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COX6A1 / Cytochrome c oxidase subunit 6A1, mitochondrial · IHC design guide
Design Immunohistochemistry for COX6A1
Plan chromogenic paraffin-section IHC for COX6A1 using the granular cytoplasmic tissue pattern as a readout (HPA tissue IHC). This guide covers the catalog antibody’s IHC dilution (datasheet A09572-1), fixation consistency and staining controls (standard IHC practice), with a caution about possible detection of protein from more than one gene (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
COX6A1 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 | Granular cytoplasm in tissue (HPA tissue IHC) | |
| Staining pattern | Granular cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A09572-1) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Antibody may detect protein from more than one gene (HPA tissue IHC) | |
| Regulation | Expression regulation not annotated (UniProt) | |
| Isoform / epitope | No isoforms annotated; mature chain spans aa 25–109 (UniProt) |
Section 1
Recommended COX6A1 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A09572-1) is paired with one published mouse liver IHC protocol (PMC12581582).
| Sample | Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A09572-1) |
| Fixation | Image fixative and duration unreported (datasheet A09572-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A09572-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A09572-1) |
| Primary antibody | Rabbit anti-COX6A1, 1:50 recommended; image 2 μg/ml (datasheet A09572-1) |
| Primary incubation | Overnight at 4 °C (datasheet A09572-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A09572-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | COX6A1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression with a granular pattern in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet: A09572-1); the published IHC method does not specify retrieval conditions (PMC12581582).
Section 2
What Is the Expected COX6A1 Staining Pattern?
COX6A1 is a cytochrome c oxidase subunit in the mitochondrial inner membrane, with one transmembrane segment at residues 35–59 (UniProt P12074 topology). In paraffin section IHC, expect granular cytoplasmic staining in many tissues, including kidney tubular and colon glandular cells, where HPA reports high staining (HPA tissue IHC). HPA rates its tissue IHC antibody Approved, pending external verification, and cautions that it targets proteins from more than one gene (HPA tissue IHC).
| Granular cytoplasmic stain in kidney tubules or colon glands, with little nuclear stain. | This matches HPA's high staining in those cell types and its granular cytoplasmic tissue profile (HPA tissue IHC). The appearance is consistent with COX6A1's inner mitochondrial membrane location (UniProt P12074 topology), but morphology alone cannot establish target specificity because the HPA antibody may recognize proteins from more than one gene (HPA tissue IHC). |
| Predominantly nuclear or sharply cell-surface stain in an otherwise interpretable section. | That compartment differs from the granular cytoplasmic IHC profile (HPA tissue IHC) and the mitochondrial inner membrane assignment (UniProt P12074 topology). Treat it as a localization mismatch; review the detection controls, morphology, and antibody specificity before scoring it as COX6A1. |
| Strong stain in adipocytes or skeletal myocytes while known-positive cells also stain. | HPA reports COX6A1 staining as not detected in those sampled cell types (HPA tissue IHC). Investigate nonspecific binding or detection background before interpreting the signal. An HPA 'not detected' result is an assay observation; it does not prove biological absence in every specimen. |
| Diffuse color across several compartments, including areas without a discernible granular cellular pattern. | The distribution does not match HPA's granular cytoplasmic profile (HPA tissue IHC). Background from the staining workflow is a possible explanation under general IHC practice; use reagent controls and inspect tissue morphology before assigning cell-specific positivity. |
| No stain in kidney tubules or colon glandular cells on the same run. | These are reported high-staining cell types (HPA tissue IHC), so a negative result calls for a run-level check of tissue preservation, retrieval, antibody application, and chromogen detection under general IHC practice. It does not, by itself, establish that the specimen lacks COX6A1. |
💡Expected COX6A1 appearanceCall a result consistent with the expected IHC pattern when granular cytoplasmic stain is evident in HPA high-staining cells such as kidney tubules or colon glands (HPA tissue IHC), while treating predominantly nuclear, cell-surface, or diffuse stain as suspect given the mitochondrial inner membrane assignment (UniProt P12074 topology).
| Target location and membrane topology | COX6A1 occupies the inner mitochondrial membrane; residues 25–34 face the matrix, 35–59 span the membrane, and 60–109 face the intermembrane space (UniProt P12074 topology). These facts support a mitochondrial interpretation but do not identify the antibody's epitope or establish which antigen retrieval condition works best. |
| Protein processing | UniProt annotates the mature chain as residues 25–109 and lists no glycosylation sites or modified residues (UniProt P12074 processing/PTMs). Without an antibody epitope map, these annotations cannot predict whether processing changes staining in a section. |
| Tissue and cell selection | HPA reports high staining in kidney tubules, colon glands, caudate neurons, and cerebral cortex neuropil, among other sites; it reports no detectable staining in adipocytes and skeletal myocytes (HPA tissue IHC). Compare the named cells, not whole-tissue averages, when assessing a control section. |
| Strength of the IHC evidence | The HPA antibody is rated Approved, with external verification pending and a warning that it targets proteins from more than one gene (HPA tissue IHC). Its reported pattern is useful for comparison, but staining with that antibody alone cannot uniquely identify COX6A1; no IHC Enhanced validation is supplied (HPA antibodies; HPA tissue IHC). |
| IF/ICC Q&A: what localization should be expected? | HPA reports supported mitochondrial localization and an additional approved principal-piece localization in ICC-IF (HPA subcellular). The principal-piece finding belongs to that imaging context; it should not be generalized to ordinary tissue IHC. HPA also cautions that the IF antibodies target proteins from multiple genes (HPA subcellular). |
| Situation | Likely cause | Next action |
|---|---|---|
| Kidney tubules and colon glands are blank in a completed IHC run. | These cells are high-staining references (HPA tissue IHC); the blank result may reflect a staining-run problem or specimen quality under general IHC practice. | Check section integrity, the run's positive control, retrieval record, primary-antibody application, and chromogen development. Resolve any run failure before interpreting the specimen as negative. |
| Stain is mainly nuclear despite acceptable morphology. | The localization conflicts with HPA's granular cytoplasmic pattern (HPA tissue IHC) and the UniProt inner mitochondrial membrane assignment (UniProt P12074 topology). | Review reagent controls and antibody specificity, then score only a reproducible cellular pattern consistent with the expected compartment. |
| Adipocytes or skeletal myocytes stain prominently. | HPA reports staining as not detected in those cell types (HPA tissue IHC); nonspecific binding or detection background is possible under general IHC practice. | Compare the same run with a high-staining cell type and appropriate reagent controls. Record the discordance instead of assigning the unexpected signal to COX6A1. |
| Color spreads through the section without cell boundaries or granules. | The appearance differs from the HPA granular cytoplasmic profile (HPA tissue IHC) and may reflect nonspecific workflow background under general IHC practice. | Examine a no-primary control and review blocking, washes, detection reagent, and chromogen development; reassess the stain only after the background is controlled. |
| A granular cytoplasmic pattern appears, but target identity is uncertain. | The HPA antibody can target proteins from more than one gene, and its IHC status remains pending external verification (HPA tissue IHC). | Describe the pattern as consistent with the reported COX6A1 distribution, and seek independent specificity evidence before claiming that the stain uniquely identifies COX6A1. |
| IF/ICC images show a principal-piece signal that is absent from the IHC section. | HPA lists the principal piece as an additional approved ICC-IF location alongside supported mitochondrial localization (HPA subcellular); the two observations have different specimen contexts. | Interpret each result within its application and sampled cell type. Use the HPA tissue IHC profile for paraffin section scoring (HPA tissue IHC). |
Experiment evidence
IHC & IF images showing COX6A1 staining
A09572-1 · IHCIHC analysis of COX6A1 using anti-COX6A1 antibody (A09572-1). COX6A1 was detected in a paraffin-embedded section of human liver cancer tissu
A09572-1 · IHCIHC analysis of COX6A1 using anti-COX6A1 antibody (A09572-1). COX6A1 was detected in a paraffin-embedded section of human liver tissue. Heat
A09572 · IHCImmunohistochemistry of paraffin-embedded rat cerebellum using COX6A1 antibody at dilution of 1:100 .Perform microwave antigen retrieval wit
A09572 · IHCImmunohistochemistry of paraffin-embedded human liver cancer using COX6A1 antibody at dilution of 1:100 .Perform microwave antigen retrieval
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000111775-COX6A1/tissue/cerebral+cortex#imid_18058231
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000111775-COX6A1/tissue/colon#imid_18058157
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000111775-COX6A1/tissue/liver#imid_18058166
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000111775-COX6A1/tissue/pancreas#imid_18058101
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA062394; green = COX6A1, blue = nucleus, red = microtubules.
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA062394; green = COX6A1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for COX6A1 IHC & IF
🧪Run colon first; its glandular cells should stain (HPA: High in colon glandular cells). Use adipose tissue adipocytes as the negative comparison (HPA: Not detected in adipocytes); on the colon slide, assess non-glandular stromal cells for low background, without assuming they are COX6A1-negative (HPA: High is assigned to glandular cells only).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show COX6A1 in MCF-7, PC-3, U2OS, Sperm, KOLF2.1J, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and rabbit IgG matched to the primary antibody’s isotype and clonality; compare with a validated COX6A1 knockout sample or, if the immunizing peptide is available, a peptide-blocked primary (caption: rabbit primary; standard IHC practice). For chromogenic colon sections, block endogenous peroxidase and inspect background before scoring DAB signal (caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A09572-1 paraffin-section caption does not state the fixative (caption: fixative unreported). Its reported staining used heat retrieval in EDTA at pH 8.0, but whether retrieval is required under other conditions is unreported (caption: heat-mediated EDTA retrieval). There is no matched comparison showing that frozen sections or IF are easier; if using IF, expect mitochondrial localisation while accounting for the HPA subcellular annotation’s multiple-gene antibody caution (HPA: mitochondria supported; multiple-gene antibody caution).
HPA tissue IHC evidence for COX6A1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Pending external verification. Caution, targets protein from more than one gene. 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuropil | High | Protein (IHC) | HPA → |
| Colon | 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 → |
| Ovary | Follicle cells | Not detected | Protein (IHC) | HPA → |
| Placenta | Decidual cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced COX6A1 IHC Tips
Troubleshoot COX6A1 staining in paraffin sections by checking retrieval, compartmental pattern, controls and scoring before interpreting chromogenic signal.
How should I retrieve COX6A1 in paraffin sections when staining is weak?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A09572-1). The selected tissue-IHC image used this retrieval before overnight primary incubation at 4°C, establishing a useful starting combination for this antibody (caption A09572-1). If staining remains weak, vary heating and cooling duration in matched sections while keeping antibody concentration and detection conditions fixed (standard IHC practice). Judge improvement by stronger granular cytoplasmic signal in expected cells without a comparable increase in tissue-wide background (HPA: granular cytoplasmic expression in most tissues; standard IHC practice). Record the retrieval conditions alongside each slide so apparent biological differences are not confounded by processing (standard IHC practice).
Can fixation explain inconsistent COX6A1 staining across paraffin blocks?
The selected paraffin-section caption does not state a fixative, so COX6A1-specific sensitivity to fixation is unknown (caption A09572-1). Record fixative identity and fixation duration for each block, then compare similarly processed sections within the same staining run (standard IHC practice). If blocks differ, test retrieval and primary-antibody conditions on representative sections before attributing a weaker result to biology (standard IHC practice). Use the reported EDTA pH 8.0 retrieval and 2 μg/ml primary concentration as documented starting conditions (caption A09572-1). Include a control section with expected granular cytoplasmic staining to distinguish a processing or staining failure from a genuinely low-scoring specimen (HPA: tissue-IHC profile; standard IHC practice).
What staining pattern supports genuine COX6A1 detection in tissue?
Look for a granular cytoplasmic pattern, consistent with mitochondrial localisation, rather than treating diffuse nuclear colour as expected COX6A1 staining (HPA: tissue-IHC profile; UniProt P12074 location). COX6A1 resides in the mitochondrial inner membrane, with residues 35–59 forming the annotated transmembrane segment (UniProt P12074 topology). Evaluate granules within identifiable cells and compare their distribution with nearby unstained structures under the same counterstain and illumination (standard IHC practice). HPA reports high staining in kidney tubular cells and adrenal glandular cells, offering tissue-level reference patterns without establishing specificity for your antibody (HPA: tissue IHC). If chromogen broadly fills nuclei, lumina or slide edges, investigate background and section quality before assigning localisation (standard IHC practice).
Could epitope position or related subunits affect my COX6A1 IHC result?
The record lists no annotated COX6A1 isoforms, while the processed chain spans residues 25–109 (UniProt P12074 processing and isoforms). Its transmembrane segment occupies 35–59; residues 25–34 face the matrix and 60–109 face the intermembrane space (UniProt P12074 topology). Check the antibody's stated immunogen before interpreting retrieval sensitivity or assuming which side of the inner membrane its epitope occupies (standard IHC practice). Complex IV can contain COX6A1 or COX6A2, and HPA cautions that its antibody evidence targets proteins from multiple genes (UniProt P12074 subunit; HPA: reliability). Therefore, a mitochondrial pattern alone cannot establish COX6A1-specific recognition; use an orthogonal specificity control when the distinction matters (standard IHC practice).
How can I adapt the localisation check to multiplex IF without misreading fluorescence?
For a separate IF experiment, pair COX6A1 with a marker identifying the expected cell population, such as kidney tubular cells, and inspect both channels independently (HPA: high in kidney tubular cells; standard IF practice). Choose fluorophores after checking the section's unstained autofluorescence, assigning the cleaner spectral channel to the weaker signal (standard IF practice). Permeabilise empirically because the epitope is unspecified and the matrix-facing 25–34 and intermembrane-facing 60–109 regions may require different access (UniProt P12074 topology; standard IF practice). HPA supports a mitochondrial main location in ICC/IF, but cautions that its antibodies target proteins from multiple genes (HPA: subcellular summary). Include single-channel and secondary-only controls to assess bleed-through and nonspecific fluorescence before calling colocalisation (standard IF practice).
How do I separate weak COX6A1 staining from chromogenic background?
Run a primary-omission control to assess secondary reagent and detection background, and inspect an adjacent section for tissue pigment (standard IHC practice). With peroxidase detection, include a peroxidase-blocking step and assess residual endogenous enzyme signal before interpreting DAB deposits (standard IHC practice). The selected caption documents goat-serum blocking at 10%, a 2 μg/ml primary incubation overnight at 4°C, and DAB development (caption A09572-1). If haze obscures granules, optimise washing, blocking and primary concentration using matched sections while preserving the reported EDTA pH 8.0 retrieval as the starting point (caption A09572-1; standard IHC practice). Interpret fine granular cytoplasmic staining in context; uniform surface staining or edge accentuation warrants investigation (HPA: tissue-IHC profile; standard IHC practice).
What should I score when comparing COX6A1 across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue compartment before scoring, because HPA reports a granular cytoplasmic pattern and differing levels among cell types (HPA: tissue IHC). For identifiable cells, record the percentage positive and an H-score based on cytoplasmic intensity; apply the same thresholds across slides (standard IHC practice). For dispersed positive cells, density per mm² of viable analysed tissue can be more informative than a whole-section average (standard IHC practice). Normalise to the relevant cell population or viable tissue area, and exclude necrosis, folds and section edges using prespecified rules (standard IHC practice). Keep retrieval, chromogen development and imaging settings consistent when comparing scores, and report any batch differences (standard IHC practice).
When does a positive COX6A1 stain warrant biological interpretation?
Give greatest weight to granular cytoplasmic staining in intact cells, consistent with COX6A1's mitochondrial inner-membrane location (HPA: tissue-IHC profile; UniProt P12074 location). Check whether the cell type fits the reference pattern: HPA reports high kidney tubular staining but no detected adipocyte staining (HPA: tissue IHC). Treat nuclear-only colour, strong slide-edge signal and deposits over necrotic areas as possible artefacts requiring review (standard IHC practice). Compare primary-omission and peroxidase-block controls before assigning a DAB signal to COX6A1 (standard IHC practice). Even convincing mitochondrial staining does not prove gene-specific detection, because HPA flags antibody recognition of proteins from multiple genes; use an independent specificity check for that claim (HPA: reliability; standard IHC practice).
Boster reagents
Best COX6A1 / Cytochrome c oxidase subunit 6A1, mitochondrial IHC Antibodies
Both anti-COX6A1 antibodies have paraffin-section IHC images (catalog IHC captions), list IF/ICC applications (catalog applications), and list human, mouse and rat reactivity (catalog reactivity).
Real IHC data
Anti-COX6A1 Antibody
Cat # A09572-1
Real IHC data
Anti-COX6A1 Antibody
Cat # A09572
A09572-1 will render with an IHC image from paraffin-embedded human liver cancer tissue; another caption documents human liver tissue (A09572-1 IHC captions). A09572 will render with an IHC image from paraffin-embedded rat cerebellum; its other captions document human liver cancer and mouse liver (A09572 IHC captions).
Which to pick: For tissue IHC using an EDTA pH 8.0 retrieval and DAB example, choose A09572-1 (A09572-1 IHC caption); the fixative is unreported (A09572-1 IHC caption). For IF/ICC, either SKU is listed for both applications, and both are rabbit polyclonals with human, mouse and rat reactivity (catalog applications, dilution_raw and reactivity); no IF image is supplied for either (catalog IF image alts). For cross-species tissue IHC, choose A09572 because its paraffin-section captions cover rat, human and mouse samples (A09572 IHC captions); the fixative is unreported (A09572 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P12074 (CX6A1_HUMAN, Cytochrome c oxidase subunit 6A1, mitochondrial).
- Human Protein Atlas. COX6A1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. COX6A1 subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the principal piece. Caution: Based on antibodies targeting proteins from multiple genes..
- Human Protein Atlas. COX6A1 antibody validation summary (1 antibodies).
- A novel palmitoylation-based molecular signature reveals COX6A1 as a key regulator in metabolic dysfunction-associated steatotic liver disease. Journal of translational medicine 2025 — PMC12581582.
- PLK1 (polo like kinase 1)-dependent autophagy facilitates gefitinib-induced hepatotoxicity by degrading COX6A1 (cytochrome c oxidase subunit 6A1). Autophagy 2021 — PMC8526032.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16541075 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for COX6A1 (Cytochrome c oxidase subunit 6A1, mitochondrial). Expected localisation Granular cytoplasm in tissue (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A09572-1). Validated IHC antibody: A09572-1 at 1:50 recommended; image 2 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A09572-1); 2. Fixation: Image fixative and duration unreported (datasheet A09572-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet A09572-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A09572-1); 8. Primary antibody: Rabbit anti-COX6A1, 1:50 recommended; image 2 μg/ml (datasheet A09572-1); 9. Primary incubation: Overnight at 4 °C (datasheet A09572-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Antibody may detect protein from more than one gene (HPA tissue IHC); Isoform / epitope: No isoforms annotated; mature chain spans aa 25–109 (UniProt); Regulation: Expression regulation not annotated (UniProt). Evidence: UniProt P12074, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the mitochondria. In addition localized to the…, Datasheet A09572-1, PMC12581582. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/COX6A1/immunohistochemistry-ihc-guide.