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ALKBH2 / DNA oxidative demethylase ALKBH2 · IHC design guide
Design Immunohistochemistry for ALKBH2
Plan ALKBH2 IHC-P using the catalog antibody at a starting concentration of 10 μg/mL (datasheet: A06325). Expect general nuclear staining and compare high-staining testis preleptotene spermatocytes with appendix glandular cells where staining was not detected (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ALKBH2 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 | Nuclear staining in tissue (HPA tissue IHC) | |
| Staining pattern | General nuclear staining across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | Appendix+3 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 | Appendix glandular cells may lack staining (HPA tissue IHC) | |
| Regulation | No expression regulation specified (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope differences unspecified (UniProt) |
Section 1
Recommended ALKBH2 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet) with a published colorectal tissue protocol (PMC7731553).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A06325); 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 Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear 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-ALKBH2, 10 μg/mL (datasheet A06325) |
| 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 | ALKBH2-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: General nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 HIER at 95–98 °C for 20 min (page retrieval rule: nuclear antigen); the published excerpt does not specify retrieval (PMC7731553).
Section 2
What Is the Expected ALKBH2 Staining Pattern?
ALKBH2 is a nuclear protein found in the nucleoplasm and nucleolus, with relocation to replication foci during S phase; it has no transmembrane segment (UniProt Q6NS38). In paraffin sections, expect nuclear staining in selected cell populations, including adipocytes, bone marrow hematopoietic cells, melanocytes and preleptotene spermatocytes (HPA: High in each). The HPA tissue profile is Approved, with medium consistency between staining and RNA expression (HPA tissue IHC).
| Distinct nuclear staining in adipocytes, marrow hematopoietic cells, melanocytes or preleptotene spermatocytes (HPA: High in each). | This fits the reported general nuclear tissue pattern (HPA tissue IHC) and ALKBH2 localization to nucleoplasm and nucleolus (UniProt Q6NS38). Compare intensity within the identified cell population: HPA levels describe observed staining, not a required intensity for every cell or section. |
| Predominantly cytoplasmic or membrane-like staining, with little discernible nuclear signal. | Review as a possible staining artefact because UniProt places ALKBH2 in the nucleus and reports no transmembrane segment (UniProt Q6NS38). HPA reports cytosol and primary cilium as uncertain additional ICC-IF locations (HPA subcellular), so those observations alone do not establish an expected paraffin IHC pattern. |
| Strong signal in appendix glandular cells or pancreatic exocrine glandular cells, especially without nuclear contrast (HPA: Not detected in those cells). | Investigate possible antibody cross-reactivity or endogenous chromogenic activity (general IHC practice). HPA's result applies to the named cells in its sampled tissues; it is not proof that every cell in either organ must be negative (HPA tissue IHC). |
| Diffuse brown deposit across cells, connective tissue or empty areas, obscuring nuclear boundaries. | The distribution is difficult to score against ALKBH2's nuclear localization (UniProt Q6NS38; HPA tissue IHC). Uneven chromogen deposition, inadequate blocking or insufficient washing can produce background (general chromogenic IHC practice); inspect controls before assigning it to ALKBH2. |
| No nuclear signal in a technically evaluable section containing an HPA High cell population (HPA tissue IHC). | Check assay performance before calling ALKBH2 absent: HPA reports high staining in specific cell populations, not uniformly across their entire tissues (HPA tissue IHC). Confirm those cells are present, then compare a positive control processed in the same run (general IHC practice). |
💡Expected ALKBH2 appearanceA convincing positive is a defined nuclear chromogenic signal in the relevant cells, potentially strong in HPA High populations; diffuse field-wide colour or dominant membrane-like staining warrants review (HPA tissue IHC; UniProt Q6NS38; general IHC practice).
| Tissue and cell selection (HPA tissue IHC) | HPA reports High staining in adipocytes, bone marrow hematopoietic cells, melanocytes and preleptotene spermatocytes, but Not detected in appendix glandular cells, lymph node germinal center cells, pancreatic exocrine glandular cells and spleen red-pulp cells (HPA tissue IHC). Select and score the named cell population, not the organ as a whole. |
| Nuclear localization and cell cycle (UniProt Q6NS38) | UniProt places ALKBH2 in nucleoplasm and nucleolus and reports relocation to replication foci during S phase (UniProt Q6NS38). Nuclear distribution can therefore vary among cells; a visible focus is not required to score an otherwise credible nuclear IHC signal. |
| Evidence and antibody validation (HPA tissue IHC; HPA antibodies) | The HPA tissue profile is Approved with medium staining-to-RNA consistency (HPA tissue IHC). CAB011198 is listed as IHC Approved, while the two listed rabbit antibodies have no IHC status in this payload (HPA antibodies). Interpret a discordant result alongside the antibody and tissue controls actually used. |
| IF/ICC Q&A: where should fluorescence localize? (HPA subcellular) | Mainly to the nucleoplasm: HPA calls that location supported (HPA subcellular). Its additional cytosol and primary-cilium assignments are uncertain, while UniProt also lists nucleolar localization (HPA subcellular; UniProt Q6NS38). This IF/ICC evidence informs compartment interpretation; it supplies no paraffin IHC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in the intended positive cells (HPA: High in the named populations). | The expected cells may be absent from the section, or the staining run may have failed (HPA tissue IHC; general IHC practice). | Verify cell identity on the counterstained section and inspect a same-run positive control; then check primary antibody application, detection reagents and assay-validated retrieval steps (general IHC practice). |
| Nuclear signal is weak despite visible, intact target cells. | Low contrast can make a true nuclear pattern hard to separate from counterstain or background (general chromogenic IHC practice). HPA's High level does not guarantee that intensity in every preparation (HPA tissue IHC). | Compare positive and omission controls from the same run; review the chosen antibody dilution, detection development and counterstain against the validated assay settings (general IHC practice). |
| Brown staining dominates cytoplasm or cell edges. | This conflicts with the main nuclear assignment and absence of a transmembrane segment (UniProt Q6NS38). Background or cross-reactivity is possible (general IHC practice). | Check whether nuclei also show discrete signal, examine the primary-omission control, and reassess blocking and washes before scoring the extranuclear colour (general IHC practice). |
| HPA Not detected cell populations show strong chromogen. | The result is discordant for those specific cells and may reflect cross-reactivity or endogenous detection activity (HPA tissue IHC; general chromogenic IHC practice). | Confirm the cell type, review a primary-omission control and the endogenous-enzyme blocking step, then compare an expected positive cell population in the same run (general IHC practice). |
| Background covers the section and obscures individual nuclei. | Nonspecific reagent binding, residual detection activity or uneven chromogen development can reduce contrast (general chromogenic IHC practice). | Inspect the omission control and background distribution; review blocking, wash stringency and chromogen development within the validated workflow before interpreting ALKBH2 intensity (general IHC practice). |
| An IF/ICC image shows cytosol or primary-cilium fluorescence. | HPA lists those additional locations as uncertain; its supported main location is nucleoplasm (HPA subcellular). | Treat the extranuclear fluorescence as provisional and assess nuclear colocalization and controls within the separate IF/ICC assay; do not transfer its distribution directly to chromogenic paraffin IHC (HPA subcellular; general IF/IHC practice). |
Experiment evidence
IHC & IF images showing ALKBH2 staining
A06325 · IHCImmunohistochemistry of ALKBH2 in human kidney tissue with ALKBH2 antibody at 10 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000189046-ALKBH2/tissue/cerebral+cortex#imid_3232328
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000189046-ALKBH2/tissue/colon#imid_3232254
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000189046-ALKBH2/tissue/liver#imid_3232247
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000189046-ALKBH2/tissue/kidney#imid_3232256
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB011198; green = ALKBH2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB011198; green = ALKBH2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ALKBH2 IHC & IF
🧪Run skin first and expect nuclear staining in melanocytes (HPA: High in melanocytes; UniProt Q6NS38: nuclear localization). Use appendix glandular cells as the negative tissue (HPA: Not detected); on the skin slide, cells without nuclear staining can serve as internal background comparators, but the supplied HPA rows do not establish a specific skin cell type as ALKBH2-negative.
Positive control tissue: Adipose tissue (Adipocytes, HPA High)
Negative control tissue: Appendix (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ALKBH2 in A-431, U2OS, HEK293, MCF-7, KOLF2.1J, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; an isotype control matched to the primary antibody’s host species, clonality and concentration; and ALKBH2 knockout material or peptide competition if the immunogen peptide is available (standard IHC practice). Block endogenous peroxidase and distinguish skin melanin from brown chromogen when scoring melanocytes (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence; the A06325 kidney IHC caption states 10 μg/mL but does not report a fixative (A06325 caption). Retrieval dependency is unreported, so assess retrieval conditions on matched paraffin sections; frozen-section IF may avoid heat retrieval, but the supplied evidence does not establish it as easier for ALKBH2 (standard IHC/IF practice). In skin, melanin can complicate chromogenic scoring of melanocytes (standard IHC practice).
HPA tissue IHC evidence for ALKBH2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Skin | Melanocytes | High | Protein (IHC) | HPA → |
| Soft tissue | Fibroblasts | Medium | Protein (IHC) | HPA → |
| Testis | Preleptotene spermatocytes | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
| Pancreas | Exocrine glandular cells | Not detected | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ALKBH2 IHC Tips
Troubleshoot ALKBH2 chromogenic IHC in paraffin sections by checking nuclear staining, tissue controls, detection background and scoring before interpreting biological differences (UniProt Q6NS38; HPA tissue IHC).
How should I adjust retrieval when ALKBH2 nuclear staining is weak?
Start with Tris-EDTA at pH 9.0 for heat-induced retrieval at 95–98 °C for 20 min (page retrieval rule: nuclear antigen). Let sections cool in retrieval buffer, then use the same wash and detection sequence across test slides so that staining differences reflect the retrieval comparison (standard IHC practice). If nuclear staining remains weak, compare a shorter heating interval on adjacent sections and check whether tissue morphology improves (standard IHC practice). Score signal within nuclei because nucleoplasmic localisation is supported, while nucleolar localisation and redistribution to replication foci are also reported (HPA subcellular; UniProt Q6NS38).
Could fixation explain loss of ALKBH2 staining in my paraffin sections?
Target-specific ALKBH2 fixation sensitivity is unknown from the supplied evidence; the kidney IHC caption reports 10 µg/mL antibody but gives no fixative (A06325 kidney IHC caption). Record the actual fixative and fixation duration for each specimen before comparing nuclear signal across blocks (standard IHC practice). On adjacent sections, keep retrieval at Tris-EDTA pH 9.0, 95–98 °C for 20 min while checking whether loss of staining tracks a fixation batch (page retrieval rule: nuclear antigen; standard IHC practice). Include an internal nuclear staining control where available, and assess morphology alongside signal; neither observation alone establishes a target-specific fixation effect (standard IHC practice).
What should I do if ALKBH2 staining appears mainly cytoplasmic?
First check whether the chromogenic deposit lies inside counterstained nuclei; ALKBH2 has supported nucleoplasmic localisation and reported nucleolar localisation (HPA subcellular; UniProt Q6NS38). Cytosol and primary-cilium signals are listed as uncertain, so predominantly cytoplasmic staining needs additional validation before it is assigned to ALKBH2 (HPA subcellular). Repeat staining with a lower antibody concentration than the starting 10 µg/mL used in the kidney image, using adjacent sections and identical detection conditions (A06325 kidney IHC caption; standard IHC practice). Review no-primary and secondary-only controls for diffuse deposit, then compare nuclear and cytoplasmic scores separately (standard IHC practice).
Can an epitope difference explain discordant ALKBH2 staining?
ALKBH2 has 2 annotated isoforms, but the supplied material does not identify the catalog antibody’s epitope or establish isoform recognition (UniProt Q6NS38; supplied A06325 caption). Check the antibody’s stated immunogen against both isoform sequences before treating discordant nuclear staining as an isoform effect (standard IHC practice). The annotated Fe2OG dioxygenase domain spans residues 152–257, while the listed protein has no transmembrane segment or annotated glycosylation sites (UniProt Q6NS38). For paraffin sections, compare adjacent-section retrieval and antibody titrations first; epitope masking and excess chromogen can produce differences without demonstrating isoform selectivity (standard IHC practice).
How can IF help check an ambiguous ALKBH2 IHC pattern?
Use IF/ICC as a separate localisation check and multiplex ALKBH2 with an independently validated marker for the cell type being assessed (standard IF practice). HPA reports high IHC staining in adipocytes, hematopoietic cells, melanocytes and preleptotene spermatocytes, but these observations do not establish an IF protocol (HPA tissue IHC). Choose a fluorophore channel with low tissue autofluorescence and inspect single-stain controls before interpreting overlap (standard IF practice). Because ALKBH2 is nuclear and has no transmembrane segment, optimise permeabilisation for access to a nuclear epitope after the chosen IF fixation, without assuming the IHC caption specifies that fixation (UniProt Q6NS38; A06325 kidney IHC caption; standard IF practice).
How do I distinguish ALKBH2 staining from chromogenic background?
Inspect no-primary and secondary-only sections beside the ALKBH2 slide to identify nonspecific detection signal (standard IHC practice). Include a peroxide block before peroxidase-based detection and develop DAB for the same measured time across compared sections; these are general workflow steps, not ALKBH2-specific validation (standard IHC practice). Titrate below the kidney image’s 10 µg/mL starting concentration if diffuse brown signal obscures nuclear boundaries (A06325 kidney IHC caption; standard IHC practice). Compare edge, fold and necrotic regions with intact tissue, and require staining to resolve within counterstained nuclei before scoring it as the supported localisation (standard IHC practice; HPA subcellular).
What is a defensible way to score ALKBH2 IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and score nuclear staining within viable, well-preserved tissue using fixed illumination and a consistent intensity threshold (standard IHC practice; HPA subcellular). Report the percentage of positive nuclei and an H-score based on the proportions at each intensity level; record cytoplasmic staining separately if present (standard IHC practice). Normalise positive-cell counts to all evaluable nuclei of the same cell population, or report positive-nucleus density per mm² of evaluable tissue (standard IHC practice). Keep retrieval, chromogen development and section thickness consistent across groups, and exclude folds and necrosis before comparing scores (standard IHC practice).
When is a positive ALKBH2 IHC result credible?
A credible pattern places chromogenic signal mainly in nuclei of morphologically identifiable cells, consistent with the supported nucleoplasmic location and HPA’s general nuclear IHC profile (HPA subcellular; HPA tissue IHC). Check the sampled cell type: HPA reports high staining in adipocytes and preleptotene spermatocytes but no detected staining in appendix glandular or pancreatic exocrine glandular cells (HPA tissue IHC). Discount deposits concentrated at section edges, folds or necrosis, and investigate signal retained in no-primary controls or attributable to endogenous peroxidase (standard IHC practice). HPA rates tissue IHC consistency with RNA expression as medium, so confirm unexpected compartment or cell-type patterns with an independent method before biological interpretation (HPA tissue IHC).
Boster reagents
Best ALKBH2 / DNA oxidative demethylase ALKBH2 IHC Antibodies
The IHC-validated ALKBH2 antibody has human kidney tissue data (catalog image: human kidney) and lists human, mouse, and rat reactivity (catalog: reactivity). IF/ICC data are absent (catalog: applications and images).
Real IHC data
Anti-ALKBH2 Antibody
Cat # A06325
A06325 will render with an IHC image of human kidney tissue at 10 μg/mL (catalog image caption). Its listed applications include IHC-P, and its listed reactivity covers human, mouse, and rat (catalog: applications and reactivity).
Which to pick: Choose A06325 for paraffin-section IHC because IHC-P is listed and its own image shows human kidney tissue staining at 10 μg/mL (catalog: applications and image caption); the fixative is unreported (catalog image caption). No IF/ICC option is supported by the listed applications or images (catalog: applications and images). A06325 lists mouse and rat reactivity, but the supplied IHC image documents human tissue only (catalog: reactivity and image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q6NS38 (ALKB2_HUMAN, DNA oxidative demethylase ALKBH2).
- Human Protein Atlas. ALKBH2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ALKBH2 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol and primary cilium..
- Human Protein Atlas. ALKBH2 antibody validation summary (3 antibodies).
- ALKBH2 promotes the Warburg effect and bladder cancer progression under hypoxic conditions via the PI3K/AKT pathway. Clinical and experimental medicine 2026 — PMC13500419.
- ALKBH2 inhibition alleviates malignancy in colorectal cancer by regulating BMI1-mediated activation of NF-κB pathway. World journal of surgical oncology 2020 — PMC7731553.
- PubMed PMID:17979886 — UniProt-cited evidence.
- PubMed PMID:16541075 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ALKBH2 (DNA oxidative demethylase ALKBH2). Expected localisation Nuclear staining in tissue (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A06325 at 10 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative Appendix. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A06325); 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 Tris-EDTA buffer, pH 9.0 , 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-ALKBH2, 10 μg/mL (datasheet A06325); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Appendix glandular cells may lack staining (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope differences unspecified (UniProt); Regulation: No expression regulation specified (UniProt). Evidence: UniProt Q6NS38, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A06325, PMC7731553. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ALKBH2/immunohistochemistry-ihc-guide.