P05023 isoforms; general IHC interpretation). |
| IF/ICC Q&A: What localisation is expected? | HPA reports approved plasma membrane and vesicle localisation in ICC-IF (HPA subcellular). This is the expectation for interpreting an IF/ICC image; the supplied ICC-IF record provides no staining protocol (HPA subcellular). |
| Situation | Likely cause | Next action |
|---|---|---|
| The known-positive collecting ducts are blank. | An assay failure is possible because HPA reports High staining there (HPA tissue IHC). | Confirm the tissue and positive control, then review retrieval, antibody incubation and detection against the chosen IHC procedure (general IHC practice). |
| Signal is mainly nuclear. | This conflicts with the supplied membrane localisation pattern (UniProt P05023 subcellular location; HPA tissue IHC: membranous expression). | Review the no-primary control and staining conditions; score membrane staining separately from nuclear signal (general IHC practice). |
| Adipocytes or cardiomyocytes appear strongly positive. | HPA reports Not detected in those named cells (HPA tissue IHC); cross-reactivity or endogenous detection activity is possible (general IHC practice). | Compare cell identity and no-primary control staining before interpreting the signal as ATP1A1 (general IHC practice). |
| Brown precipitate or haze obscures cell borders. | Background or excess chromogen can obscure a membrane pattern (general IHC practice). | Check the no-primary control and review blocking, washes and chromogen development (general IHC practice). |
| Only weak staining appears in lung alveolar cells. | HPA lists alveolar cells as Low, so weak staining can fit the tissue profile (HPA tissue IHC). | Compare with a documented high-staining cell population and score each cell type separately (HPA tissue IHC; general IHC practice). |
| A membrane-negative result follows a retrieval change. | The supplied sources do not establish ATP1A1-specific retrieval sensitivity (UniProt P05023; HPA tissue IHC). | Compare assay controls and the antibody's documented IHC conditions before attributing the change to ATP1A1 biology (general IHC practice). |
Experiment evidence
IHC & IF images showing ATP1A1 staining
PB9482 · IHCIHC analysis of ATP1A1 using anti-ATP1A1 antibody (PB9482). ATP1A1 was detected in a paraffin-embedded section of human ovarian cancer tissu
PB9482 · IHCIHC analysis of ATP1A1 using anti-ATP1A1 antibody (PB9482). ATP1A1 was detected in a paraffin-embedded section of human colon cancer tissue.
PB9482 · IFIF analysis of ATP1A1 using anti-ATP1A1 antibody (A05223-3). ATP1A1 was detected in a paraffin-embedded section of human colon cancer tissue
M00956 · IHCImmunohistochemical analysis of paraffin-embedded Rat cerebral cortex, using the Antibody at 1:100 dilution.
M00956 · IHCImmunohistochemical analysis of paraffin-embedded Rat heart, using the Antibody at 1:100 dilution.
M00956 · IFImmunofluorescent analysis using the Antibody at 1:50 dilution.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000163399-ATP1A1/tissue/cerebral+cortex#imid_18603580
HPA · Parathyroid glandHuman Protein Atlas IHC, Parathyroid gland. Source: https://www.proteinatlas.org/ENSG00000163399-ATP1A1/tissue/parathyroid+gland#imid_18625159
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000163399-ATP1A1/tissue/colon#imid_18625186
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000163399-ATP1A1/tissue/liver#imid_18625187
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB018702; green = ATP1A1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB018702; green = ATP1A1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ATP1A1 IHC & IF
🧪Run adrenal gland first: glandular cells should stain strongly (HPA: High in adrenal gland glandular cells). Use adipose tissue as the negative tissue, focusing on adipocytes (HPA: Not detected in adipocytes); any negative cells within the adrenal section should show counterstain without distinct membrane staining (UniProt P05023: cell membrane), but the supplied HPA row does not identify a specific adrenal cell type as negative.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ATP1A1 in A-431, U-251MG, U2OS, with annotated localisation: Vesicles (approved), Plasma membrane (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only section and a concentration-matched rabbit IgG isotype control (selected PB9482 IHC caption: rabbit primary antibody). Use ATP1A1-knockout material or peptide competition, if the immunizing peptide is available, as a biological specificity control; quench endogenous peroxidase and check native pigment against the no-primary section when interpreting brown DAB in adrenal tissue (selected PB9482 IHC caption: HRP/DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected PB9482 tissue-IHC caption does not state the fixative (selected PB9482 IHC caption: fixative not stated). Heat retrieval in EDTA at pH 8.0 was used for the paraffin-section example, but retrieval dependence has not been established (selected PB9482 IHC caption: EDTA retrieval). Frozen-section IHC or IF cannot be judged easier from these data; ICC-IF images do support assessing plasma-membrane and vesicle localization in A-431, U-251MG and U2OS cells (HPA subcellular: approved plasma membrane and vesicles; ICC-IF image cell lines).
HPA tissue IHC evidence for ATP1A1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Endocrine cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Cerebellum | Granular cells - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Colon | Endocrine cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ATP1A1 IHC Tips
Troubleshoot ATP1A1 staining by checking retrieval, membrane localisation, antibody specificity, and cell type before interpreting chromogenic signal.
What retrieval should I try first for weak ATP1A1 staining in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet PB9482). This is the condition reported for ATP1A1 staining in the selected human ovarian cancer section, although its fixative was not stated (caption PB9482). If staining is weak, compare a carefully controlled alternative retrieval condition on adjacent sections while holding detection and the 2 μg/ml primary concentration constant (caption PB9482; standard IHC practice). Check whether stronger retrieval restores crisp cell boundaries or instead damages morphology and increases diffuse staining (standard IHC practice). Include the same positive control in each retrieval run so a change in signal can be assigned to retrieval rather than run variation (standard IHC practice).
How should I assess whether fixation caused weak or uneven ATP1A1 IHC staining?
ATP1A1-specific fixation sensitivity is unknown from the supplied evidence, and the selected paraffin-section caption does not identify a fixative (caption PB9482). Record the actual fixative and processing history for each specimen, then compare sections processed with consistent retrieval and detection settings (standard IHC practice). If matched material with different fixation histories is available, stain it in one run with EDTA at pH 8.0 and the same primary concentration (datasheet PB9482; standard IHC practice). Evaluate membrane definition alongside tissue morphology because weak staining alone cannot establish a fixation effect (standard IHC practice). Avoid assigning differences between specimens to ATP1A1 biology until section quality and run controls have been reviewed (standard IHC practice).
Where should convincing ATP1A1 staining appear in tissue sections?
Look first for staining that outlines cells, with basolateral enrichment where epithelial polarity is discernible (UniProt P05023 localisation; HPA: membranous expression in most tissues). ATP1A1 is also annotated at the sarcolemma and in axons, so the expected boundary depends on the cell being examined (UniProt P05023 localisation). HPA reports high staining in kidney collecting ducts, making them a useful tissue comparison for membrane pattern and intensity (HPA: High in kidney collecting ducts). Vesicular signal can occur, but an entirely diffuse cytoplasmic pattern needs additional scrutiny because HPA also approves a plasma-membrane location (HPA subcellular: vesicles and plasma membrane). Compare the stained structures with the counterstain and neighboring cells before calling weak outlines positive (standard IHC practice).
How can epitope position change interpretation of ATP1A1 staining?
Confirm the antibody's stated immunogen or epitope before treating staining as specific to every ATP1A1 variant; the supplied record lists 4 isoforms without defining antibody recognition (UniProt P05023 isoforms; supplied product evidence). ATP1A1 has 10 transmembrane segments, a large cytoplasmic region at residues 343–772, and extracellular loops including 871–917 (UniProt P05023 topology). Retrieval may expose these regions differently in processed sections, so an intensity change does not by itself identify an isoform (standard IHC practice). The record also lists N-terminal acetylation and phosphorylation sites; their effect on this antibody's staining is unestablished (UniProt P05023 modified residues; supplied product evidence). Use documented epitope coverage and an independent specificity control before making an isoform-level claim (standard IHC practice).
How can IF help investigate an ambiguous ATP1A1 IHC pattern?
Use IF as a separate validation experiment and compare ATP1A1 signal with a marker for the expected cell population, such as a collecting-duct marker when examining kidney (HPA: High in kidney collecting ducts; standard IF practice). HPA places ATP1A1 at the plasma membrane and vesicles, so assess both boundary signal and intracellular puncta in the appropriate cells (HPA subcellular). Choose fluorophores after checking the section's autofluorescence, and favor a spectrally separated, longer-wavelength channel when background obscures the target (standard IF practice). Match permeabilisation to the documented epitope: cytoplasmic regions require antibody access across the membrane, whereas extracellular loops may be assessed with gentler conditions (UniProt P05023 topology; standard IF practice). The supplied IF evidence does not establish a target-specific fixation condition (HPA subcellular; supplied product evidence).
What should I check when ATP1A1 DAB staining looks diffuse or widespread?
First inspect a no-primary control and apply an endogenous-peroxidase block before DAB development, since either nonspecific detection or tissue enzyme activity can produce misleading chromogen (standard IHC practice). The selected section used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and a peroxidase-linked secondary (caption PB9482). If background persists, compare primary concentrations around that reported condition while keeping section processing and development time identical (caption PB9482; standard IHC practice). Assess whether signal follows cell boundaries rather than uniformly filling tissue spaces (UniProt P05023 localisation; standard IHC practice). Review damaged edges and necrotic areas separately because staining there can be misleading (standard IHC practice).
How should I score ATP1A1 IHC across samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and score membrane-associated DAB in viable cells, because ATP1A1 is reported at the plasma membrane and basolateral membrane (UniProt P05023 localisation). Report the percentage of positive cells with intensity categories, or calculate an H-score from percentages at 0–3 intensity for a 0–300 range (standard IHC practice). If counting positive cells per mm², normalise to the measured viable tissue area and report the cell population analyzed (standard IHC practice). Keep retrieval, primary concentration, chromogen development, imaging, and threshold rules consistent across slides (standard IHC practice). Separate membrane scores from diffuse cytoplasmic signal rather than combining patterns that have different interpretive strength (HPA: membranous expression in most tissues; standard IHC practice).
How do I distinguish true ATP1A1 staining from artefact?
Favor reproducible cell-boundary staining in the expected population: HPA reports membranous expression in most tissues and high staining in kidney collecting ducts (HPA tissue IHC). Question isolated nuclear staining, diffuse necrotic staining, or signal concentrated only at section edges against the reported membrane location and routine artefact checks (UniProt P05023 localisation; standard IHC practice). Check a no-primary control and endogenous-peroxidase blocking when DAB appears in unexpected areas (standard IHC practice). The PB9482 image concerns ovarian cancer tissue, whereas HPA reports ATP1A1 undetected in normal ovarian follicle cells; these are different populations and should be interpreted separately (caption PB9482; HPA: Not detected in ovarian follicle cells). Require agreement between morphology, appropriate controls, and localisation before calling a faint signal positive (standard IHC practice).
Boster reagents
Best ATP1A1 / Sodium/potassium-transporting ATPase subunit alpha-1 IHC Antibodies
ATP1A1 antibodies have paraffin-section IHC images from human and rat tissues and an IF image with no sample specified (catalog image captions). Both list Human, Mouse and Rat reactivity (catalog).
Real IHC data
Anti-alpha 1 Sodium Potassium ATPase/ATP1A1 Antibody ®
Cat # PB9482
Real IHC data
Anti-Sodium Potassium ATPase ATP1A1 Rabbit Monoclonal Antibody
Cat # M00956
PB9482 lists IHC and IF applications and Human, Mouse and Rat reactivity; its IHC images show human paraffin sections (catalog applications, reactivity and IHC image captions). M00956 lists IHC, IF and ICC applications and Human, Mouse and Rat reactivity; its IHC images show rat and human paraffin sections (catalog applications, reactivity and IHC image captions).
Which to pick: For tissue IHC, choose PB9482 for its documented EDTA pH 8.0 retrieval and 2 μg/ml primary incubation on a human paraffin section (PB9482 IHC image caption); choose M00956 if a rabbit monoclonal is preferred, with rat and human paraffin-section images (M00956 catalog clone and IHC image captions). For IF/ICC, M00956 lists both applications, whereas PB9482 lists IF (catalog applications). Both list Human, Mouse and Rat reactivity (catalog); the fixative is unreported in each SKU’s IHC captions (catalog IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P05023 (AT1A1_HUMAN, Sodium/potassium-transporting ATPase subunit alpha-1).
- Human Protein Atlas. ATP1A1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ATP1A1 subcellular location (ICC-IF): Localized to the plasma membrane and vesicles..
- Human Protein Atlas. ATP1A1 antibody validation summary (2 antibodies).
- LncDARS-AS1 Regulates ATP1A1 Stability and Enhances Na(+)/K(+) ATPase Activity to Promote Osteosarcoma Metastasis. Advanced science (Weinheim, Baden-Wurttemberg, Germany) 2025 — PMC12442677.
- Hypomethylation of ATP1A1 Is Associated with Poor Prognosis and Cancer Progression in Triple-Negative Breast Cancer. Cancers 2024 — PMC11083557.
- Expression Regulation of Water Reabsorption Genes and Transcription Factors in the Kidneys of Lepus yarkandensis. Frontiers in physiology 2022 — PMC9204326.
- Molecular architecture underlying fluid absorption by the developing inner ear. eLife 2017 — PMC5634787.
- PubMed PMID:2430951 — UniProt-cited evidence.
- PubMed PMID:7536695 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ATP1A1 (Sodium/potassium-transporting ATPase subunit alpha-1). Expected localisation Membranous in tissue (HPA tissue IHC); basolateral location annotated (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9482). Validated IHC antibody: PB9482 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human ovarian cancer tissue; fixative not specified (datasheet PB9482); 2. Fixation: Image fixative and duration unreported (datasheet PB9482); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet PB9482); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9482); 8. Primary antibody: Rabbit anti-ATP1A1, 2-5 μg/ml (datasheet PB9482); 9. Primary incubation: Overnight at 4 °C (datasheet PB9482). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image PB9482); Caveat: Cell-type variation: cardiomyocytes not detected (HPA tissue IHC); Isoform / epitope: 4 isoforms; cytoplasmic and extracellular regions are annotated, but variant epitope coverage is unknown (UniProt); Regulation: No expression regulator annotated (UniProt). Evidence: UniProt P05023, HPA tissue IHC · Enhanced, HPA ICC-IF · plasma membrane and vesicles., Datasheet PB9482, PMC12442677, PMC11083557. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ATP1A1/immunohistochemistry-ihc-guide.