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COX6B1 / Cytochrome c oxidase subunit 6B1 · IHC design guide
Design Immunohistochemistry for COX6B1
Plan COX6B1 paraffin-section IHC around the granular cytoplasmic pattern seen across tissues (HPA tissue IHC). Compare strong staining in glandular and neuronal cells with low staining in smooth muscle cells when choosing controls (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
COX6B1 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 (HPA tissue IHC); inner mitochondrial membrane (UniProt) | |
| Staining pattern | Widespread granular cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A10285-2) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Bone marrow+1 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Smooth muscle cells may stain weakly (HPA tissue IHC) | |
| Regulation | Specific regulators are not annotated (UniProt) | |
| Isoform / epitope | No isoforms annotated; mature chain spans aa 2–86 (UniProt) |
Section 1
Recommended COX6B1 IHC & IF Protocols
The catalog antibody has an IHC-P protocol (datasheet A10285-2). Two published COX6B1 studies provide additional paraffin-section protocols (PMC12800125; PMC10965397).
| Sample | Paraffin-embedded human placenta tissue; fixative not specified (datasheet A10285-2) |
| Fixation | Image fixative and duration unreported (datasheet A10285-2); 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 A10285-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A10285-2) |
| Primary antibody | Rabbit anti-COX6B1, 2 μg/ml (datasheet A10285-2) |
| Primary incubation | Overnight at 4 °C (datasheet A10285-2) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A10285-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | COX6B1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression with a granular pattern. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet A10285-2). The published GBM protocol used boiling citrate buffer (PMC10965397).
Section 2
What Is the Expected COX6B1 Staining Pattern?
COX6B1 is a cytochrome c oxidase subunit associated with the mitochondrial inner membrane and has no transmembrane segment (UniProt P14854: location, topology). In paraffin-section IHC, expect granular cytoplasmic staining across many tissues, with strong staining in selected glandular, epithelial and neuronal cells (HPA: ubiquitous granular cytoplasmic profile; High in listed cells). HPA rates the tissue pattern Supported, with medium consistency between staining and RNA expression (HPA: reliability).
| Granular cytoplasmic staining in adrenal glandular cells, bronchial respiratory epithelium or cerebellar Purkinje cells. | This fits the expected compartment and cell distribution (HPA: granular cytoplasmic profile; High in these cells). Score the named cell population, not the entire section: neighboring populations can differ in intensity (HPA: cell-specific staining levels). |
| Predominantly crisp nuclear staining, with little granular cytoplasmic signal. | Treat the compartment as discordant: the expected IHC profile is cytoplasmic and granular (HPA: tissue IHC), while the protein is associated with the mitochondrial inner membrane (UniProt P14854: location). Review the counterstain and detection controls before calling it specific. |
| Strong signal in bone-marrow hematopoietic cells or ovarian stroma cells. | These populations were Not detected in the supplied tissue IHC record (HPA: bone marrow; ovarian stroma). Recheck cell identification and controls; cross-reactivity or endogenous detection activity is possible in chromogenic IHC (general IHC practice). HPA's Supported rating does not make every negative observation universal (HPA: medium consistency). |
| Uniform diffuse color across cells and surrounding tissue, without discernible cytoplasmic granules. | The distribution does not match the reported granular cytoplasmic profile (HPA: tissue IHC). Background from detection chemistry or insufficient blocking can obscure a real signal (general IHC practice); compare with a matched no-primary control. |
| No signal in an adequately sampled HPA High cell population. | Absent staining in a documented positive population, such as colon glandular cells, conflicts with the supplied IHC observation (HPA: High in colon glandular cells). Check section quality, antigen-retrieval and detection controls before interpreting the result as biological absence (general IHC practice). |
💡Expected COX6B1 appearanceCall a positive result when the relevant glandular, epithelial or neuronal cells show clear granular cytoplasmic staining (HPA: High in listed cells; granular profile); isolated nuclear color or uniform haze is discordant with that profile (HPA: tissue IHC).
| Cell population and tissue | HPA reports High staining in several glandular, respiratory epithelial and neuronal populations, Low staining in smooth muscle cells, fibroblasts, adipocytes and splenic white-pulp cells, and Not detected in bone-marrow hematopoietic and ovarian stroma cells (HPA: tissue IHC). Choose comparisons by cell population. |
| Expected compartment | The granular cytoplasmic IHC pattern is consistent with mitochondrial localization (HPA: tissue IHC and subcellular profile; UniProt P14854: inner-membrane location). The no-transmembrane-segment annotation describes topology; it does not establish a retrieval condition or fixation effect (UniProt P14854: topology). |
| Strength of tissue evidence | The tissue IHC pattern is Supported, with medium consistency against RNA expression; the listed antibody HPA004192 is Supported for IHC and ICC (HPA: reliability; antibody validation). Use this as pattern support, while evaluating unexpected staining with controls. |
| IF/ICC Q: Where should fluorescence appear? | A: Mainly in mitochondria; an additional principal-piece location is reported in sperm (HPA: subcellular profile, supported mitochondrial and approved principal-piece locations). This localization answer does not supply an IF/ICC protocol. |
| Chromogenic detection | If peroxidase-based detection is used, endogenous enzyme activity can add color unrelated to the primary antibody (general IHC practice). Assess this with appropriate detection controls; the supplied HPA and UniProt records do not identify a COX6B1-specific enzyme artifact. |
| Situation | Likely cause | Next action |
|---|---|---|
| Granular signal is weak in a candidate section. | The sampled population may normally stain at a lower level, or the staining run may have low sensitivity (HPA: Low in specified cell populations; general IHC practice). | Compare with a listed High population in the same run, and verify antigen retrieval, primary-antibody use and detection controls (HPA: High cell populations; general IHC practice). |
| No stain appears in a listed High population. | A staining-run failure or inadequate sampling is possible; absence conflicts with the HPA High observation for that population (HPA: tissue IHC; general IHC practice). | Confirm the cell population on the section, then review retrieval, antibody application and chromogen development using run controls (general IHC practice). Do not infer a target-specific fixation effect. |
| Nuclei dominate the staining pattern. | The compartment conflicts with granular cytoplasmic IHC and mitochondrial localization (HPA: tissue and subcellular profiles). Counterstain or nonspecific color may complicate reading (general IHC practice). | Compare a no-primary control and review the chromogen and counterstain separately; require convincing cytoplasmic granules before scoring COX6B1 (HPA: tissue IHC; general IHC practice). |
| Staining is strong in a population listed as Not detected. | Cell misidentification, cross-reactivity or endogenous detection activity may explain discordant color (HPA: bone-marrow hematopoietic and ovarian stroma cells Not detected; general IHC practice). | Recheck morphology and compare no-primary and detection controls. Interpret the HPA negative observations with its medium-consistency reliability rating (HPA: reliability; general IHC practice). |
| Diffuse color hides cell boundaries and granules. | Background from blocking, washing or detection chemistry may obscure the expected granular profile (HPA: tissue IHC; general IHC practice). | Check the no-primary control and review blocking, washes and chromogen development for the run (general IHC practice). Score only resolved cellular signal. |
| A low-staining population appears negative beside a strong one. | Staining intensity varies by cell population; HPA lists both High and Low populations (HPA: tissue IHC). | Score each population separately against its reported level and the run controls (HPA: tissue IHC; general IHC practice). Avoid applying a High-population threshold to Low populations. |
Experiment evidence
IHC & IF images showing COX6B1 staining
A10285-2 · IHCIHC analysis of COX6B1 using anti-COX6B1 antibody (A10285-2). COX6B1 was detected in a paraffin-embedded section of human placenta tissue. H
A10285-2 · IHCIHC analysis of COX6B1 using anti-COX6B1 antibody (A10285-2). COX6B1 was detected in a paraffin-embedded section of human colorectal cancer
A10285-2 · IFIF analysis of COX6B1 using anti-COX6B1 antibody (A10285-2). COX6B1 was detected in an immunocytochemical section of A549 cells. Enzyme anti
A10285-1 · IFFluorescent image of A549 cells stained with COX6B1 Antibody (N-term). A10285-1 was diluted at 1:25 dilution. An Alexa Fluor 488-conjugated
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000126267-COX6B1/tissue/cerebral+cortex#imid_1525664
HPA · StomachHuman Protein Atlas IHC, Stomach. Source: https://www.proteinatlas.org/ENSG00000126267-COX6B1/tissue/stomach+1#imid_1525556
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000126267-COX6B1/tissue/colon#imid_1525590
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000126267-COX6B1/tissue/liver#imid_1525599
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA004192; green = COX6B1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA004192; green = COX6B1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for COX6B1 IHC & IF
🧪Run adrenal gland first: its glandular cells should stain (HPA: High in adrenal glandular cells). Use bone marrow hematopoietic cells as the negative tissue (HPA: Not detected); on the adrenal slide, nonglandular cells should show only background, but the supplied HPA rows do not establish a validated internal negative cell population.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show COX6B1 in A-431, U-251MG, U2OS, Sperm, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host- and isotype-matched rabbit IgG controls (selected-SKU IHC caption: rabbit primary antibody), plus a validated COX6B1-knockout specimen or peptide competition if the immunizing peptide is available (standard IHC practice). For adrenal sections, quench endogenous peroxidase and check endogenous biotin before interpreting DAB staining (selected-SKU IHC caption: biotinylated secondary and SABC/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A10285-2 paraffin-section caption does not state the fixative (selected-SKU IHC caption). That caption uses heat retrieval in EDTA at pH 8.0 for placenta, but does not establish retrieval dependence in adrenal tissue (selected-SKU IHC caption). The supplied evidence does not establish that frozen sections or IF are easier; HPA supports mitochondrial localization by ICC-IF, and adrenal DAB interpretation requires checking endogenous pigment and peroxidase background (HPA: mitochondria supported; standard IHC practice).
HPA tissue IHC evidence for COX6B1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
Section 3
Advanced COX6B1 IHC Tips
Troubleshoot COX6B1 staining in paraffin sections by checking retrieval, cellular pattern, controls, and scoring against its mitochondrial localization.
What retrieval should I start with for COX6B1 paraffin sections?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A10285-2). The selected placenta image used 2 μg/ml primary antibody overnight at 4°C; retain those conditions while comparing retrieval on serial sections, since the caption gives no heating duration (datasheet A10285-2). If staining remains weak, vary heating duration and inspect tissue integrity before trying another buffer as a fallback, with a no-primary section for each condition (standard IHC practice). Judge improvement by sharper granular cytoplasmic signal in intact cells, rather than stronger diffuse DAB or damaged edges (HPA tissue IHC; standard IHC practice).
Could fixation explain weak COX6B1 staining?
The selected paraffin-section caption does not state its fixative, so target-specific fixation sensitivity is unknown (datasheet A10285-2). Record each sample’s fixative, concentration, duration, and processing delay; compare matched sections using the documented EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody (datasheet A10285-2; standard IHC practice). If one batch loses signal, compare its morphology and staining with an independently processed positive control before adjusting antibody concentration (standard IHC practice). Avoid diagnosing overfixation from a weak image alone; neither the reported tissue staining nor COX6B1 topology establishes a fixation effect (HPA tissue IHC; UniProt P14854 topology).
Where should convincing COX6B1 staining appear?
Expect a granular cytoplasmic pattern in tissue sections, consistent with the reported mitochondrial localization of COX6B1 (HPA tissue IHC; HPA subcellular; UniProt P14854). Examine intact cells at high magnification and compare punctate staining with cell outlines and the counterstained nuclei before scoring (standard IHC practice). Strong nuclear-only or continuous cell-surface DAB should prompt review of controls and staining conditions, because those patterns do not match the reported predominant location (HPA subcellular; standard IHC practice). A matched mitochondrial marker on an adjacent section can support the compartment assignment, although agreement alone does not prove antibody specificity (UniProt P14854; standard IHC practice).
How should epitope uncertainty affect antibody validation?
The COX6B1 record lists 0 annotated isoforms, a mature chain spanning residues 2–86, and a CHCH domain at 27–73 (UniProt P14854). It also lists acetylation at residue 2; the supplied caption does not locate this antibody’s epitope, so that modification cannot explain a staining change on its own (UniProt P14854; datasheet A10285-2). Request the immunogen or epitope range when investigating discrepant staining, and compare an independent antibody or orthogonal expression evidence where available (standard IHC practice). Do not assume recognition of COX6B2 or discriminate it from COX6B1 without sequence and antibody-specific validation (UniProt P14854; standard IHC practice).
How can IF help check a disputed COX6B1 IHC pattern?
For a separate IF experiment, pair COX6B1 with a marker of the expected cell population, such as an epithelial marker in bronchus, where respiratory epithelial staining is reported high (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and place the weaker signal in a channel with less tissue autofluorescence, checking unstained and single-stained controls (standard IF practice). COX6B1 is assigned to the mitochondrial inner membrane but has no annotated transmembrane segment; the supplied topology does not establish which side contains the antibody epitope (UniProt P14854). Compare mild permeabilisation conditions empirically and assess mitochondrial signal and cell morphology; the paraffin IHC caption supplies no IF fixation or permeabilisation conditions (datasheet A10285-2; standard IF practice).
What causes diffuse brown background in COX6B1 IHC?
First inspect a no-primary section for DAB deposition and include a peroxidase block in the chromogenic workflow, since endogenous enzyme activity can mimic staining (standard IHC practice). The selected image used 10% goat serum block, biotinylated secondary antibody, a streptavidin–biotin complex, and DAB; assess secondary-only and detection-reagent controls when background persists (datasheet A10285-2; standard IHC practice). Review washing, primary concentration, and retrieval damage while retaining the documented 2 μg/ml condition as the comparison point (datasheet A10285-2; standard IHC practice). Score discrete granular cytoplasmic staining separately from diffuse haze, pigment, and section-edge deposits (HPA tissue IHC; standard IHC practice).
How should COX6B1 IHC staining be quantified across samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and viable tissue area before scoring, then use a consistent threshold for granular cytoplasmic DAB (HPA tissue IHC; standard IHC practice). An H-score can combine the percentage of cells at intensity levels 0–3 into a 0–300 score; report percent-positive cells separately when intensity is unreliable (standard IHC practice). Normalize counts to the number of eligible cells, or staining area to viable tissue area in mm², and apply identical imaging and analysis settings (standard IHC practice). If comparing biological expression, account for differences in mitochondrial abundance with a separately validated mitochondrial marker, since COX6B1 resides in mitochondria (UniProt P14854; standard IHC practice).
How can I distinguish true COX6B1 signal from artefact?
Favor granular cytoplasmic staining in intact cells, matching the reported tissue pattern and mitochondrial localization, over nuclear-only or extracellular deposits (HPA tissue IHC; HPA subcellular). Compare the specimen with a positive control and no-primary control processed together, and inspect section edges and necrotic areas for nonspecific DAB (standard IHC practice). Check endogenous peroxidase activity when brown staining survives omission of primary antibody, especially in a chromogenic assay (standard IHC practice). Cell identity also matters: reported high staining in bronchial respiratory epithelium and undetected staining in ovarian stromal cells provide context, but the HPA tissue assessment has only supported reliability (HPA tissue IHC).
Boster reagents
Best COX6B1 / Cytochrome c oxidase subunit 6B1 IHC Antibodies
The catalog includes COX6B1 staining data for human paraffin tissue sections (A10285-2 IHC image caption) and human A549 cells (A10285-1 IF image caption).
Real IHC data
Anti-COX6B1 Antibody ®
Cat # A10285-2
Real IF data
Anti-COX6B1 Antibody (N-term)
Cat # A10285-1
A10285-2 will render with an IHC image of a human placenta paraffin section (A10285-2 IHC image caption). A10285-1 will render with an IF image of human A549 cells stained at 1:25 (A10285-1 IF image caption; catalog reactivity).
Which to pick: Choose A10285-2 for chromogenic IHC of paraffin sections: its human placenta image used EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody; the fixative is unreported (A10285-2 IHC image caption). For IF in A549 cells, A10285-1 has an image at 1:25, while A10285-2 also lists ICC/IF and has an A549 IF image (A10285-1 IF image caption; A10285-2 catalog applications and IF image caption). A10285-2 lists human, mouse and rat reactivity, but its supplied tissue IHC images show human samples only (A10285-2 catalog reactivity and IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P14854 (CX6B1_HUMAN, Cytochrome c oxidase subunit 6B1).
- Human Protein Atlas. COX6B1 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. COX6B1 subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the principal piece..
- Human Protein Atlas. COX6B1 antibody validation summary (1 antibodies).
- Identification of potential biomarkers associated with mitochondrial oxidative stress in idiopathic pulmonary arterial hypertension via bioinformatic and experimental analysis. Scientific reports 2025 — PMC12800125.
- Glioma cells are resistant to inflammation‑induced alterations of mitochondrial dynamics. International journal of oncology 2020 — PMC7646598.
- Integrative analysis of metabolism subtypes and identification of prognostic metabolism-related genes for glioblastoma. Bioscience reports 2024 — PMC10965397.
- PubMed PMID:2537962 — UniProt-cited evidence.
- PubMed PMID:2172092 — UniProt-cited evidence.
- PubMed PMID:1651883 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for COX6B1 (Cytochrome c oxidase subunit 6B1). Expected localisation Granular cytoplasm (HPA tissue IHC); inner mitochondrial membrane (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A10285-2). Validated IHC antibody: A10285-2 at 2 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Bone marrow. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human placenta tissue; fixative not specified (datasheet A10285-2); 2. Fixation: Image fixative and duration unreported (datasheet A10285-2); 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 A10285-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A10285-2); 8. Primary antibody: Rabbit anti-COX6B1, 2 μg/ml (datasheet A10285-2); 9. Primary incubation: Overnight at 4 °C (datasheet A10285-2). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Smooth muscle cells may stain weakly (HPA tissue IHC); Isoform / epitope: No isoforms annotated; mature chain spans aa 2–86 (UniProt); Regulation: Specific regulators are not annotated (UniProt). Evidence: UniProt P14854, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the mitochondria. In addition localized to the…, Datasheet A10285-2, PMC12800125, PMC10965397. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/COX6B1/immunohistochemistry-ihc-guide.