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ATP1B2 / Sodium/potassium-transporting ATPase subunit beta-2 · IHC design guide
Design Immunohistochemistry for ATP1B2
Plan chromogenic IHC for ATP1B2 using retinal cell subsets and nerve fibers as expected staining patterns (HPA tissue IHC). This guide covers fixation consistency, antibody dilution, detection, and interpretation alongside the catalog antibody’s paraffin-section protocol (datasheet A07027-2).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ATP1B2 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 | Retinal cell membranes and cytoplasm; retinal and brain fibers (HPA tissue IHC) | |
| Staining pattern | Retinal cell subsets: membranous and cytoplasmic; nerve fibers (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A07027-2) | |
| Positive control | Retina+1 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). Selected-image fixative and duration unreported (datasheet A07027-2); verify before use. | |
| Caveat | Retinal staining is limited to cell subsets; field choice matters (HPA tissue IHC) | |
| Regulation | Brain/retina group-enriched RNA (HPA RNA) | |
| Isoform / epitope | No isoforms; epitope side: cytoplasmic 1–39 vs external 68–290 (UniProt) |
Section 1
Recommended ATP1B2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A07027-2) is followed by two published paraffin-section IHC protocols (PMC5555477; PMC12203461).
| Sample | Paraffin-embedded human glioma tissue; fixative not specified (datasheet A07027-2) |
| Fixation | Image fixative and duration unreported (datasheet A07027-2); 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 A07027-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A07027-2) |
| Primary antibody | Rabbit anti-ATP1B2, 2-5 μg/ml (datasheet A07027-2) |
| Primary incubation | Overnight at 4 °C (datasheet A07027-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A07027-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ATP1B2-positive staining in inner nuclear layer of retina (HPA tissue IHC: High). HPA tissue profile: Membranous and cytoplasmic expression in subsets of cells in retina. Additional expression in nerve fibers in retina and brain. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet: A07027-2); adjust retrieval conditions for the chosen sample using the published methods (PMC5555477; PMC12203461).
Section 2
What Is the Expected ATP1B2 Staining Pattern?
ATP1B2 is a cell-membrane protein with a cytoplasmic N terminus and extracellular C terminus (UniProt P14415 topology). In paraffin-section IHC, expect membranous and cytoplasmic staining in subsets of retinal cells, including strong staining in the inner nuclear layer, plus staining of nerve fibers in retina and brain (HPA tissue IHC: Supported; HPA: Retina inner nuclear layer High).
| Strong, selective staining in the retinal inner nuclear layer, with membranous and some cytoplasmic signal. | This fits the reported retinal pattern (HPA tissue IHC: Supported; HPA: inner nuclear layer High). Some cytoplasmic staining is expected in retinal cell subsets, so score it alongside its location and cell distribution (HPA tissue IHC). |
| Moderate staining in cerebral-cortex neuropil or staining along retinal and brain nerve fibers. | These are reported structures, although cortex neuropil is less intense than the listed retinal positive (HPA: cerebral-cortex neuropil Medium; HPA tissue IHC: nerve fibers). Compare the same anatomical structures across sections before calling a difference in intensity. |
| Predominantly nuclear staining, or uniform staining across cells and structures beyond the reported distribution. | Nuclear localisation conflicts with the membrane assignment (UniProt P14415 subcellular location). An unusually widespread pattern needs review for background or nonspecific binding (general IHC practice); do not dismiss reported retinal cytoplasmic staining as artefact (HPA tissue IHC). |
| Clear signal in a cell type listed as not detected, such as adrenal glandular cells. | That differs from the listed HPA result for that cell type (HPA: adrenal glandular cells Not detected). Check specificity and endogenous detection activity before interpreting it as ATP1B2; the HPA entry does not classify every cell in the organ. |
| Diffuse staining throughout the section, or no staining in the retinal inner nuclear layer. | Diffuse signal makes the reported cell and structure pattern hard to identify (HPA tissue IHC). An absent retinal positive is inconsistent with the listed High result (HPA: inner nuclear layer High); first assess section quality and the IHC detection workflow (general IHC practice). |
💡Expected ATP1B2 appearanceA convincing positive shows High staining in retinal inner nuclear-layer cells, with membranous and some cytoplasmic signal; cortex neuropil may be Medium (HPA tissue IHC). Strong nuclear or indiscriminate staining warrants investigation (UniProt P14415 subcellular location; general IHC practice).
| Tissue and cell selection | Retinal inner nuclear layer is the listed High positive; cerebral-cortex neuropil is Medium (HPA tissue IHC). Listed negatives refer to specified cell types, such as adrenal glandular cells, not entire tissues (HPA tissue IHC). |
| Membrane topology and epitope location | ATP1B2 spans residues 40–67; residues 1–39 are cytoplasmic and 68–290 extracellular (UniProt P14415 topology). The supplied record gives no antibody epitope, so it cannot predict which side an antibody recognizes or whether retrieval is needed. |
| Glycosylation and processing | Seven glycosylation sites are annotated in the extracellular region, at residues 96, 118, 153, 159, 193, 197 and 238 (UniProt P14415 glycosylation; topology). No cleavage or shedding is annotated in the supplied record; staining cannot establish either event (UniProt P14415 processing). |
| Strength and scope of validation | HPA rates the tissue IHC pattern Supported, citing consistency between staining and RNA expression (HPA tissue IHC: Supported). The listed antibody HPA010698 has IHC Supported status (HPA antibodies); that does not validate every reagent or every tissue. |
| What about IF/ICC? | The HPA subcellular summary says Membrane, but gives no main location or ICC-IF image cell lines, and the listed antibody has no ICC status (HPA subcellular; HPA antibodies). Use the membrane assignment as an interpretation guide, not as evidence of an IF/ICC protocol or validated cell-line result. |
| Situation | Likely cause | Next action |
|---|---|---|
| Retinal inner nuclear layer has no detectable signal. | A listed High positive is missing (HPA: retinal inner nuclear layer High); section quality or an IHC workflow step may have failed (general IHC practice). | Confirm the anatomical layer and tissue preservation, then review reagent activity, detection and the catalog antibody's IHC-P instructions (general IHC practice). Do not assign an ATP1B2-specific fixation effect from this result. |
| Signal is broadly nuclear. | A dominant nuclear pattern conflicts with the recorded cell-membrane localisation (UniProt P14415 subcellular location). Background or nonspecific antibody binding is possible (general IHC practice). | Compare with a no-primary control and the retinal positive pattern; review blocking and detection conditions (general IHC practice; HPA: retinal inner nuclear layer High). |
| A listed negative cell type stains strongly. | The result differs from the HPA listing for that specific cell type (HPA: adrenal glandular cells Not detected). Nonspecific binding or endogenous chromogenic activity could contribute (general IHC practice). | Identify the stained cell type precisely; inspect no-primary and detection-only controls, then compare the signal with a known-positive section (general IHC practice; HPA: retinal inner nuclear layer High). |
| Brown precipitate or diffuse color obscures cell boundaries. | Nonspecific background or detection activity may prevent assessment of the reported membranous and cytoplasmic retinal pattern (general IHC practice; HPA tissue IHC). | Check no-primary controls, blocking, washes, chromogen development and counterstain; adjust using the detection system's instructions (general IHC practice). Reassess whether the expected structures remain distinguishable (HPA tissue IHC). |
| Cortex neuropil appears weaker than retinal inner nuclear layer. | That difference can agree with the listed Medium cortex neuropil and High retinal inner nuclear-layer levels (HPA tissue IHC). | Score each listed structure against its own expected level, using comparable staining conditions (HPA tissue IHC; general IHC practice). Investigate only if its signal or anatomical distribution is otherwise inconsistent. |
| Cytoplasmic staining is seen in retinal cell subsets. | HPA explicitly reports cytoplasmic as well as membranous expression in subsets of retinal cells (HPA tissue IHC); compartment alone does not make this an artefact. | Check whether the signal remains confined to the reported retinal subsets and accompanies a plausible membrane pattern (HPA tissue IHC; UniProt P14415 subcellular location). Use controls if it becomes diffuse or widespread (general IHC practice). |
Experiment evidence
IHC & IF images showing ATP1B2 staining
A07027-2 · IHCIHC analysis of ATP1B2 using anti-ATP1B2 antibody (A07027-2). ATP1B2 was detected in a paraffin-embedded section of human glioma tissue. Hea
A07027-1 · IHCFormalin-fixed and paraffin-embedded human hepatocarcinoma reacted with ATP1B2 Antibody (Center), which was peroxidase-conjugated to the sec
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000129244-ATP1B2/tissue/cerebral+cortex#imid_21067323
HPA · RetinaHuman Protein Atlas IHC, Retina. Source: https://www.proteinatlas.org/ENSG00000129244-ATP1B2/tissue/retina#imid_1586
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000129244-ATP1B2/tissue/colon#imid_21067241
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000129244-ATP1B2/tissue/liver#imid_21067258
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ATP1B2 IHC & IF
🧪Run retina first; its inner nuclear layer should stain for ATP1B2 (HPA: High in retina inner nuclear layer). Use appendix glandular cells as the negative tissue (HPA: Not detected); on the retina slide, assess internal background only in cells independently confirmed to lack target signal, which should show counterstain without specific DAB deposition (standard IHC practice).
Positive control tissue: Retina (Inner nuclear layer, 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 ATP1B2; derive a cell-line control from the positive tissue's cell type (Inner nuclear layer) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control, a concentration-matched rabbit IgG isotype control of corresponding clonality, and ATP1B2 knockout tissue or a validated peptide competition control (caption: rabbit primary antibody; standard IHC practice). Quench endogenous peroxidase for DAB detection and assess retinal pigment as a possible source of apparent signal (caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the selected A07027-2 paraffin-section caption does not state a fixative (caption: fixative unreported). The reported IHC conditions use heat-mediated EDTA retrieval at pH 8.0, but a strict retrieval dependency is unreported (caption: EDTA retrieval). Whether frozen sections or IF are easier is unreported; validate those preparations separately and distinguish retinal pigment from chromogenic signal (standard IHC practice).
HPA tissue IHC evidence for ATP1B2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — High consistency between antibody staining and RNA expression data. Antibody staining in cells/structures not annotated, view images.). 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 |
|---|---|---|---|---|
| Retina | Inner nuclear layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuropil | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| 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 → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ATP1B2 IHC Tips
Troubleshoot ATP1B2 staining in paraffin sections by checking retrieval, compartment, tissue context, and controls before comparing signal intensity.
What retrieval should I try first if ATP1B2 staining is weak?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 (datasheet A07027-2). That condition preceded ATP1B2 detection in a paraffin section of human glioma using 2 μg/ml primary antibody overnight at 4°C (datasheet A07027-2). If staining remains weak, compare adjusted heating and cooling times on matched sections while holding primary concentration and detection constant (standard IHC practice). Score membrane-associated signal separately from diffuse background, because ATP1B2 is a cell-membrane protein and tissue staining can also appear cytoplasmic (UniProt P14415 localisation; HPA tissue IHC).
Could fixation explain weak or variable ATP1B2 staining?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state its fixative, and no fixation comparison is supplied (datasheet A07027-2). Record each sample’s fixative and fixation duration, then compare sections processed with the same retrieval and detection conditions (standard IHC practice). Use heat-mediated EDTA retrieval at pH 8.0 as the documented starting condition, with 2 μg/ml primary antibody overnight at 4°C if reproducing the caption (datasheet A07027-2). If staining differs between batches, check processing records and a shared control section before attributing the difference to ATP1B2 abundance (standard IHC practice).
Where should I expect ATP1B2 staining in a tissue section?
Look first for membrane-associated staining: ATP1B2 is annotated at the cell membrane, with a transmembrane segment at residues 40–67 (UniProt P14415 localisation and topology). Membranous and cytoplasmic staining occurs in subsets of retinal cells, with additional staining in retinal and brain nerve fibers (HPA tissue IHC). The retinal inner nuclear layer is reported high and cerebral cortex neuropil medium, so compare like structures when assessing a run (HPA tissue IHC). A diffuse signal across unrelated structures needs control review; the glioma caption reports detection but does not establish a universal cellular staining pattern (datasheet A07027-2; standard IHC practice).
How can epitope location affect ATP1B2 staining?
Ask for the antibody’s epitope map before interpreting a compartment-specific failure; the supplied product caption does not identify its binding site (datasheet A07027-2). ATP1B2 has a cytoplasmic region at residues 1–39, a membrane span at 40–67, and an extracellular region at 68–290 (UniProt P14415 topology). Seven glycosylation sites are annotated in the extracellular region, so altered epitope accessibility is a possibility to investigate, not a demonstrated cause of weak staining (UniProt P14415 glycosylation; standard IHC practice). No isoforms are annotated in the supplied record; do not assign a staining difference to a specific isoform without independent evidence (UniProt P14415 isoform record).
How should I adapt ATP1B2 staining for multiplex immunofluorescence?
Treat multiplex IF as a separate optimization: the supplied subcellular record lists no ICC/IF image-bearing cell lines (HPA subcellular). In retina, pair ATP1B2 with a marker that identifies the cells being scored in the inner nuclear layer, where staining is reported high (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores, favor a far-red channel if the section is autofluorescent, and inspect single-stain and unstained controls before merging channels (standard IF practice). If the epitope is cytoplasmic, optimize permeabilisation; if extracellular, test minimal permeabilisation, because the two regions lie on opposite sides of the membrane (UniProt P14415 topology; standard IF practice).
What controls help identify ATP1B2 IHC background?
Include a no-primary section to reveal detection-system signal, and inspect tissue regions expected to stain weakly alongside the target region (standard IHC practice). The caption used 10% goat serum before 2 μg/ml rabbit primary antibody, followed by a peroxidase-linked secondary and DAB development (datasheet A07027-2). Block endogenous peroxidase and assess whether DAB appears without primary antibody; these are general chromogenic IHC controls (standard IHC practice). Appendix glandular cells and bone-marrow hematopoietic cells were reported as not detected, but their status in your assay still requires verification (HPA tissue IHC; standard IHC practice).
How should I quantify ATP1B2 across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the anatomical region and scoring compartment before analysis, since ATP1B2 staining may occur at membranes, in cytoplasm, and along nerve fibers (HPA tissue IHC). For cells, report the percentage positive and an H-score from 0–300; for fiber-rich regions, report positive area or signal density per mm² (standard IHC practice). Normalize cell counts to eligible cells and fiber measurements to the measured region area, using the same threshold and exposure or scan settings across groups (standard IHC practice). Keep retinal inner nuclear layer and cerebral cortex neuropil results separate because their reported staining levels and structures differ (HPA tissue IHC).
How do I distinguish convincing ATP1B2 staining from artefact?
A convincing result matches the sampled structure and shows membrane-associated staining or the reported retinal and brain patterns, with low signal in the no-primary control (UniProt P14415 localisation; HPA tissue IHC; standard IHC practice). Retinal inner nuclear layer staining and cerebral cortex neuropil staining have different reported levels, so interpret them against their own tissue context (HPA tissue IHC). Review staining confined to section edges or necrotic areas as possible processing artefact, and check for endogenous-peroxidase signal before assigning DAB deposition to ATP1B2 (standard IHC practice). The glioma image documents detection under its stated conditions; it does not establish that every stained glioma cell is positive (datasheet A07027-2).
Boster reagents
Best ATP1B2 / Sodium/potassium-transporting ATPase subunit beta-2 IHC Antibodies
Two anti-ATP1B2 antibodies have human paraffin-section IHC images (catalog captions). Neither has an IF/ICC image or listed IF/ICC application (catalog applications and image records).
Real IHC data
Anti-ATP1B2 Antibody ®
Cat # A07027-2
Real IHC data
Anti-ATP1B2 Antibody (Center)
Cat # A07027-1
A07027-2 has an IHC image from a paraffin-embedded human glioma section; its listed reactivity is human, mouse and rat (A07027-2 caption; catalog reactivity). A07027-1 has an IHC-P image from formalin-fixed, paraffin-embedded human hepatocarcinoma and lists human reactivity (A07027-1 caption; catalog applications and reactivity).
Which to pick: For human tissue IHC, choose A07027-2 for a paraffin-section workflow with documented EDTA retrieval and 2 μg/ml primary antibody; its caption does not report the fixative (A07027-2 caption). Choose A07027-1 when documented fixation matters; it is a rabbit polyclonal antibody with a listed IHC-P dilution of 1:50–1:100 (A07027-1 caption; catalog dilution record). Neither SKU lists IF/ICC validation; A07027-2 lists mouse and rat reactivity, but its supplied tissue IHC image documents human tissue only (catalog applications and reactivity; A07027-2 caption). The selected A07027-2 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image A07027-2).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P14415 (AT1B2_HUMAN, Sodium/potassium-transporting ATPase subunit beta-2).
- Human Protein Atlas. ATP1B2 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. ATP1B2 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. ATP1B2 antibody validation summary (1 antibodies).
- A SINE Insertion in ATP1B2 in Belgian Shepherd Dogs Affected by Spongy Degeneration with Cerebellar Ataxia (SDCA2). G3 (Bethesda, Md.) 2017 — PMC5555477.
- Overexpression of ATP1B2 promotes cancer cell migration and inhibits apoptosis in patients with esophageal squamous cell carcinoma. Oncology reports 2025 — PMC12203461.
- PubMed PMID:8305453 — UniProt-cited evidence.
- PubMed PMID:8918259 — UniProt-cited evidence.
- PubMed PMID:9524271 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ATP1B2 (Sodium/potassium-transporting ATPase subunit beta-2). Expected localisation Retinal cell membranes and cytoplasm; retinal and brain fibers (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A07027-2). Validated IHC antibody: A07027-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Retina (Inner nuclear layer); negative Adrenal gland. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human glioma tissue; fixative not specified (datasheet A07027-2); 2. Fixation: Image fixative and duration unreported (datasheet A07027-2); 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 A07027-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A07027-2); 8. Primary antibody: Rabbit anti-ATP1B2, 2-5 μg/ml (datasheet A07027-2); 9. Primary incubation: Overnight at 4 °C (datasheet A07027-2). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet A07027-2); verify before use.; Caveat: Retinal staining is limited to cell subsets; field choice matters (HPA tissue IHC); Isoform / epitope: No isoforms; epitope side: cytoplasmic 1–39 vs external 68–290 (UniProt); Regulation: Brain/retina group-enriched RNA (HPA RNA). Evidence: UniProt P14415, HPA tissue IHC · Supported, HPA ICC-IF · Membrane, Datasheet A07027-2, PMC5555477, PMC12203461. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ATP1B2/immunohistochemistry-ihc-guide.