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ANKRD26 / Ankyrin repeat domain-containing protein 26 · IHC design guide
Design Immunohistochemistry for ANKRD26
Colon glandular cells and cerebral cortex neurons offer high-staining IHC references for ANKRD26 (HPA tissue IHC). This guide pairs those references with the catalog antibody’s 1:100–1:300 IHC dilution range (datasheet) and consistent paraffin-section fixation.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ANKRD26 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, often granular in tissue (HPA tissue IHC) | |
| Staining pattern | Glandular and neuronal cells: cytoplasmic, often granular (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Appendix+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 paraffin-section fixation consistent across samples (standard IHC practice; not target-specific) | |
| Caveat | Medium IHC–RNA concordance; external verification pending (HPA tissue IHC) | |
| Regulation | No expression induction annotated (UniProt) | |
| Isoform / epitope | 2 isoforms; check antibody epitope coverage (UniProt) |
Section 1
Recommended ANKRD26 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published ANKRD26 staining protocols for zebrafish paraffin sections (PMC12067082) and mouse brain and pituitary cryosections (PMC4601608).
| Sample | Paraffin-embedded human brain tissue; fixative not specified (datasheet A09289-1) |
| Fixation | Image fixative and duration unreported (datasheet A09289-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-ANKRD26, 1:100-1:300 (datasheet A09289-1) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ANKRD26-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression, which often had a granular pattern. No signal in the no-primary control. |
💡Decision noteFor paraffin sections, start with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen-retrieval setting); the published zebrafish excerpt does not specify retrieval (PMC12067082).
Section 2
What Is the Expected ANKRD26 Staining Pattern?
ANKRD26 shows generally cytoplasmic, often granular staining in tissue IHC, with High staining reported in several glandular cell populations and cerebral cortex neuronal cells (HPA: tissue IHC). It has no annotated transmembrane segment (UniProt Q9UPS8 topology). Treat this as a reference pattern, not an absolute rule: HPA rates the tissue staining Approved, with medium consistency against RNA data and external verification pending (HPA: tissue IHC reliability).
| Granular cytoplasmic staining in appendix, colon or duodenum glandular cells. | This fits the reported tissue pattern; those cell populations are rated High (HPA: tissue IHC). Score the cells and compartment together, then compare intensity within the same staining run. Granularity can vary across cells, so an exact puncta count is not an established acceptance criterion (HPA: tissue IHC profile). |
| Strong staining confined to nuclei, cell borders or extracellular material. | These compartments do not match the reported general cytoplasmic pattern (HPA: tissue IHC). Review morphology, counterstain and detection controls before interpreting the signal as ANKRD26; HPA tissue IHC does not establish an expected nuclear or surface pattern. |
| Prominent staining of adipocytes or bone marrow hematopoietic cells. | Both are reported as Not detected (HPA: adipose tissue; bone marrow). Check whether the signal follows a cell population, tissue pigment or endogenous detection activity. Persistent staining may indicate cross-reactivity; a negative HPA observation alone cannot prove its cause. |
| Diffuse colour across cells and surrounding tissue, with little cellular contrast. | This is difficult to score as the reported granular cytoplasmic pattern (HPA: tissue IHC). Inspect a no-primary control and the distribution of precipitate; excess primary antibody, incomplete blocking or detection background are general IHC possibilities, not established ANKRD26-specific effects. |
| No interpretable signal in the glandular cells of an expected positive section. | Appendix, colon, duodenum and several other glandular populations are rated High (HPA: tissue IHC). First confirm that the expected cells are present and that the staining run worked. Then review antibody dilution, retrieval and detection as general IHC-P checks; HPA supplies no ANKRD26-specific retrieval condition here. |
💡Expected ANKRD26 appearanceCall the section positive when identifiable glandular cells or cerebral cortex neuronal cells show strong, predominantly granular cytoplasmic staining (HPA: High tissue IHC; HPA: cytoplasmic profile); isolated nuclear staining or broad colour in reported negative cells is suspect.
| Tissue and cell selection | Use a reported High population as a positive reference and a reported Not detected population as a contrast (HPA: tissue IHC). Interpret each named cell type, rather than treating an entire organ as uniformly positive or negative. |
| Antibody evidence | HPA040654 and HPA041460 are IHC Approved (HPA: antibody validation). The tissue profile has medium agreement with RNA and awaits external verification (HPA: tissue IHC reliability), so unexpected staining merits an independent control. |
| Antigen retrieval | Retrieval can be optimised against positive and negative sections as general IHC-P practice. No target-specific retrieval condition or fixation sensitivity is supplied; neither the HPA staining levels nor UniProt topology establishes one. |
| IF/ICC expectation? | For the separate IF/ICC guide, HPA reports mainly Golgi localisation, with vesicles and cytosol also approved (HPA: subcellular ICC-IF). This supports an IF interpretation; it does not require organelle-level resolution in chromogenic tissue IHC. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive glandular cells have no visible stain. | The selected cells may be absent, or the staining run may have failed; the listed glandular populations are High in HPA tissue IHC. | Confirm cell identity on the counterstain and inspect a working positive control. Then review dilution, retrieval and detection as general IHC-P steps; do not infer a target-specific fixation effect. |
| Only nuclei appear strongly positive. | A nuclear-only result conflicts with the general cytoplasmic tissue profile (HPA: tissue IHC). | Check morphology and no-primary staining, then reassess with the IHC-validated antibody. Keep the result unassigned if the cytoplasmic reference pattern cannot be reproduced. |
| Reported negative cells stain prominently. | For example, adipocytes and bone marrow hematopoietic cells are Not detected (HPA: tissue IHC); cross-reactivity or endogenous detection activity is possible. | Compare matched positive tissue and a no-primary control. If the negative-cell signal persists, investigate specificity and endogenous enzyme activity before scoring it as ANKRD26. |
| Colour is widespread and masks cell boundaries. | Diffuse background prevents evaluation of the reported granular cytoplasmic pattern (HPA: tissue IHC). | Inspect no-primary and detection controls; adjust blocking, washes or antibody dilution using standard chromogenic IHC practice. Reassess only where cell morphology remains clear. |
| A section shows fewer positive cell types than expected. | HPA reports Low tissue specificity at RNA level but cell-specific IHC intensities (HPA: tissue IHC); whole-section appearance can obscure the relevant population. | Identify the named cell population before comparing results. Record its compartment and intensity separately from nearby cells, and use the HPA tissue observations as reference rather than a universal cutoff. |
| A Golgi-like IF image seems different from the tissue IHC slide. | Golgi, vesicle and cytosol assignments come from ICC-IF (HPA: subcellular); tissue IHC is described more broadly as cytoplasmic and often granular (HPA: tissue IHC). | Assess each application against its own reported pattern. Do not reject otherwise concordant chromogenic cytoplasmic staining solely because individual Golgi structures cannot be resolved. |
Experiment evidence
IHC & IF images showing ANKRD26 staining
A09289-1 · IHCImmunohistochemistry analysis of paraffin-embedded human brain, using ANKRD26 Antibody. The picture on the right is blocked with the synthes
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000107890-ANKRD26/tissue/cerebral+cortex#imid_10595477
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000107890-ANKRD26/tissue/colon#imid_10595411
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000107890-ANKRD26/tissue/liver#imid_10595404
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000107890-ANKRD26/tissue/kidney#imid_10595405
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA040654; green = ANKRD26, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA040654; green = ANKRD26, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ANKRD26 IHC & IF
🧪Run cerebral cortex first: neuronal cells should stain (HPA: cerebral cortex neuronal cells High). Use bone marrow hematopoietic cells as the biological negative (HPA: bone marrow hematopoietic cells Not detected); compare non-neuronal cells on the cortex slide for background, without assuming they are ANKRD26-negative (HPA: cerebral cortex neuronal cells High).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ANKRD26 in A-431, U-251MG, U2OS, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Golgi apparatus (approved) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and concentration-matched isotype controls appropriate to the primary antibody’s host species and clonality (standard IHC practice); use a knockout specimen or matched peptide block as a target-specific control (caption: A09289-1 brain IHC image includes synthesized-peptide blocking). For cerebral cortex, quench endogenous peroxidase and distinguish intrinsic pigment from chromogen staining (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the fixative for the selected paraffin-section image is unreported (caption: A09289-1 human brain paraffin IHC). Retrieval dependence is unreported; optimize antigen retrieval for paraffin IHC (standard IHC practice). The supplied evidence does not establish frozen sections or IF/ICC as easier; neuronal pigment can complicate chromogenic scoring in cerebral cortex (standard IHC practice).
HPA tissue IHC evidence for ANKRD26
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Pending external verification.). 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | 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 → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ANKRD26 IHC Tips
Troubleshoot ANKRD26 staining in paraffin sections by checking retrieval, cellular pattern, controls and scoring before interpreting differences between samples.
What retrieval should I try first when ANKRD26 staining is weak?
Start with citrate pH 6.0 heat induced epitope retrieval at 95–98 °C for 20 min (page retrieval rule). Let slides cool in buffer for about 20 min, then compare a cerebral cortex control across runs (standard IHC practice; HPA: High in cerebral cortex neuronal cells). If staining remains weak, test EDTA pH 9.0 on an adjacent section while keeping antibody concentration and detection conditions fixed (standard IHC practice). Compare cytoplasmic signal with tissue integrity and no primary control staining; stronger diffuse background alone does not establish improved ANKRD26 detection (HPA: general cytoplasmic pattern; standard IHC practice).
How should I troubleshoot inconsistent ANKRD26 staining between fixed tissue blocks?
ANKRD26 specific sensitivity to fixation is unknown from the supplied evidence; the A09289-1 image describes paraffin embedded human brain but does not state its fixative (catalog caption: A09289-1). For new specimens, document fixative, fixation duration and processing, then compare sections of equal thickness under one retrieval and detection run (standard IHC practice). Include a similarly processed positive control and a no primary control to separate tissue handling from detection background (HPA: High in cerebral cortex neuronal cells; standard IHC practice). If blocks differ, interpret intensity cautiously until matched processing shows that the difference persists (standard IHC practice).
Which cellular staining pattern supports ANKRD26 in chromogenic sections?
Look first for cytoplasmic staining, sometimes granular, in the expected cells rather than treating any brown deposit as positive (HPA: general cytoplasmic expression, often granular). Golgi, vesicle and cytosol localisation in cultured cell imaging provides a useful comparison, but section morphology may not resolve those structures (HPA subcellular: approved Golgi, vesicles and cytosol; standard IHC practice). Neuronal cells in cerebral cortex and glandular cells in appendix are useful positive tissue contexts (HPA: High in both). Score unexpected nuclear only or extracellular staining separately and check adjacent sections and no primary controls before assigning it to ANKRD26 (standard IHC practice).
Could ANKRD26 isoforms or epitope accessibility explain discordant staining?
ANKRD26 has 2 listed isoforms and several annotated phosphoserines, so an antibody's recognised sequence matters when comparing staining patterns (UniProt Q9UPS8: isoforms and modified residues). Check the catalog antibody's immunogen and epitope information against each isoform before claiming either isoform is detected (standard IHC practice). The A09289-1 paraffin brain caption reports inhibition by a synthesised peptide, which supports recognition of that peptide under the pictured conditions but does not establish isoform specificity (catalog caption: A09289-1). If staining differs across tissues, compare retrieval conditions and controls before attributing the difference to splicing or phosphorylation (standard IHC practice).
How can IF help assess an ambiguous ANKRD26 IHC pattern?
On a separately validated IF specimen, multiplex ANKRD26 with a marker for the expected cell type, such as a validated neuronal marker when evaluating cerebral cortex (HPA: High in cerebral cortex neuronal cells; standard IF practice). Choose a spectrally separated far red fluorophore for the weaker target channel when tissue autofluorescence affects shorter wavelengths, and image single stain controls (standard IF practice). ANKRD26 has no annotated transmembrane segment, so test mild permeabilisation for access to intracellular epitopes while checking that cellular structure remains intact (UniProt Q9UPS8: topology; standard IF practice). Compare puncta with Golgi, vesicle and cytosol expectations without treating IF colocalisation as proof of IHC antibody specificity (HPA subcellular: approved locations; standard IF practice).
What should I change if ANKRD26 staining is diffusely brown?
First inspect a no primary control and confirm that endogenous peroxidase was blocked before chromogenic detection (standard IHC practice). If background persists, test a stronger protein block, more thorough washes and a lower primary antibody concentration on matched sections, changing one condition at a time (standard IHC practice). Examine DAB development time and counterstain intensity so weak cytoplasmic granules are distinguishable from diffuse deposit (HPA: often granular cytoplasmic pattern; standard IHC practice). Use high staining glandular or neuronal controls beside low or undetected cell contexts, but remember the HPA tissue profile has medium agreement with RNA and awaits external verification (HPA: tissue profile and reliability).
How should I quantify ANKRD26 IHC across samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the expected cell population and scoring compartment before measuring signal; the reported tissue pattern is generally cytoplasmic and sometimes granular (HPA: tissue IHC profile). For each region, record the percentage of positive target cells and an intensity weighted H-score from 0–300, using fixed thresholds across slides (standard IHC practice). If cell numbers vary, report positive cells per mm² or normalise positive counts to all evaluable cells of that type (standard IHC practice). Exclude folds, necrosis and section edges by prespecified rules, and report control performance and batch alongside the scores (standard IHC practice).
When is an apparent ANKRD26 positive result likely artefactual?
A plausible result places predominantly cytoplasmic, sometimes granular signal in appropriate cells, including neuronal cells in cerebral cortex or glandular cells in appendix (HPA: tissue IHC profile). Isolated nuclear or extracellular deposits, staining confined to section edges or necrotic regions, and colour present in a no primary control warrant investigation (HPA: cytoplasmic profile; standard IHC practice). Check endogenous enzyme blocking and DAB exposure when brown signal appears broadly across unrelated cell types (standard IHC practice). The A09289-1 peptide blocked brain image is supporting evidence for that pictured assay, while HPA rates its tissue staining Approved with medium RNA agreement and pending external verification (catalog caption: A09289-1; HPA: reliability).
Boster reagents
Best ANKRD26 / Ankyrin repeat domain-containing protein 26 IHC Antibodies
A09289-1 lists human and mouse reactivity and IHC/IF applications (catalog: applications/reactivity). Its IHC figure shows paraffin-embedded human brain (image caption); no IF figure is supplied (catalog: IF images).
Real IHC data
Anti-ANKRD26 Antibody
Cat # A09289-1
A09289-1 will render with its own paraffin-embedded human brain IHC image and peptide-blocked comparison (image caption). It lists IHC, IF and ELISA applications and human and mouse reactivity (catalog: applications/reactivity).
Which to pick: For tissue IHC, choose A09289-1: its figure documents paraffin-embedded human brain (image caption), and the fixative is unreported (image caption). For IF, A09289-1 is listed at 1:50 (datasheet: IF dilution), but no IF image or ICC application is supplied (catalog: applications/IF images). For cross-species work, A09289-1 lists human and mouse reactivity (catalog: reactivity); its displayed IHC evidence is limited to human brain (image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UPS8 (ANR26_HUMAN, Ankyrin repeat domain-containing protein 26).
- Human Protein Atlas. ANKRD26 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ANKRD26 subcellular location (ICC-IF): Mainly localized to the Golgi apparatus. In addition localized to vesicles and cytosol..
- Human Protein Atlas. ANKRD26 antibody validation summary (2 antibodies).
- Modeling ANKRD26 5'-UTR mutation-related thrombocytopenia. Disease models & mechanisms 2025 — PMC12067082.
- A novel form of ciliopathy underlies hyperphagia and obesity in Ankrd26 knockout mice. Brain structure & function 2015 — PMC4601608.
- PubMed PMID:10470851 — UniProt-cited evidence.
- PubMed PMID:15164054 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ANKRD26 (Ankyrin repeat domain-containing protein 26). Expected localisation Cytoplasmic, often granular in tissue (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A09289-1 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human brain tissue; fixative not specified (datasheet A09289-1); 2. Fixation: Image fixative and duration unreported (datasheet A09289-1); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-ANKRD26, 1:100-1:300 (datasheet A09289-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep paraffin-section fixation consistent across samples (standard IHC practice; not target-specific); Caveat: Medium IHC–RNA concordance; external verification pending (HPA tissue IHC); Isoform / epitope: 2 isoforms; check antibody epitope coverage (UniProt); Regulation: No expression induction annotated (UniProt). Evidence: UniProt Q9UPS8, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the Golgi apparatus. In addition localized to…, Datasheet A09289-1, PMC12067082, PMC4601608. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ANKRD26/immunohistochemistry-ihc-guide.