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ATP1B1 / Sodium/potassium-transporting ATPase subunit beta-1 · IHC design guide
Design Immunohistochemistry for ATP1B1
Plan ATP1B1 chromogenic IHC on paraffin sections using 2–5 μg/ml as the catalog antibody’s starting range (datasheet A03469-1). Kidney collecting ducts provide a high-staining control, and membranous staining is expected in many tissues (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ATP1B1 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 staining in many tissues (HPA tissue IHC) | |
| Staining pattern | Membranous staining in glandular cells and collecting ducts (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03469-1) | |
| Positive control | Duodenum+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A03469-1) | |
| Caveat | Antibody staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Kidney-enhanced RNA expression (HPA tissue RNA) | |
| Isoform / epitope | 2 isoforms; check cytoplasmic versus extracellular epitope (UniProt) |
Section 1
Recommended ATP1B1 IHC & IF Protocols
The catalog antibody has an IHC-P protocol (datasheet A03469-1). Published IHC examples include rat CA1 sections (PMC5970211) and lung cancer tissue microarrays (PMC7174014).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A03469-1) |
| Fixation | Image fixative and duration unreported (datasheet A03469-1); 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 A03469-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03469-1) |
| Primary antibody | Rabbit anti-ATP1B1, 2-5 μg/ml (datasheet A03469-1) |
| Primary incubation | Overnight at 4 °C (datasheet A03469-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A03469-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ATP1B1-positive staining in glandular cells of duodenum (HPA tissue IHC: High). HPA tissue profile: Membranous expression in many tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet A03469-1). The tissue microarray article reports antigen retrieval without specifying conditions (PMC7174014).
Section 2
What Is the Expected ATP1B1 Staining Pattern?
ATP1B1 is a cell membrane protein with residues 1–34 cytoplasmic, 35–62 transmembrane, and 63–303 extracellular (UniProt P05026 topology). In paraffin section IHC, expect membranous staining in many tissues, especially kidney collecting ducts and glandular cells of duodenum, gallbladder, salivary gland, and small intestine (HPA tissue IHC: High). HPA rates the tissue IHC pattern Enhanced, with medium consistency between staining and RNA data (HPA tissue IHC reliability).
| Membrane outlines in kidney collecting ducts or duodenal glandular cells, with strong chromogenic signal (HPA: High in these cells). | This fits the reported cell types, intensity, and membrane pattern (HPA tissue IHC). Compare staining within the named cells; a high tissue rating does not mean every cell in that section should stain equally (HPA tissue IHC). |
| Predominantly nuclear staining, with no convincing membrane outline in an otherwise positive cell population. | A nuclear dominant pattern conflicts with ATP1B1 membrane localisation (UniProt P05026 subcellular location; HPA tissue IHC: membranous expression). Review staining controls and morphology before scoring it as specific ATP1B1 signal (general IHC practice). |
| Strong signal in adipocytes or esophageal squamous epithelial cells. | HPA reports ATP1B1 as not detected in those specific cell populations (HPA tissue IHC). Check for nonspecific antibody binding or endogenous detection activity (general IHC practice); the HPA result does not label every cell in those tissues negative. |
| Uniform brown haze across cells, stroma, and empty areas, without cell membrane definition. | This distribution obscures the reported membranous pattern (HPA tissue IHC). Assess background with a negative reagent control and review blocking, washing, and chromogen development (general IHC practice). |
| No staining in kidney collecting ducts, despite visible, intact ducts and an otherwise interpretable section. | This misses a reported High population (HPA tissue IHC: kidney collecting ducts). First confirm assay controls and tissue identity; absent staining alone cannot distinguish technical failure from sample variation (general IHC practice). |
💡Expected ATP1B1 appearanceCall a positive result when the IHC-validated antibody gives clear membrane staining in kidney collecting ducts or listed high expressing glandular cells (HPA tissue IHC: High; UniProt P05026 subcellular location); diffuse haze or dominant nuclear signal is inconsistent with that pattern (HPA tissue IHC; UniProt P05026).
| Membrane topology and epitope location | ATP1B1 has one membrane span and a large extracellular region at residues 63–303 (UniProt P05026 topology). The payload does not identify the catalog antibody epitope, so topology alone cannot set retrieval or permeabilisation conditions. |
| Tissue and cell selection | Kidney collecting ducts and the listed high expressing glandular populations offer observed positive patterns (HPA tissue IHC: High). HPA also reports medium neuropil staining in cerebral cortex and no detection in selected glial populations; score the annotated cell type, not the tissue name (HPA tissue IHC). |
| Evidence strength | HPA describes membranous expression in many tissues and rates its tissue IHC reliability Enhanced, while noting medium agreement with RNA (HPA tissue IHC). One listed antibody, HPA012911, has IHC Enhanced status (HPA antibodies); these summaries do not prove specificity in every specimen. |
| Isoforms and modification | UniProt lists 2 isoforms and glycosylation sites at residues 158, 193, and 265 (UniProt P05026). Their effect on this antibody's staining is unreported; do not infer an isoform specific or glycosylation dependent pattern from these annotations. |
| IF/ICC Q&A: What localisation should an IF result support? | A membrane pattern is consistent with UniProt and the HPA subcellular summary (UniProt P05026; HPA ICC-IF: Membrane). HPA supplies no ICC-IF image cell lines or main location here, and the listed antibody has no ICC validation status (HPA ICC-IF; HPA antibodies). |
| Situation | Likely cause | Next action |
|---|---|---|
| Kidney collecting ducts show no chromogenic signal (HPA tissue IHC: High). | The run may have failed, or the section may lack assessable collecting ducts (general IHC practice). | Confirm duct identity and section quality; inspect assay controls, then review retrieval, antibody incubation, and detection steps (general IHC practice). No ATP1B1 specific retrieval condition is established by these sources. |
| The entire section is brown, including areas without recognisable cells. | Background from the staining workflow can conceal membrane outlines (general IHC practice). | Compare a negative reagent control, check blocking and wash steps, and shorten excessive chromogen development if indicated (general IHC practice). Reassess whether cell membranes remain distinguishable (HPA tissue IHC: membranous expression). |
| Nuclei dominate the signal in a reported positive population. | This conflicts with ATP1B1's reported cell membrane localisation (UniProt P05026; HPA tissue IHC). | Check counterstain and chromogen appearance, examine a negative reagent control, and score only a reproducible membrane pattern in the expected cells (general IHC practice; HPA tissue IHC). |
| Adipocytes or esophageal squamous epithelial cells stain strongly. | Those populations are reported as not detected (HPA tissue IHC); nonspecific staining or endogenous detection activity is possible (general IHC practice). | Verify cell identity, inspect negative controls, and review blocking and detection chemistry before interpreting the signal as ATP1B1 (general IHC practice). |
| A whole organ is called negative because one cell population lacks staining. | HPA observations apply to named cells: for example, liver cholangiocytes are not detected, while kidney collecting ducts are High (HPA tissue IHC). | Record tissue compartment and cell type alongside intensity; compare like cell populations before classifying a result (HPA tissue IHC; general IHC practice). |
| An IF/ICC image is treated as validation for the paraffin section result. | The supplied HPA subcellular entry has no ICC-IF image cell lines, and HPA012911 has no listed ICC status (HPA ICC-IF; HPA antibodies). | Judge the paraffin section against the HPA tissue IHC membrane pattern and cell specific observations; keep IF/ICC interpretation within its own guide (HPA tissue IHC; HPA ICC-IF). |
Experiment evidence
IHC & IF images showing ATP1B1 staining
A03469-1 · IHCIHC analysis of ATP1B1 using anti-ATP1B1 antibody (A03469-1). ATP1B1 was detected in a paraffin-embedded section of human breast cancer tiss
A03469-1 · IHCIHC analysis of ATP1B1 using anti-ATP1B1 antibody (A03469-1). ATP1B1 was detected in a paraffin-embedded section of human colonic adenocarci
A03469-1 · IFIF analysis of ATP1B1 using anti-ATP1B1 antibody (A03469-1). ATP1B1 was detected in an immunocytochemical section of SiHa cells. Enzyme anti
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000143153-ATP1B1/tissue/cerebral+cortex#imid_20780888
HPA · Parathyroid glandHuman Protein Atlas IHC, Parathyroid gland. Source: https://www.proteinatlas.org/ENSG00000143153-ATP1B1/tissue/parathyroid+gland#imid_20780779
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000143153-ATP1B1/tissue/colon#imid_20780806
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000143153-ATP1B1/tissue/liver#imid_20780823
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ATP1B1 IHC & IF
🧪Run kidney first; collecting-duct cells should stain (HPA: High in kidney collecting ducts). Use adipose tissue adipocytes as the negative (HPA: Not detected in adipocytes); unstained background on the kidney slide provides an internal background reference, but the supplied HPA rows do not identify a kidney cell type as negative.
Positive control tissue: Duodenum (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for ATP1B1; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control; a matched nonimmune rabbit IgG control (caption: rabbit primary antibody); and an ATP1B1-knockout biological negative, if available (standard IHC specificity controls). For kidney chromogenic IHC, quench endogenous peroxidase and assess endogenous biotin background when using the biotin-based detection shown in the caption (caption: biotinylated secondary, SABC and DAB; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A03469-1 paraffin-section caption does not state a fixative (caption: fixative unreported). Heat retrieval in EDTA at pH 8.0 is documented, but whether retrieval is required has not been established (caption: EDTA retrieval). The supplied evidence does not establish whether frozen sections or IF are easier; in kidney, check for endogenous biotin background with the caption’s biotin-based detection (caption: SABC/DAB; standard IHC practice).
HPA tissue IHC evidence for ATP1B1
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 |
|---|---|---|---|---|
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Kidney | Collecting ducts | High | Protein (IHC) | HPA → |
| Salivary gland | Glandular cells | High | Protein (IHC) | HPA → |
| Small intestine | 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 → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ATP1B1 IHC Tips
Troubleshoot ATP1B1 chromogenic IHC by checking retrieval, membrane localisation, staining controls and cell specific scoring.
How should I troubleshoot weak ATP1B1 staining after antigen retrieval?
Start with heat mediated retrieval in EDTA, pH 8.0, for paraffin sections (datasheet A03469-1). The documented tissue IHC example used this retrieval before applying 2 μg/ml antibody overnight at 4°C (caption A03469-1). If staining is weak, compare retrieval durations on adjacent sections while holding antibody concentration and detection conditions constant (standard IHC practice). Include a section with expected membranous staining, such as kidney collecting ducts (HPA: High in kidney collecting ducts; HPA: membranous expression in many tissues). Excessive retrieval can damage tissue morphology, so assess membrane definition alongside signal intensity (standard IHC practice).
Could fixation explain weak or uneven ATP1B1 IHC staining?
ATP1B1 sensitivity to a particular fixative or fixation duration is unknown from the supplied evidence (caption A03469-1: fixative not stated). The documented image shows a paraffin embedded section, but its caption does not establish how the tissue was fixed (caption A03469-1). Record the fixative and processing history, then compare similarly processed sections when investigating uneven staining (standard IHC practice). Keep retrieval at EDTA, pH 8.0, and the primary antibody at 2 μg/ml initially, matching the documented example (caption A03469-1). Assess tissue preservation and membrane definition before attributing weak staining to fixation (standard IHC practice; HPA: membranous expression in many tissues).
Where should convincing ATP1B1 chromogenic staining appear?
Prioritise sharply defined cell membrane staining, including the apical membrane where appropriate (UniProt P05026 localisation; HPA: membranous expression in many tissues). ATP1B1 has an extracellular region at residues 63–303 and one membrane spanning segment at 35–62 (UniProt P05026 topology). In epithelia, examine cell boundaries because ATP1B1 contributes to lateral pump polarisation and cell adhesion (UniProt P05026 function). Kidney collecting ducts and duodenal glandular cells provide examples with high tissue IHC staining (HPA: High in kidney collecting ducts; HPA: High in duodenal glandular cells). Diffuse nuclear colour without corresponding membrane staining warrants review of detection controls and tissue morphology (standard IHC practice; UniProt P05026 localisation).
How could epitope position or isoforms affect ATP1B1 IHC?
ATP1B1 has 2 annotated isoforms, but the supplied caption does not identify the antibody epitope or establish isoform selectivity (UniProt P05026 isoforms; caption A03469-1). The protein has a cytoplasmic segment at residues 1–34, a transmembrane segment at 35–62, and an extracellular segment at 63–303 (UniProt P05026 topology). Glycosylation is annotated at residues 158, 193, and 265; these annotations alone do not show that glycosylation changes this antibody's staining (UniProt P05026 glycosylation; caption A03469-1). Check the antibody's mapped immunogen or epitope before interpreting variable staining as an isoform difference (standard IHC practice). Compare adjacent sections under identical retrieval and detection conditions to separate staining variability from tissue distribution (standard IHC practice).
How should I adapt ATP1B1 localisation checks to multiplex IF?
For multiplex IF, pair ATP1B1 with a validated marker of the expected cell type, such as a collecting duct cell marker in kidney sections (HPA: High in kidney collecting ducts; standard IF practice). Choose spectrally separated fluorophores and favour a longer wavelength channel for weak ATP1B1 signal when tissue autofluorescence interferes (standard IF practice). Determine the antibody epitope before setting permeabilisation: access to residues 1–34 on the cytoplasmic side may require it, while residues 63–303 are extracellular (UniProt P05026 topology; standard IF practice). Check single stain and secondary only controls for bleed through and background (standard IF practice). The supplied tissue caption documents chromogenic IHC, not an IF validation or IF fixation condition (caption A03469-1).
What causes diffuse brown background in ATP1B1 IHC?
First compare a no primary control with the stained section to distinguish detection background from antibody associated signal (standard IHC practice). Apply a peroxidase block before DAB development and examine whether endogenous enzyme activity persists in the control (standard chromogenic IHC practice). The documented method used 10% goat serum, a biotinylated secondary antibody, a streptavidin biotin complex, and DAB (caption A03469-1). If background remains, check blocking, washing, secondary antibody binding, and detection reagent exposure on adjacent sections (standard IHC practice). Score only anatomically resolved membrane staining; ATP1B1 is reported at cell membranes, while diffuse brown deposits can obscure that pattern (UniProt P05026 localisation; standard IHC practice).
How should I quantify ATP1B1 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue compartment and cell population before measuring membranous ATP1B1 staining (HPA: membranous expression in many tissues; standard IHC practice). For an H-score, grade membrane intensity from 0–3, multiply each grade by its percentage of cells, and sum to a 0–300 score (standard IHC scoring practice). Alternatively, report the percentage of membrane positive cells or positive cell density per mm² within a predefined region (standard IHC scoring practice). Normalise counts to eligible cells or measured tissue area, and apply the same threshold and exclusion rules across sections (standard IHC practice). Record cell type because HPA reports high staining in kidney collecting ducts but no detected staining in adipocytes (HPA tissue IHC).
How can I distinguish true ATP1B1 signal from artefact?
A credible positive follows cell membranes in an appropriate cell population, such as kidney collecting ducts or duodenal glandular cells (UniProt P05026 localisation; HPA: High in both populations). Treat isolated nuclear colour, tissue edge intensification, and staining confined to necrotic areas as reasons to inspect morphology and controls (standard IHC practice). Compare a no primary control to identify endogenous enzyme activity or other detection background before calling diffuse DAB colour positive (standard chromogenic IHC practice). Check the cell type as well as the organ: HPA reports no detected staining in adipocytes, despite broad ATP1B1 tissue distribution (HPA: Not detected in adipocytes; UniProt P05026 tissue specificity). HPA reports medium consistency between staining and RNA expression, so interpret unexpected patterns with appropriate controls (HPA: Enhanced reliability description; standard IHC practice).
Boster reagents
Best ATP1B1 / Sodium/potassium-transporting ATPase subunit beta-1 IHC Antibodies
A03469-1 has illustrated IHC in human paraffin sections and IF in SiHa cells and mouse and rat kidney sections (catalog image captions).
Real IHC data
Anti-ATP1B1 Antibody ®
Cat # A03469-1
A03469-1 is the SKU with a rendered card and its own IHC figure, showing staining in a human breast cancer paraffin section (card caption). Its catalog images also show IHC in human colonic adenocarcinoma, gall bladder adenosquamous carcinoma and liver cancer sections, plus IF in SiHa cells and mouse and rat kidney sections (catalog image captions).
Which to pick: Choose A03469-1 for tissue IHC: its own figure uses a human paraffin section, EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody; the fixative is unreported (card caption). Choose A03469-1 for IF/ICC or illustrated cross-species work: its catalog images show IF in SiHa cells and mouse and rat kidney sections, while its application list includes IHC, IF and ICC with human, mouse and rat reactivity (catalog image captions; catalog applications and reactivity). M03469 is a rabbit monoclonal listed for IHC in those three species, but it has no supplied IHC image caption to establish sample processing (catalog: clone 23A21, applications and reactivity; catalog image alts).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P05026 (AT1B1_HUMAN, Sodium/potassium-transporting ATPase subunit beta-1).
- Human Protein Atlas. ATP1B1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ATP1B1 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. ATP1B1 antibody validation summary (1 antibodies).
- Neuroglobin mediates neuroprotection of hypoxic postconditioning against transient global cerebral ischemia in rats through preserving the activity of Na(+)/K(+) ATPases. Cell death & disease 2018 — PMC5970211.
- Na/K-ATPase Gene Expression in the Human Cochlea: A Study Using mRNA in situ Hybridization and Super-Resolution Structured Illumination Microscopy. Frontiers in molecular neuroscience 2022 — PMC9009265.
- Immunohistochemical validation study of 15-gene biomarker panel predictive of benefit from adjuvant chemotherapy in resected non-small-cell lung cancer: analysis of JBR.10. ESMO open 2020 — PMC7174014.
- Genome-wide association study identifies three novel loci in Fuchs endothelial corneal dystrophy. Nature communications 2017 — PMC5379100.
- PubMed PMID:3008098 — UniProt-cited evidence.
- PubMed PMID:2559024 — UniProt-cited evidence.
- PubMed PMID:7536695 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ATP1B1 (Sodium/potassium-transporting ATPase subunit beta-1). Expected localisation Membranous staining in many tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03469-1). Validated IHC antibody: A03469-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Duodenum (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A03469-1); 2. Fixation: Image fixative and duration unreported (datasheet A03469-1); 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 A03469-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03469-1); 8. Primary antibody: Rabbit anti-ATP1B1, 2-5 μg/ml (datasheet A03469-1); 9. Primary incubation: Overnight at 4 °C (datasheet A03469-1). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A03469-1); Caveat: Antibody staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 2 isoforms; check cytoplasmic versus extracellular epitope (UniProt); Regulation: Kidney-enhanced RNA expression (HPA tissue RNA). Evidence: UniProt P05026, HPA tissue IHC · Enhanced, HPA ICC-IF · Membrane, Datasheet A03469-1, PMC5970211, PMC7174014. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ATP1B1/immunohistochemistry-ihc-guide.