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ALKBH8 / tRNA (carboxymethyluridine(34)-5-O)-methyltransferase ALKBH8 · IHC design guide
Design Immunohistochemistry for ALKBH8
Plan paraffin IHC around cytoplasmic staining in most tissues (HPA tissue IHC). The catalog antibody has an IHC dilution range of 1:50–1:200 (datasheet); colon glandular cells show medium staining, while appendix glandular cells show no detected staining (HPA tissue IHC).
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 staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in most 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+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | No staining-linked regulator reported (UniProt) | |
| Isoform / epitope | 4 isoforms; epitope coverage is unknown (UniProt) |
Section 1
Recommended ALKBH8 IHC & IF Protocols
The catalog antibody IHC-P protocol is accompanied by published ALKBH8 protocols for brain and GBM sections (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 HIER at 95–98 °C for 20 min (page antigen retrieval); compare citrate pH 6.0 for 30 min (PMC8965608).
Section 2
What Is the Expected ALKBH8 Staining Pattern?
In paraffin section IHC, expect predominantly cytoplasmic ALKBH8 staining across many tissues, including medium staining in adipocytes and several glandular or epithelial cell populations (HPA: tissue IHC). UniProt also lists nuclear localization and describes ALKBH8 as predominantly cytoplasmic; it has no transmembrane segment (UniProt Q96BT7: localization and topology). The HPA tissue pattern is Approved, with medium consistency between antibody staining and RNA expression (HPA: tissue IHC reliability).
| Cytoplasmic staining in adipocytes, or in bronchial respiratory epithelial or colon glandular cells. | This fits the reported paraffin section pattern; HPA records Medium staining in each of these cell populations (HPA: tissue IHC). Interpret the compartment and the stained cell population together, rather than treating any brown signal in the tissue as confirmation. |
| Predominantly membrane outlining, with little cytoplasmic staining. | This differs from the reported cytoplasmic IHC pattern and from ALKBH8's lack of a transmembrane segment (HPA: tissue IHC; UniProt Q96BT7: topology). Review morphology and background before scoring it as target staining. Nuclear signal alone needs more caution: UniProt includes the nucleus, while HPA tissue IHC emphasizes cytoplasm. |
| Strong staining in a cell population listed as Not detected, such as appendix glandular cells. | Check for cross-reactivity or endogenous detection activity before assigning the signal to ALKBH8 (HPA: appendix glandular cells, Not detected). A negative HPA observation is a useful comparator, not proof that every specimen or assay must be blank. |
| Diffuse color across cells, stroma, and the rest of the section without a clear intracellular pattern. | Treat this as background until a no-primary control and inspection of the detection step clarify its source (general IHC practice). It cannot establish the predominantly cytoplasmic ALKBH8 pattern reported for tissue IHC (HPA: tissue IHC). |
| No staining in adipocytes or bronchial respiratory epithelial cells expected to provide a comparison. | HPA reports Medium staining in these cells, so an entirely blank result warrants review of the antibody, retrieval, and detection workflow (HPA: tissue IHC; general IHC practice). Their reported level does not guarantee a positive result in every section. |
💡Expected ALKBH8 appearanceA convincing positive result is discernible, predominantly cytoplasmic staining in a reported positive cell population, such as Medium staining in adipocytes or bronchial respiratory epithelium; diffuse section-wide color or membrane outlining alone is suspect (HPA: tissue IHC; UniProt Q96BT7: topology).
| Tissue and cell population | HPA describes cytoplasmic expression in most tissues but records different levels by cell population: bronchial respiratory epithelial cells are Medium, lung alveolar cells are Low, and appendix glandular cells are Not detected (HPA: tissue IHC). Select comparison cells by their reported identity. |
| IHC versus ICC-IF localization | In tissue IHC, HPA summarizes a cytoplasmic pattern; in ICC-IF, it reports supported nucleoplasmic localization and approved additional microtubule localization (HPA: tissue IHC; HPA: subcellular ICC-IF). Q: Should nucleoplasmic IF dictate the IHC scoring pattern? A: No; assess each assay against its own reported pattern. |
| Evidence strength | The tissue IHC profile is Approved, with medium consistency between antibody staining and RNA expression (HPA: tissue IHC reliability). The listed antibodies HPA038724, HPA038725, and HPA061514 each have Approved IHC status (HPA: antibody validation). These labels support comparison, while preserving the reported reliability caveat. |
| Cellular distribution | UniProt calls ALKBH8 predominantly cytoplasmic, also lists the nucleus, and reports no transmembrane segment (UniProt Q96BT7: localization and topology). Thus, some nuclear signal can be evaluated in context; a membrane-only result conflicts with the supplied localization evidence. |
| Isoforms and processing | UniProt lists 4 isoforms and one chain spanning residues 1–664, with no signal peptide or propeptide (UniProt Q96BT7: isoforms and processing). The supplied records do not map an antibody epitope to an isoform, so they cannot predict isoform-specific staining. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells are blank. | The staining run may have failed, or the signal may be below detection; HPA reports Medium staining in adipocytes and bronchial respiratory epithelial cells (HPA: tissue IHC). | Check a reported positive cell population on the run, then review the IHC-validated antibody's documented conditions, retrieval, and detection controls (general IHC practice). |
| Color covers the section without a cell-specific cytoplasmic pattern. | Background from detection chemistry or nonspecific binding may obscure the reported tissue pattern (HPA: tissue IHC; general IHC practice). | Inspect a no-primary control, blocking, washes, and detection reagent activity; score only interpretable cell-associated signal (general IHC practice). |
| Membranes appear to be the main positive compartment. | This conflicts with predominantly cytoplasmic localization and the absence of a transmembrane segment (HPA: tissue IHC; UniProt Q96BT7: topology). | Recheck cellular boundaries and compare the suspected signal with a reported positive cell population before assigning ALKBH8 localization. |
| A reported Not detected cell population stains strongly. | Cross-reactivity or endogenous detection activity is possible; HPA lists appendix glandular cells as Not detected (HPA: tissue IHC; general IHC practice). | Compare a no-primary control and a reported positive population in the same run; investigate persistent staining before interpreting it as ALKBH8. |
| Nuclear staining seems inconsistent with cytoplasmic tissue IHC. | UniProt also lists nuclear localization, and HPA ICC-IF reports supported nucleoplasmic localization, although its tissue IHC summary is cytoplasmic (UniProt Q96BT7: localization; HPA: subcellular ICC-IF and tissue IHC). | Record nuclear and cytoplasmic staining separately. Judge the paraffin section against the tissue IHC pattern and cell population, without applying the ICC-IF pattern as an IHC criterion. |
| Staining differs between two tissue sections. | HPA reports cell-dependent levels, from Medium in colon glandular cells to Not detected in appendix glandular cells; its tissue IHC reliability notes medium staining-to-RNA consistency (HPA: tissue IHC). | Identify the scored cell populations before comparing intensity, and document the difference without assuming that whole tissues share one staining level. |
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 stain at a Medium level (HPA: bronchus respiratory epithelial cells, Medium). Use appendix glandular cells as the negative tissue (HPA: appendix glandular cells, Not detected); on the bronchus slide, assess adjacent non-epithelial cells for background staining, but do not treat them as confirmed ALKBH8-negative cells because the supplied HPA row specifies only respiratory epithelial cells (HPA: bronchus respiratory epithelial cells, Medium).
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 control and a primary-matched isotype control (same host species, immunoglobulin class and concentration; use host-matched normal IgG for a polyclonal primary), plus identically processed ALKBH8 knockout tissue if available or a validated immunizing-peptide block (standard IHC specificity controls). Block endogenous peroxidase for chromogenic detection and inspect bronchial inflammatory cells for residual peroxidase signal (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and retrieval dependency are unreported in the supplied evidence; optimize antigen retrieval on matched paraffin sections (supplied evidence: no fixation-window or retrieval data). The catalog antibody caption shows paraffin-section mouse lung staining at 1:100, but its fixative is unreported (A09491 caption: mouse lung, paraffin, 1:100; fixative not stated). The supplied evidence does not establish that frozen sections or IF are easier; in bronchus, residual inflammatory-cell peroxidase can complicate chromogenic scoring (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 by checking retrieval, cellular compartment, controls and scoring before interpreting chromogenic signal.
How should I adjust retrieval when ALKBH8 staining is weak in paraffin sections?
Start with Tris-EDTA pH 9.0 heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval specification). Allow sections to cool consistently, then compare matched sections with the same antibody dilution and detection conditions so retrieval is the only changed variable (standard IHC practice). If staining remains weak, test a shorter heating time or citrate pH 6.0 as a fallback, recording tissue morphology alongside signal (standard IHC practice). The catalog image shows staining in paraffin-embedded mouse lung at 1:100, but its caption does not report retrieval conditions (A09491 caption).
Could fixation explain weak or uneven ALKBH8 staining?
ALKBH8-specific fixation sensitivity is unknown from the supplied evidence; the mouse lung caption identifies paraffin embedding but does not state a fixative (A09491 caption). Record fixative, fixation duration and tissue thickness for each specimen before comparing staining across batches (standard IHC practice). When fixation histories differ, stain sections from each batch together with the same retrieval, antibody incubation and detection settings to assess batch effects (standard IHC practice). Compare intact tissue away from cut edges with its own controls, because uneven processing can change morphology and apparent chromogenic intensity (standard IHC practice). Do not assign a weak result to ALKBH8-specific fixation sensitivity without a controlled comparison.
Should ALKBH8 appear in nuclei or cytoplasm?
Expect predominantly cytoplasmic staining in tissue: UniProt describes ALKBH8 as predominantly cytoplasmic, and HPA reports cytoplasmic expression in most tissues (UniProt Q96BT7; HPA tissue IHC). Nuclear staining warrants inspection rather than automatic rejection, since UniProt also lists the nucleus and HPA reports supported nucleoplasmic localisation in cell imaging (UniProt Q96BT7; HPA subcellular). Score nuclear and cytoplasmic signal separately in intact cells under the same counterstain and exposure conditions (standard IHC practice). Treat staining confined to an acellular edge or damaged region as suspect until it persists in well-preserved cells and survives appropriate controls (standard IHC practice).
How can isoforms and epitope location affect an ALKBH8 IHC result?
ALKBH8 has 4 listed isoforms, but the supplied evidence does not locate the catalog antibody’s epitope or establish isoform coverage (UniProt Q96BT7; supplied product evidence). Its annotated RRM spans residues 43–120, and its Fe2OG dioxygenase domain spans 220–337 (UniProt Q96BT7). Check the antibody’s stated immunogen and mapped epitope, if available, against the isoform sequences before interpreting absent staining as absent protein (standard antibody validation practice). The record lists no signal peptide, transmembrane segment or glycosylation sites, so those features provide no basis for assigning an extracellular epitope or glycan-dependent retrieval effect (UniProt Q96BT7).
How should I investigate ALKBH8 localisation with IF alongside this IHC guide?
Use the separate IF/ICC guide for assay conditions; the supplied mouse lung image documents paraffin-section IHC, with its fixative unreported (A09491 caption). For multiplex IF, pair ALKBH8 with a validated marker of the cell population under study and check that secondary antibodies do not cross-react (standard IF practice). Select spectrally separated fluorophores and inspect unstained tissue channels before assigning signal, because tissue autofluorescence can obscure weak IF staining (standard IF practice). ALKBH8 has no annotated transmembrane segment and is reported in cytoplasm and nucleus, so choose permeabilisation that permits intracellular antibody access, then assess both compartments (UniProt Q96BT7; standard IF practice).
What should I check when chromogenic ALKBH8 staining looks diffuse?
First compare the stained section with a matched no-primary control and inspect whether colour follows cells, pigment or damaged tissue (standard IHC practice). Include a peroxidase block before HRP-based detection and control DAB development time; these are general chromogenic workflow steps, not ALKBH8-specific findings (standard IHC practice). Titrate the primary antibody and blocking conditions while holding retrieval and detection constant, recording both background and cellular signal (standard IHC practice). The catalog image used 1:100 in paraffin-embedded mouse lung, which is a documented image condition rather than a validated dilution for every tissue (A09491 caption).
How should I score ALKBH8 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because tissue IHC is reported as mostly cytoplasmic while nuclear localisation is also recorded (HPA tissue IHC; UniProt Q96BT7). For cellular staining, report the percentage of positive cells and an intensity-weighted H-score using fixed intensity bins across sections (standard IHC quantification practice). If counting distinct positive cells, report density per mm² of viable tissue and normalise counts to the measured area or relevant cell population (standard IHC quantification practice). Keep retrieval, DAB development, image acquisition and scoring thresholds consistent, and exclude folds, necrosis and section edges using preset criteria (standard IHC quantification practice).
How can I distinguish convincing ALKBH8 staining from artefact?
A convincing pattern has cellular staining in the expected compartment, with cytoplasm the main tissue reference and nucleus a documented possible location (HPA tissue IHC; UniProt Q96BT7). Compare cell identity with the sampled tissue: HPA reports medium staining in bronchial respiratory epithelial cells but low staining in lung alveolar cells (HPA tissue IHC). Distrust signal limited to section edges, necrosis, pigment or no-primary controls, and check the peroxidase block when colour appears without specific primary binding (standard IHC practice). HPA assigns tissue staining Approved reliability with medium RNA–protein consistency, so corroborate an unexpected result with an independent antibody or a suitable loss-of-target control where available (HPA tissue IHC; standard IHC validation practice).
Boster reagents
Best ALKBH8 / tRNA (carboxymethyluridine(34)-5-O)-methyltransferase ALKBH8 IHC Antibodies
A09491 has paraffin-section IHC images from mouse lung and human gastric cancer, plus an IF image from A549 cells (catalog image captions). Human and mouse reactivity is listed (catalog).
Real IHC data
Anti-ALKBH8 Antibody
Cat # A09491
A09491 is listed for IHC and IF in human and mouse samples (catalog applications and reactivity). Its images show paraffin-section IHC in mouse lung and human gastric cancer, and IF in A549 cells (catalog image captions).
Which to pick: For tissue IHC, choose A09491: its own images document paraffin sections of mouse lung and human gastric cancer (catalog IHC image captions); the fixative is unreported (catalog IHC image captions). For IF/ICC, A09491 has listed IF use and an A549-cell IF image, while ICC validation is unreported (catalog applications and IF image caption). For work across human and mouse samples, A09491 is the listed polyclonal option with both species in its reactivity profile (catalog reactivity and dilution record).
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 staining 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 conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 4 isoforms; epitope coverage is unknown (UniProt); Regulation: No staining-linked regulator reported (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.