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ALKBH8 / tRNA (carboxymethyluridine(34)-5-O)-methyltransferase ALKBH8 · IHC design guide
Design Immunohistochemistry for ALKBH8
Plan chromogenic ALKBH8 IHC in paraffin sections around the cytoplasmic tissue pattern (HPA tissue IHC). Colon glandular cells show medium staining, while appendix glandular cells are not detected (HPA tissue IHC); the catalog antibody’s IHC dilution is 1:50–1:200 (datasheet).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ALKBH8 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 in most tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in most tissues (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+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | No stimulus-specific regulation established (UniProt) | |
| Isoform / epitope | 4 isoforms; check antibody epitope coverage (UniProt) |
Section 1
Recommended ALKBH8 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published ALKBH8 protocols for FFPE brain and GBM tissue (PMC8965608) and colorectal cancer tissue arrays (PMC12518840).
| Sample | Paraffin-embedded mouse lung tissue; fixative not specified (datasheet A09491) |
| Fixation | Image fixative and duration unreported (datasheet A09491); 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-ALKBH8, 1:50-1:200 (datasheet A09491) |
| 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 | ALKBH8-positive staining in adipocytes of adipose tissue (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 at 95–98 °C for 20 min (page retrieval); citrate pH 6.0 for 30 min is a published alternative (PMC8965608).
Section 2
What Is the Expected ALKBH8 Staining Pattern?
ALKBH8 is predominantly cytoplasmic, with nuclear localization also reported (UniProt Q96BT7). In tissue IHC, expect cytoplasmic staining in many cell types, including colon glandular cells and bronchial respiratory epithelial cells (HPA: cytoplasmic expression in most tissues; Medium in both examples). HPA rates its tissue staining Approved, with medium consistency against RNA data (HPA: tissue IHC). ALKBH8 has no transmembrane segment (UniProt Q96BT7 topology).
| Cytoplasmic staining in colon glandular cells or bronchial respiratory epithelial cells. | This fits the reported IHC pattern; both examples are Medium, so judge signal against adjacent background rather than requiring intense staining (HPA: tissue IHC). |
| A predominantly crisp membrane rim, with little cytoplasmic signal. | This is discordant with the reported localization and warrants an artefact check (HPA: cytoplasmic IHC; UniProt Q96BT7 topology: no transmembrane segment). Nuclear signal alone needs separate judgment because nuclear localization is reported (UniProt Q96BT7). |
| Strong staining in appendix glandular cells while a positive control stains. | Consider cross-reactivity or endogenous detection activity before assigning ALKBH8 expression (HPA: appendix glandular cells Not detected; general IHC practice). HPA's result is a reference pattern, not proof that every specimen must be negative. |
| Uniform color over cells, stroma, and empty spaces. | Diffuse background prevents a cell-level call. Check the detection-only control and blocking, then reassess whether cytoplasmic staining remains distinct (general IHC practice; HPA: cytoplasmic IHC pattern). |
| No signal in colon glandular cells or bronchial respiratory epithelial cells. | These are reported Medium reference populations, so absence raises a control or assay-performance question (HPA: tissue IHC). It does not establish biological absence until tissue quality, detection, and antibody conditions are checked (general IHC practice). |
💡Expected ALKBH8 appearanceCall a positive IHC result when discernible, typically medium-level cytoplasmic staining appears in HPA-positive cell populations such as colon glandular cells; isolated membrane rims or uniform background are suspect (HPA: tissue IHC; UniProt Q96BT7 topology; general IHC practice).
| Tissue and cell choice | HPA reports Medium staining in colon glandular cells and bronchial respiratory epithelial cells, but Not detected in appendix glandular cells (HPA: tissue IHC). Use cell-resolved comparisons; a whole-tissue label alone can obscure the relevant population. |
| Antibody evidence | HPA rates HPA038724, HPA038725, and HPA061514 Approved for IHC (HPA: antibody validation). Its tissue profile has medium RNA–staining consistency, so treat a discrepant specimen as a finding to investigate rather than a definitive expression change (HPA: tissue IHC). |
| Isoforms and epitope | UniProt lists four ALKBH8 isoforms (UniProt Q96BT7). Without an epitope location for the antibody in this payload, isoform-specific staining or failure cannot be predicted; review the antibody's documented epitope before making that interpretation. |
| Topology and processing | ALKBH8 has no transmembrane segment or signal peptide, and the recorded chain spans residues 1–664 (UniProt Q96BT7). Those facts support scrutiny of an isolated surface pattern; they do not establish antigen-retrieval needs or fixation sensitivity. |
| IF/ICC question: should signal match tissue IHC? | HPA reports mainly nucleoplasmic ICC-IF localization, with supported nucleoplasm and approved microtubule localization; mitotic spindle and primary cilium assignments are uncertain (HPA: subcellular ICC-IF). Interpret that assay separately from HPA's predominantly cytoplasmic tissue IHC profile. |
| Situation | Likely cause | Next action |
|---|---|---|
| A positive reference section is blank. | The assay may have failed at staining or detection; HPA reports Medium staining in colon glandular cells and bronchial respiratory epithelial cells (HPA: tissue IHC). | Check section integrity, detection reagents, and the antibody's documented IHC conditions; repeat alongside a reported positive cell population (general IHC practice; HPA: tissue IHC). |
| All structures show similar diffuse color. | Nonspecific background or endogenous detection activity can obscure cell boundaries (general IHC practice). This appearance does not resolve HPA's cytoplasmic reference pattern (HPA: tissue IHC). | Compare a detection-only control; review blocking, washing, and detection chemistry, then score only distinct cellular staining (general IHC practice). |
| A membrane rim dominates the signal. | That location conflicts with the expected tissue pattern and the absence of a transmembrane segment (HPA: tissue IHC; UniProt Q96BT7 topology). | Inspect morphology and negative controls, and seek corroboration with another IHC-approved antibody if needed (general IHC practice; HPA: antibody validation). |
| Appendix glandular cells stain strongly. | Cross-reactivity or endogenous activity is possible because those cells are Not detected in the HPA reference (HPA: tissue IHC; general IHC practice). | Compare controls and the positive cell population in the same run; avoid calling the signal ALKBH8 solely from color intensity (general IHC practice; HPA: tissue IHC). |
| Nuclear signal accompanies cytoplasmic IHC staining. | A nuclear component is biologically plausible (UniProt Q96BT7: nucleus), although HPA describes tissue IHC as cytoplasmic in most tissues (HPA: tissue IHC). | Record nuclear and cytoplasmic compartments separately; compare matched controls before treating the nuclear component as specific (general IHC practice). |
| IF/ICC is nucleoplasmic while IHC is cytoplasmic. | The two HPA assay summaries report different dominant compartments (HPA: nucleoplasmic ICC-IF; cytoplasmic tissue IHC). | Interpret each assay against its own reference and controls; do not transfer an ICC-IF localization call directly to chromogenic paraffin IHC (general IHC practice; HPA: assay-specific profiles). |
Experiment evidence
IHC & IF images showing ALKBH8 staining
A09491 · IHCImmunohistochemistry of paraffin-embedded mouse lung using ALKBH8 antibody at dilution of 1:100 (x40 lens).
A09491 · IHCImmunohistochemistry of paraffin-embedded human gastric cancer using ALKBH8 antibody at dilution of 1:100 (x40 lens).
A09491 · IFImmunofluorescence analysis of A549 cell using ALKBH8 antibody. Blue: DAPI for nuclear staining.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000137760-ALKBH8/tissue/cerebral+cortex#imid_9800588
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000137760-ALKBH8/tissue/colon#imid_9794170
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000137760-ALKBH8/tissue/liver#imid_9794171
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000137760-ALKBH8/tissue/kidney#imid_9794172
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA038725; green = ALKBH8, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA038725; green = ALKBH8, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ALKBH8 IHC & IF
🧪Run bronchus first: respiratory epithelial cells should show medium staining (HPA: Bronchus, respiratory epithelial cells, Medium). Use appendix glandular cells as a negative tissue comparator (HPA: Appendix, glandular cells, Not detected); on the bronchus slide, treat any unstained neighboring cells as background comparators without assuming they are ALKBH8-negative.
Positive control tissue: Adipose tissue (Adipocytes, HPA Medium)
Negative control tissue: Appendix (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ALKBH8 in A-431, U-251MG, U2OS, KOLF2.1J, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) slide, a host- and isotype-matched control IgG for a monoclonal antibody or host-matched normal IgG for a polyclonal antibody, and ALKBH8 knockout tissue if available (standard IHC controls). For bronchus, quench endogenous peroxidase before chromogenic detection and check for residual background (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and retrieval dependency are unreported in the supplied evidence; optimize antigen retrieval empirically (standard IHC practice). The selected A09491 caption shows paraffin-section staining in mouse lung at 1:100, but its fixative is unreported (selected SKU tissue-IHC caption). This evidence does not establish that frozen sections or IF are easier; airway mucus or trapped chromogen can complicate interpretation of bronchial staining (standard IHC practice).
HPA tissue IHC evidence for ALKBH8
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 | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Adipocytes | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Caudate | Glial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Placenta | Trophoblastic cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ALKBH8 IHC Tips
Troubleshoot ALKBH8 staining in paraffin sections using compartment, tissue pattern and control evidence; assess IF separately.
Which antigen retrieval should I try first when ALKBH8 staining is weak?
Start with Tris-EDTA at pH 9.0, heated to 95–98 °C for 20 min (page retrieval rule). Allow sections to cool consistently, then compare a known positive section processed in the same run; inconsistent heating or cooling can change chromogenic intensity (standard IHC practice). The selected antibody has a paraffin-section mouse lung image at 1:100, but its caption reports no retrieval method, so that image cannot establish retrieval conditions (catalog antibody A09491 caption). If staining remains weak, compare a gentler retrieval condition on adjacent sections while holding antibody dilution and detection constant, and judge signal against tissue morphology and a no-primary control (standard IHC practice).
Could fixation explain weak or uneven ALKBH8 staining in paraffin sections?
Target-specific fixation sensitivity is unknown: the selected mouse lung caption identifies paraffin embedding but does not state the fixative (catalog antibody A09491 caption). Record fixative, fixation duration and tissue processing for each specimen, then compare sections prepared together before attributing a weak result to ALKBH8 abundance (standard IHC practice). Uneven staining can reflect variable fixation or processing, so inspect tissue morphology and repeat staining on a separately processed section when available (standard IHC practice). Do not infer a fixation tolerance from ALKBH8’s lack of a transmembrane segment or listed glycosylation sites, because those annotations do not test fixation effects (UniProt Q96BT7 topology and glycosylation; standard IHC practice).
How should I evaluate cytoplasmic versus nuclear ALKBH8 staining?
Assess both compartments separately: ALKBH8 is annotated in cytoplasm and nucleus, with predominantly cytoplasmic localisation (UniProt Q96BT7 subcellular annotation). Tissue IHC reports cytoplasmic expression in most tissues, whereas cell-based IF reports mainly nucleoplasmic localisation; these are different assay contexts and should be reported as such (HPA tissue IHC; HPA subcellular). Score cytoplasmic signal only within intact cell boundaries and nuclear signal against a counterstain, using matched no-primary sections to assess detection background (standard IHC practice). A nuclear-only or diffuse extracellular pattern warrants review of morphology, retrieval and antibody specificity before it is treated as an ALKBH8 redistribution (UniProt Q96BT7 subcellular annotation; standard IHC practice).
Can ALKBH8 isoforms or epitope placement change the IHC result?
ALKBH8 has 4 annotated isoforms, but the supplied antibody evidence does not identify its epitope or establish which isoforms it detects (UniProt Q96BT7 isoforms; catalog antibody A09491 caption). The protein has an RRM at residues 43–120 and a Fe2OG dioxygenase domain at 220–337; those coordinates do not locate this antibody’s binding site (UniProt Q96BT7 domains). Before comparing specimens, obtain epitope or immunogen information for the exact antibody and document any claimed isoform coverage (standard IHC practice). If staining differs despite matched processing, use an independent epitope or a suitable loss-of-target control where available rather than assigning the difference to a specific isoform from IHC alone (standard IHC practice).
How can I check an ALKBH8 IHC pattern by multiplex IF?
Treat IF as a separate assay and pair ALKBH8 with a validated marker for the cell population being scored in the IHC section; record marker-positive and marker-negative cells separately (standard IF practice). Choose spectrally separated fluorophores and inspect unstained tissue in each channel, especially when autofluorescence could resemble a weak ALKBH8 signal (standard IF practice). ALKBH8 has no transmembrane segment and is annotated in cytoplasm and nucleus, so assess permeabilisation for access to intracellular epitopes, including the nucleus, without assuming the antibody’s epitope is known (UniProt Q96BT7 topology and subcellular annotation; standard IF practice). Compare localisation with nuclear counterstain and single-colour controls; cell-based IF reports mainly nucleoplasmic ALKBH8, while tissue IHC reports mainly cytoplasmic staining (HPA subcellular; HPA tissue IHC).
What should I check when ALKBH8 chromogenic staining is widespread or granular?
Run a no-primary control alongside the ALKBH8 section to separate detection-system deposits from antibody-associated staining, and inspect unstained tissue pigment before interpreting brown signal (standard IHC practice). For a peroxidase-based DAB workflow, check that endogenous peroxidase was blocked and compare development time across sections; these are general chromogenic controls, not ALKBH8-specific validation (standard IHC practice). Review primary-antibody concentration and wash stringency if the entire section darkens; the selected mouse lung paraffin image used 1:100, which is an image condition rather than a universal optimum (catalog antibody A09491 caption; standard IHC practice). Give greatest weight to signal within intact cells in a plausible compartment, since HPA tissue IHC describes predominantly cytoplasmic staining (HPA tissue IHC).
How should I quantify ALKBH8 IHC across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define cell type and compartment before scoring, because tissue IHC describes predominantly cytoplasmic ALKBH8 while the protein also has a nuclear annotation (HPA tissue IHC; UniProt Q96BT7 subcellular annotation). Within comparable regions, report percentage of positive cells and an H-score based on predefined intensity categories, or report positive-cell density per mm² when cell abundance itself is the question (standard IHC practice). Normalise positive counts to the number of evaluable cells of the same type, or to measured viable tissue area for density, and exclude folds, edges and necrotic regions (standard IHC practice). Keep retrieval, antibody dilution, DAB development and image thresholds consistent across groups; document any batch effects before comparing scores (standard IHC practice).
How do I distinguish true ALKBH8 staining from artefact?
Prioritise signal in intact cells with interpretable cytoplasmic or nuclear boundaries: ALKBH8 is annotated in both compartments, and tissue IHC most often shows cytoplasmic staining (UniProt Q96BT7 subcellular annotation; HPA tissue IHC). Compare the same cell population across sections rather than declaring every unstained cell a failure; HPA reports medium staining in bronchial respiratory epithelial cells but low staining in lung alveolar cells (HPA tissue IHC). Discount edge-only deposits, necrotic areas and signal reproduced by a no-primary control, and check peroxidase blocking when DAB staining appears independent of the primary antibody (standard IHC practice). Finally, weigh positive staining against matched controls and morphology, since HPA rates tissue-IHC consistency with RNA expression as medium (HPA tissue IHC reliability).
Boster reagents
Best ALKBH8 / tRNA (carboxymethyluridine(34)-5-O)-methyltransferase ALKBH8 IHC Antibodies
A09491 has IHC images from paraffin-embedded mouse lung and human gastric cancer, plus IF data from A549 cells (catalog image captions). Human and mouse reactivity is listed (catalog: reactivity).
Real IHC data
Anti-ALKBH8 Antibody
Cat # A09491
A09491 is listed for IHC and IF in human and mouse samples (catalog: applications, reactivity). Its images show IHC in paraffin-embedded mouse lung and human gastric cancer, and IF in A549 cells (catalog image captions).
Which to pick: Choose A09491 for tissue IHC: its own captions show paraffin-embedded mouse lung and human gastric cancer at 1:100; the fixative is unreported (catalog IHC image captions). For IF/ICC, A09491 has an A549-cell IF image and a listed dilution of 1:50–1:100 (catalog IF image caption; catalog: IF dilution). For cross-species work, A09491 is a rabbit polyclonal listed for human and mouse IHC and IF (catalog: host, clonality, reactivity, applications).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q96BT7 (ALKB8_HUMAN, tRNA (carboxymethyluridine(34)-5-O)-methyltransferase ALKBH8).
- Human Protein Atlas. ALKBH8 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ALKBH8 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the microtubules, mitotic spindle and primary cilium..
- Human Protein Atlas. ALKBH8 antibody validation summary (3 antibodies).
- The AlkB Family: Potential Prognostic Biomarkers and Therapeutic Targets in Glioblastoma. Frontiers in oncology 2022 — PMC8965608.
- ALKBH8-mediated codon-specific translation promotes colorectal tumorigenesis. Nature communications 2025 — PMC12518840.
- PubMed PMID:17979886 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16554811 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ALKBH8 (tRNA (carboxymethyluridine(34)-5-O)-methyltransferase ALKBH8). Expected localisation Cytoplasmic in most tissues (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A09491 at 1:50-1:200 (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: Paraffin-embedded mouse lung tissue; fixative not specified (datasheet A09491); 2. Fixation: Image fixative and duration unreported (datasheet A09491); 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-ALKBH8, 1:50-1:200 (datasheet A09491); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 4 isoforms; check antibody epitope coverage (UniProt); Regulation: No stimulus-specific regulation established (UniProt). Evidence: UniProt Q96BT7, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A09491, PMC8965608, PMC12518840. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ALKBH8/immunohistochemistry-ihc-guide.