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ATP1A2 / Sodium/potassium-transporting ATPase subunit alpha-2 · IHC design guide
Design Immunohistochemistry for ATP1A2
Use cerebellar molecular-layer neuropil as a high-staining reference for ATP1A2 paraffin-section IHC (HPA tissue IHC). This guide covers fixation consistency, chromogenic detection and interpretation of cytoplasmic tissue staining alongside ATP1A2’s cell-membrane annotation (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ATP1A2 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 staining in several tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining, strongest in CNS neuropil (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A02064-1) | |
| Positive control | Cerebellum+4 more · see all | |
| Negative control | Adrenal gland+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) | |
| Caveat | Staining and RNA expression have medium consistency (HPA tissue IHC) | |
| Regulation | Alpha-isoform expression varies by tissue (UniProt) | |
| Isoform / epitope | No isoforms annotated; epitope side depends on topology (UniProt) |
Section 1
Recommended ATP1A2 IHC & IF Protocols
The catalog antibody protocol uses EDTA retrieval (datasheet A02064-1). Published chromogenic IHC protocols cover serous ovarian cancer tissue (PMC8607279) and aortic disease tissue (PMC9097831).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A02064-1) |
| Fixation | Image fixative and duration unreported (datasheet A02064-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A02064-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A02064-1) |
| Primary antibody | Rabbit anti-ATP1A2, 2-5 μg/ml (datasheet A02064-1) |
| Primary incubation | Overnight at 4 °C (datasheet A02064-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A02064-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ATP1A2-positive staining in molecular layer - neuropil of cerebellum (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues, most abundant in CNS. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet A02064-1). Sodium citrate is a published alternative for ovarian tissue (PMC8607279).
Section 2
What Is the Expected ATP1A2 Staining Pattern?
ATP1A2 is a cell-membrane pump with 10 transmembrane segments (UniProt P50993 topology). In paraffin-section IHC, expect the strongest reported staining in cerebellar molecular-layer and cerebral-cortex neuropil, with medium staining in selected glial, glandular, endocrine and macrophage populations (HPA tissue IHC). HPA describes the tissue pattern as cytoplasmic and rates its reliability Enhanced, while noting medium consistency with RNA data (HPA tissue IHC).
| Strong staining in cerebellar molecular-layer or cerebral-cortex neuropil. | This matches HPA's High staining in those compartments (HPA tissue IHC). Read the signal within the tissue architecture: neuropil contains closely packed cellular processes, so a membrane-associated target need not appear as a crisp outline of individual cells (UniProt P50993 topology; general IHC interpretation). |
| Moderate staining in caudate or hippocampal glial cells, lung macrophages, or pancreatic endocrine cells. | These are reported Medium cell-specific patterns (HPA tissue IHC). Appendix and duodenal glandular cells are also Medium (HPA tissue IHC). Compare the named cell population rather than treating an entire organ as uniformly positive (HPA tissue IHC). |
| Predominantly nuclear staining, without the expected neuropil or cell-associated pattern. | Nuclear localisation conflicts with UniProt's cell-membrane assignment and HPA's cytoplasmic tissue profile (UniProt P50993 subcellular location; HPA tissue IHC). Treat it as suspect staining and check controls before scoring it as ATP1A2 (general IHC practice). |
| Strong staining in a population HPA reports as not detected, or broad colour across many unrelated cells. | For example, HPA reports adrenal glandular and bone-marrow hematopoietic cells as Not detected (HPA tissue IHC). Such staining warrants checks for antibody cross-reactivity, endogenous detection activity and background; an HPA negative is cell-specific, not proof that every cell in that tissue must be blank (general IHC practice; HPA tissue IHC). |
| Diffuse deposit across tissue and background, or no signal in a known-positive section. | Diffuse deposit is difficult to assign to the reported cells or compartment and may reflect nonspecific staining (general IHC practice; HPA tissue IHC). A blank result in high-staining CNS neuropil calls for a control and workflow check before concluding that ATP1A2 is absent (HPA tissue IHC; general IHC practice). |
💡Expected ATP1A2 appearanceCall the stain positive when high CNS neuropil signal, or the reported medium signal in the specified cell populations, is identifiable within tissue architecture (HPA tissue IHC); predominantly nuclear staining or strong signal in a reported negative cell population is suspect (UniProt P50993 subcellular location; HPA tissue IHC).
| Tissue and cell selection | Cerebellar molecular-layer and cerebral-cortex neuropil provide High reference patterns; caudate and hippocampal glia are Medium (HPA tissue IHC). HPA's Not detected calls apply to specified cells, not whole organs (HPA tissue IHC). |
| Compartment and epitope | ATP1A2 spans the membrane 10 times and has both cytoplasmic and extracellular regions (UniProt P50993 topology). The antibody epitope is unspecified here, so its accessibility in a section cannot be inferred from topology alone. |
| Strength of IHC evidence | HPA rates tissue IHC reliability Enhanced but reports only medium consistency with RNA expression; antibody CAB022230 has Enhanced IHC validation (HPA tissue IHC; HPA antibodies). Interpret unexpected staining against controls and tissue morphology (general IHC practice). |
| Antigen retrieval | Retrieval conditions are a general paraffin-section IHC variable (general IHC practice). No ATP1A2-specific fixation sensitivity or retrieval requirement is supplied by UniProt or HPA; do not infer one from topology, modifications or staining intensity. |
| IF/ICC: what pattern is supported? | HPA summarises ATP1A2 as Membrane in its subcellular record, but lists no main location or ICC-IF image-bearing cell line (HPA subcellular). UniProt also assigns it to the cell membrane (UniProt P50993 subcellular location). These records do not establish a cell-line-specific IF pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in cerebellar molecular-layer or cerebral-cortex neuropil. | These are HPA High reference sites, so a blank slide leaves assay performance unresolved (HPA tissue IHC). | Check section integrity, retrieval, primary-antibody application, detection reagents and a concurrently stained positive control (general IHC practice). Do not label the sample negative until the control works. |
| Strong nuclear signal dominates the slide. | Nuclear staining does not fit the reported membrane location or cytoplasmic tissue profile (UniProt P50993 subcellular location; HPA tissue IHC). | Review a no-primary control and tissue morphology; reassess antibody specificity before interpreting the nuclear signal as ATP1A2 (general IHC practice). |
| Colour appears in reported negative cells or across unrelated cell populations. | This conflicts with the cell-specific HPA comparison and may reflect nonspecific binding or endogenous chromogen-generating activity (HPA tissue IHC; general IHC practice). | Compare the same cell type with HPA's reported level; inspect no-primary and detection controls, and confirm endogenous-activity blocking where applicable (general IHC practice). |
| Diffuse background obscures cell boundaries and neuropil. | The deposit prevents comparison with HPA's specified cellular pattern (HPA tissue IHC); inadequate blocking, washing or detection control can contribute (general IHC practice). | Review no-primary background, blocking, washes and detection exposure, then rescore only where the relevant tissue structures are readable (general IHC practice). |
| Signal looks cytoplasmic rather than like a sharp membrane outline. | HPA describes cytoplasmic tissue staining, while UniProt assigns ATP1A2 to the cell membrane (HPA tissue IHC; UniProt P50993 subcellular location). Dense neuropil can limit membrane resolution in chromogenic IHC (general IHC interpretation). | Judge whether staining follows the reported cells or neuropil and whether controls are clean; avoid rejecting a matching pattern solely because individual membranes are unresolved (HPA tissue IHC; general IHC practice). |
| A low-staining population is blank. | HPA reports Low staining in populations including liver hepatocytes and heart-muscle cardiomyocytes (HPA tissue IHC). A blank weak reference is less informative than a blank High reference (general IHC interpretation). | Check a high-staining CNS section in the same run, then interpret the low-staining population within the assay's demonstrated sensitivity (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing ATP1A2 staining
A02064-1 · IHCIHC analysis of ATP1A2 using anti-ATP1A2 antibody (A02064-1). ATP1A2 was detected in a paraffin-embedded section of human breast cancer tiss
A02064-1 · IHCIHC analysis of ATP1A2 using anti-ATP1A2 antibody (A02064-1). ATP1A2 was detected in a paraffin-embedded section of mouse cerebellum tissue.
A02064 · IHCImmunohistochemical analysis of paraffin-embedded human meningioma. 1, Antibody was diluted at 1:200(4° overnight). 2, Tris-EDTA,pH9.0 was u
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000018625-ATP1A2/tissue/cerebral+cortex#imid_5779476
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000018625-ATP1A2/tissue/colon#imid_5779418
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000018625-ATP1A2/tissue/liver#imid_5779419
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000018625-ATP1A2/tissue/kidney#imid_5779404
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ATP1A2 IHC & IF
🧪Run cerebellum first: its molecular-layer neuropil should stain (HPA: High in cerebellar molecular-layer neuropil). Run adrenal gland as the negative tissue, scoring its glandular cells (HPA: Not detected in adrenal gland glandular cells); any internal negative cells on the cerebellar slide should show counterstain without specific DAB signal, although the supplied HPA rows do not identify a cerebellar cell type as negative.
Positive control tissue: Cerebellum (Molecular layer - neuropil, HPA High)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for ATP1A2; derive a cell-line control from the positive tissue's cell type (Molecular layer - neuropil) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) control, a matched rabbit IgG isotype control, and an ATP1A2-knockout biological negative if available (catalog caption: rabbit anti-ATP1A2; standard IHC practice). Quench endogenous peroxidase before HRP/DAB detection and compare cerebellar neuropil with the no-primary slide for background (catalog caption: HRP/DAB; HPA: High in cerebellar molecular-layer neuropil).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A02064-1 paraffin-section caption does not state the fixative (catalog caption). That caption used heat retrieval in EDTA at pH 8.0, but it does not establish that retrieval is required or that frozen sections or IF are easier (catalog caption). Cerebellar autofluorescence can complicate IF interpretation, so assess it with a no-primary image before scoring neuropil signal (standard IF practice; HPA: High in cerebellar molecular-layer neuropil).
HPA tissue IHC evidence for ATP1A2
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 |
|---|---|---|---|---|
| Cerebellum | Molecular layer - neuropil | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuropil | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Caudate | Glial cells | Medium | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ATP1A2 IHC Tips
Troubleshoot ATP1A2 staining in paraffin section IHC using the catalog antibody’s tissue caption, protein topology, and tissue expression references.
Which retrieval condition should I start with for ATP1A2 paraffin sections?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 (datasheet A02064-1). This condition preceded ATP1A2 detection in a paraffin section of human breast cancer tissue, with 2 μg/mL primary antibody incubated overnight at 4°C (datasheet A02064-1). If staining is weak, compare heating durations on adjacent sections while keeping antibody concentration and DAB development constant (standard IHC practice). Assess signal against cerebellar molecular-layer neuropil and adrenal glandular cells as high-expression and undetected references, respectively (HPA: high in cerebellar neuropil; not detected in adrenal glandular cells). Preserve tissue morphology when selecting a retrieval duration (standard IHC practice).
How should I handle variable fixation across ATP1A2 IHC specimens?
ATP1A2-specific fixation sensitivity is unknown because the selected tissue caption does not report a fixative (datasheet A02064-1). Record fixative, fixation duration, and processing history before comparing staining across blocks (standard IHC practice). For uneven archival staining, compare sections with similar processing histories and change retrieval or primary incubation one variable at a time (standard IHC practice). The documented EDTA pH 8.0 heat retrieval, 2 μg/mL primary concentration, and overnight incubation at 4°C provide starting conditions for that comparison, but establish no preferred fixative (datasheet A02064-1). Include a consistently processed reference section in each staining run (standard IHC practice).
Should ATP1A2 IHC signal be membranous or cytoplasmic?
Prioritise cell-border staining when assessing localisation because ATP1A2 is annotated at the cell membrane and has 10 transmembrane segments (UniProt P50993: subcellular location and topology). HPA describes cytoplasmic staining in several tissues, most abundant in the CNS, so document cytoplasmic signal separately rather than treating it as proof of plasma-membrane localisation (HPA: tissue IHC profile; UniProt P50993: cell membrane). Compare staining within the same cell population and inspect section morphology at high magnification (standard IHC practice). Cerebellar molecular-layer neuropil and cortical neuropil have high HPA staining and offer useful spatial references (HPA: high in cerebellar and cortical neuropil).
How can epitope location affect ATP1A2 staining and specificity?
Confirm the antibody’s immunogen or mapped epitope before interpreting a negative section; the supplied caption does not identify its epitope (datasheet A02064-1). ATP1A2 has 10 transmembrane segments and alternating cytoplasmic and extracellular regions, so epitope accessibility can depend on processing and retrieval (UniProt P50993: topology; standard IHC practice). For example, residues 337–769 are cytoplasmic, whereas residues 864–915 are extracellular (UniProt P50993: topology). The record lists 0 ATP1A2 isoforms but describes multiple sodium-pump alpha-subunit isoforms; assess cross-reactivity using antibody-specific validation rather than the ATP1A2 isoform count alone (UniProt P50993: isoforms and subunit).
How should I adapt ATP1A2 tissue IHC observations to multiplex IF?
Treat multiplex IF conditions as an assay to optimise: the supplied antibody evidence documents chromogenic paraffin-section IHC, with no IF image set provided (datasheet A02064-1; HPA: subcellular IF images unavailable). Pair ATP1A2 with a validated marker for the cell population being assessed, such as a glial marker when examining the HPA-positive glial populations in caudate or hippocampus (HPA: medium in caudate and hippocampal glial cells; standard IF practice). Choose fluorophores after checking tissue autofluorescence and include single-label controls (standard IF practice). Match permeabilisation to the mapped epitope: intracellular epitopes require access across the membrane, while extracellular epitopes may be assessed without that step (UniProt P50993: topology; standard IF practice).
What should I check when ATP1A2 DAB staining looks diffuse?
Run a no-primary control to assess secondary-reagent and DAB background, and apply an endogenous-peroxidase block for chromogenic IHC (standard IHC practice). The selected caption used 10% goat-serum block, a peroxidase-conjugated secondary, and DAB development (datasheet A02064-1). If diffuse staining persists, compare primary-antibody concentrations around the documented 2 μg/mL condition and shorten DAB development while keeping reference sections in the run (datasheet A02064-1; standard IHC practice). Evaluate background separately from potentially genuine cytoplasmic staining, which HPA reports in several tissues (HPA: tissue IHC profile). Inspect folds, damaged edges, and precipitate before assigning a cellular score (standard IHC practice).
How should I score ATP1A2 staining across tissue groups? ⚠ ANSWER MARKED FOR VERIFICATION
Define the compartment and cell population before scoring, because UniProt places ATP1A2 at the cell membrane while HPA also reports cytoplasmic tissue staining (UniProt P50993: subcellular location; HPA: tissue IHC profile). For identifiable cells, record percent positive and intensity, then calculate an H-score on a 0–300 scale using the same thresholds across slides (standard IHC practice). For neuropil, measure stained area or optical density per mm² of annotated neuropil rather than forcing a cell count (HPA: high in cerebellar and cortical neuropil; standard image-analysis practice). Normalise to the measured area or eligible cell count and compare sections processed in the same run (standard IHC practice).
How do I distinguish credible ATP1A2 staining from artefact?
Look for reproducible staining in the intended compartment and cell population: ATP1A2 is a cell-membrane protein, while HPA reports high staining in cerebellar and cortical neuropil (UniProt P50993: subcellular location; HPA: tissue IHC). Interpret purely nuclear staining cautiously because neither source identifies the nucleus as an ATP1A2 location (UniProt P50993: subcellular location; HPA: subcellular summary). Compare adrenal glandular cells, where HPA reports no detection, with positive reference regions on the same run (HPA: adrenal glandular cells not detected; high in cerebellar neuropil). Exclude edge staining, necrotic areas, and signal retained in a no-primary control from positive scoring (standard IHC practice). HPA labels its tissue-IHC reliability Enhanced, with medium staining–RNA consistency (HPA: reliability description).
Boster reagents
Best ATP1A2 / Sodium/potassium-transporting ATPase subunit alpha-2 IHC Antibodies
Both antibodies have paraffin-section IHC images from human tissue; A02064-1 also has mouse and rat cerebellum images (catalog captions). Neither lists IF/ICC validation (catalog applications).
Real IHC data
Anti-ATP1A2 Antibody ®
Cat # A02064-1
Real IHC data
Anti-ATP1A2 Antibody
Cat # A02064
A02064-1 has IHC images from human breast cancer tissue and mouse and rat cerebellum (A02064-1 captions). A02064 has an IHC image from human meningioma; its listed reactivity also includes monkey, mouse and rat (A02064 caption; catalog reactivity).
Which to pick: For tissue IHC across human, mouse and rat, choose A02064-1 because its paraffin-section captions document all three species (A02064-1 captions). For monkey reactivity, consider the polyclonal A02064, but its IHC image documents only human meningioma (A02064 catalog reactivity; dilution_raw; caption). Neither SKU lists IF/ICC, so neither has a supported IF/ICC recommendation; both captions describe paraffin sections without reporting the fixative (catalog applications; IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P50993 (AT1A2_HUMAN, Sodium/potassium-transporting ATPase subunit alpha-2).
- Human Protein Atlas. ATP1A2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ATP1A2 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. ATP1A2 antibody validation summary (1 antibodies).
- Identification of Three Potential Prognostic Genes in Platinum-Resistant Ovarian Cancer via Integrated Bioinformatics Analysis. Cancer management and research 2021 — PMC8607279.
- Comprehensive analysis identified a reduction in ATP1A2 mediated by ARID3A in abdominal aortic aneurysm. Journal of cellular and molecular medicine 2022 — PMC9097831.
- Targeted ASO-mediated Atp1a2 knockdown in astrocytes reduces SOD1 aggregation and accelerates disease onset in mutant SOD1 mice. PloS one 2023 — PMC10683999.
- Comprehensive analysis of the expression of sodium/potassium-ATPase α subunits and prognosis of ovarian serous cystadenocarcinoma. Cancer cell international 2020 — PMC7362554.
- PubMed PMID:2477373 — UniProt-cited evidence.
- PubMed PMID:9872452 — UniProt-cited evidence.
- PubMed PMID:16710414 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ATP1A2 (Sodium/potassium-transporting ATPase subunit alpha-2). Expected localisation Cytoplasmic staining in several tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A02064-1). Validated IHC antibody: A02064-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (Molecular layer - neuropil); negative Adrenal gland. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A02064-1); 2. Fixation: Image fixative and duration unreported (datasheet A02064-1); 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 A02064-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A02064-1); 8. Primary antibody: Rabbit anti-ATP1A2, 2-5 μg/ml (datasheet A02064-1); 9. Primary incubation: Overnight at 4 °C (datasheet A02064-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining and RNA expression have medium consistency (HPA tissue IHC); Isoform / epitope: No isoforms annotated; epitope side depends on topology (UniProt); Regulation: Alpha-isoform expression varies by tissue (UniProt). Evidence: UniProt P50993, HPA tissue IHC · Enhanced, HPA ICC-IF · Membrane, Datasheet A02064-1, PMC8607279, PMC9097831. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ATP1A2/immunohistochemistry-ihc-guide.