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COX5B / Cytochrome c oxidase subunit 5B, mitochondrial · IHC design guide
Design Immunohistochemistry for COX5B
Plan chromogenic COX5B IHC in paraffin sections using its inner mitochondrial membrane location (UniProt) and widespread tissue staining (HPA tissue IHC). Use the catalog antibody’s paraffin-section protocol (datasheet A06090-2), and account for low staining in chondrocytes when comparing cell types (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
COX5B 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 | Inner mitochondrial membrane; tissue signal is widespread (UniProt; HPA tissue IHC) | |
| Staining pattern | Mitochondrial staining across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A06090-2) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
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 | Chondrocytes show low staining despite broad expression (HPA tissue IHC) | |
| Regulation | Heart and skeletal muscle have enhanced RNA (HPA tissue RNA) | |
| Isoform / epitope | No isoforms annotated; mature chain spans residues 32–129 (UniProt) |
Section 1
Recommended COX5B IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet: A06090-2) with two published COX5B chromogenic IHC protocols (PMC12774556; PMC11588429).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A06090-2) |
| Fixation | Image fixative and duration unreported (datasheet A06090-2); 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 A06090-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A06090-2) |
| Primary antibody | Rabbit anti-COX5B, 2-5 μg/ml (datasheet A06090-2) |
| Primary incubation | Overnight at 4 °C (datasheet A06090-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A06090-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | COX5B-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous mitochondrial expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet: A06090-2); evaluate citrate retrieval if adapting either published protocol (PMC12774556; PMC11588429).
Section 2
What Is the Expected COX5B Staining Pattern?
COX5B is a component of mitochondrial complex IV in the inner membrane, with no annotated transmembrane segment (UniProt P10606 topology and subunit). Expect predominantly cytoplasmic mitochondrial staining across many cell types; HPA describes ubiquitous mitochondrial expression and high IHC staining in several glandular, epithelial, hematopoietic and neural cell populations (HPA: tissue IHC). Treat intensity comparisons cautiously: HPA rates tissue IHC reliability as Supported, with medium consistency between staining and RNA data (HPA: reliability).
| Granular or punctate cytoplasmic staining in expected cells. | This fits a mitochondrial pattern (UniProt P10606: inner membrane; HPA: ubiquitous mitochondrial expression). Judge the pattern within cells as well as its overall intensity; chromogenic IHC cannot resolve the inner membrane itself (standard IHC interpretation). |
| Dominant nuclear staining or a sharply defined cell-surface rim. | These compartments do not fit the annotated mitochondrial location (UniProt P10606: inner membrane; HPA: mitochondria enhanced in ICC-IF). Review controls and the staining workflow before calling such a pattern COX5B specific (standard IHC practice). |
| Strong staining only in cells outside the expected reference population. | Compare adjacent morphology and a matched reference section before suspecting cross-reactivity or endogenous detection activity (standard IHC practice). HPA reports broad expression, so an unlisted cell type alone is not evidence of a false positive (HPA: ubiquitous mitochondrial expression). |
| Uniform haze across cells and surrounding section. | Diffuse staining obscures the granular mitochondrial pattern expected for COX5B (HPA: ubiquitous mitochondrial expression; HPA: mitochondria enhanced in ICC-IF). It can reflect background from the antibody or detection system; assess a matched negative control (standard IHC practice). |
| Little or no signal in a reference population reported as High. | Check tissue preservation, antibody and detection controls before interpreting absence as biology (standard IHC practice). HPA lists High staining in adrenal gland glandular cells and bronchus respiratory epithelial cells, but its IHC reliability is Supported with medium RNA agreement (HPA: tissue IHC; HPA: reliability). |
💡Expected COX5B appearanceCall an IHC result positive when cells show appreciable granular cytoplasmic staining consistent with mitochondria, especially in HPA High reference populations; dominant nuclear staining or diffuse section-wide color is suspect (UniProt P10606: inner membrane; HPA: tissue IHC; standard IHC interpretation).
| Cell and tissue context | HPA reports High IHC staining in breast glandular cells, bone marrow hematopoietic cells, caudate neuronal cells and other listed populations, but Low staining in soft tissue chondrocytes (HPA: tissue IHC). Use those observations as reference patterns, not absolute thresholds. |
| Strength of tissue evidence | The tissue profile is Supported, with medium consistency between antibody staining and RNA expression (HPA: reliability). HPA supplies no negative tissue example, so absence in an unlisted tissue has no validated negative-reference status (HPA: tissue IHC). |
| Antibody validation | HPA034517 has Supported IHC status and Enhanced ICC status (HPA: antibody validation). These are application-specific assessments; the ICC rating does not upgrade the IHC evidence or establish a staining threshold for every tissue (HPA: antibody validation). |
| Protein location and processing | UniProt places COX5B in the inner membrane, annotates no transmembrane segment and identifies a mature chain spanning residues 32–129 (UniProt P10606: topology and processing). Epitope location is unspecified, so these annotations cannot predict retrieval requirements (UniProt P10606: processing). |
| Antigen retrieval in paraffin IHC | Use a retrieval condition appropriate to the IHC-validated antibody and compare it with the supplied positive reference and controls (standard IHC-P practice). No COX5B-specific retrieval condition or fixation-sensitivity result is supplied (UniProt P10606; HPA: tissue IHC). |
| IF/ICC Q&A: what localisation should I expect? | Predominantly mitochondrial signal; HPA also reports acrosome localisation in ICC-IF (HPA: subcellular ICC-IF). That additional finding is an ICC-IF observation, not an expected feature of every paraffin tissue section or an IHC protocol instruction (HPA: subcellular ICC-IF). |
| Situation | Likely cause | Next action |
|---|---|---|
| A High-reference section is blank. | An ineffective antibody, retrieval or chromogenic detection step is possible; blank staining alone does not establish absent COX5B (standard IHC practice; HPA: High in adrenal gland glandular cells). | Verify the IHC-validated antibody and detection controls, then assess retrieval using its IHC-P instructions (standard IHC-P practice). Compare with a matched HPA High reference population (HPA: tissue IHC). |
| Signal is mostly nuclear. | Nuclear dominance conflicts with mitochondrial localisation (UniProt P10606: inner membrane; HPA: mitochondria enhanced in ICC-IF). | Check the negative control and whether counterstain or chromogen has been mistaken for specific signal; reassess the cytoplasmic pattern at higher magnification (standard IHC practice). |
| Color appears throughout the section. | Diffuse background can arise from nonspecific binding or the detection chemistry (standard IHC practice). It cannot be assigned to COX5B from intensity alone (HPA: ubiquitous mitochondrial expression). | Compare a no-primary control, review blocking and washes, and adjust antibody concentration according to the IHC-P instructions (standard IHC-P practice). Retain the condition that best preserves cellular detail. |
| Strong signal appears in unexpected cells. | Cross-reactivity or endogenous detection activity is possible (standard IHC practice). HPA's ubiquitous profile means an unlisted cell population is not automatically negative (HPA: tissue IHC). | Compare morphology with an HPA High reference and a no-primary control; if using enzyme detection, check the corresponding endogenous-activity block (standard IHC practice; HPA: tissue IHC). |
| Chondrocytes stain weakly. | Low staining in soft tissue chondrocytes is reported by HPA and need not indicate a failed run (HPA: Low in chondrocytes). | Assess the same run in a High-reference population before changing conditions; use controls to distinguish weak specific staining from background (HPA: tissue IHC; standard IHC practice). |
| IF/ICC and tissue IHC patterns differ. | The applications have separate evidence ratings, and ICC-IF includes an additional acrosome observation (HPA: antibody validation; HPA: subcellular ICC-IF). | Judge paraffin IHC against tissue IHC references and its own controls; use the separate IF/ICC guide for fluorescence-specific setup (HPA: tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing COX5B staining
A06090-2 · IHCIHC analysis of COX5B using anti-COX5B antibody (A06090-2). COX5B was detected in a paraffin-embedded section of human breast cancer tissue.
A06090-2 · IHCIHC analysis of COX5B using anti-COX5B antibody (A06090-2). COX5B was detected in a paraffin-embedded section of human endometrial carcinoma
A06090-2 · IFIF analysis of COX5B using anti-COX5B antibody (A06090-2). COX5B was detected in an immunocytochemical section of A549 cells. Enzyme antigen
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000135940-COX5B/tissue/cerebral+cortex#imid_8306497
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000135940-COX5B/tissue/colon#imid_8306438
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000135940-COX5B/tissue/liver#imid_8306423
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000135940-COX5B/tissue/kidney#imid_8306440
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA034517; green = COX5B, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA034517; green = COX5B, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for COX5B IHC & IF
🧪Run breast first and expect staining in glandular cells (High; HPA: breast glandular cells). HPA detects COX5B in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and treat unstained nuclei and background on the positive slide as internal negative areas rather than assuming a negative cell population (HPA: no negative tissue rows; UniProt P10606: mitochondrial inner membrane).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: COX5B is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show COX5B in A-431, U-251MG, U2OS, Sperm, U2OS, siRNA 1 (10x), U2OS, siRNA 1 (40x), U2OS, siRNA 2 (10x), U2OS, siRNA 2 (40x), U2OS, scrambled (10x), U2OS, scrambled (40x), with annotated localisation: Mitochondria (enhanced) (HPA subcellular).
Technical controls: For the rabbit catalog antibody, include a no-primary, secondary-only slide, a matched rabbit IgG isotype control, and a COX5B knockout specimen if available as a biological negative (caption: rabbit primary; standard IHC controls). Block endogenous peroxidase before HRP–DAB detection and check the breast section for residual background (caption: breast section with HRP–DAB; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A06090-2 paraffin-section caption does not state the fixative (caption: fixative unreported). The caption uses heat-mediated retrieval in EDTA, pH 8.0, but does not establish that retrieval is required (caption: EDTA retrieval). Paraffin IHC is demonstrated in breast tissue, and mitochondrial ICC-IF images are available; the supplied evidence does not establish whether frozen sections or IF are easier or document a breast-specific artefact (caption: breast IHC; HPA: mitochondrial ICC-IF).
HPA tissue IHC evidence for COX5B
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 | Endocrine cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: COX5B is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced COX5B IHC Tips
Troubleshoot COX5B chromogenic IHC in paraffin sections using the catalog antibody’s tissue staining conditions and mitochondrial localisation as reference points.
Which retrieval condition should I try first for weak COX5B staining?
Start with heat mediated antigen retrieval in EDTA, pH 8.0 (datasheet A06090-2). This condition preceded COX5B detection in a paraffin section of human breast cancer tissue with the catalog antibody (datasheet A06090-2). Keep section thickness, heating duration, cooling time and detection chemistry consistent while titrating retrieval duration, so changes in staining are interpretable (standard IHC practice). Compare each condition with a concurrently processed positive control and inspect tissue integrity; stronger diffuse staining or damaged morphology does not establish better retrieval (standard IHC practice). The caption specifies the buffer and pH but gives no heating duration, so determine that setting on your equipment (datasheet A06090-2).
Could fixation explain variable COX5B staining between paraffin blocks?
The selected paraffin section caption does not state a fixative, and target specific COX5B fixation sensitivity is unknown from the supplied evidence (datasheet A06090-2). Record the fixative, fixation duration, tissue thickness and processing history for each block before attributing differences to biology (standard IHC practice). Compare blocks using the same EDTA, pH 8.0 retrieval and detection run, with a consistent control section included each time (datasheet A06090-2; standard IHC practice). If a block remains weak, examine morphology and staining in adjacent tissue before changing retrieval or antibody concentration (standard IHC practice). Do not infer fixation tolerance from mitochondrial localisation or acetylation annotations (UniProt P10606).
Where should convincing COX5B staining appear in an IHC section?
Assess COX5B as cytoplasmic staining compatible with mitochondria, since the protein resides in the mitochondrial inner membrane (UniProt P10606 topology). A fine granular or organelle rich cytoplasmic pattern is more plausible than isolated nuclear staining, although chromogenic IHC cannot resolve the inner membrane itself (UniProt P10606 topology; standard IHC practice). Compare signal within identifiable cells, because HPA reports ubiquitous mitochondrial expression and high staining in breast glandular cells, among other cells (HPA: tissue IHC profile; HPA: High in breast glandular cells). Use morphology and a suitable mitochondrial reference on a serial section when localisation is ambiguous (standard IHC practice). Interpret uniform nuclear or extracellular deposits cautiously (UniProt P10606 topology; standard IHC practice).
How should I investigate unexpected staining when the antibody epitope is unspecified?
Check the antibody immunogen or epitope documentation before assigning a staining difference to epitope masking; the supplied tissue caption does not locate that epitope (datasheet A06090-2). COX5B has one annotated mature chain spanning residues 32–129 and no listed isoforms, so an isoform specific interpretation is unsupported here (UniProt P10606 processing; UniProt P10606 isoforms). Acetyllysine is annotated at residues 68, 86 and 121, but the payload does not show that these modifications affect antibody binding (UniProt P10606 modified residues). Compare retrieval conditions and independent antibodies with documented epitopes, if available, while requiring the expected cytoplasmic mitochondrial distribution (standard IHC practice; UniProt P10606 topology).
How can I check COX5B localisation by multiplex IF?
Treat IF as a separate application requiring its own antibody validation; the supplied A06090-2 evidence describes chromogenic IHC in a paraffin section (datasheet A06090-2). For a breast section, pair COX5B with a glandular epithelial marker and a nuclear counterstain to identify the expected high staining cells (HPA: High in breast glandular cells; standard IF practice). Choose spectrally separated fluorophores, place the weaker signal in a far red channel when tissue autofluorescence interferes, and include single stain controls (standard IF practice). COX5B is associated with the inner mitochondrial membrane but has no annotated transmembrane segment; establish epitope accessibility and titrate permeabilisation for the epitope’s membrane side rather than assuming its orientation (UniProt P10606 topology; standard IF practice).
What should I change when COX5B DAB staining appears diffuse?
First inspect a no primary antibody control for detection reagent binding or endogenous peroxidase activity, and apply a peroxidase block as a general chromogenic IHC step (standard IHC practice). The selected example used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and DAB detection (datasheet A06090-2). Titrate primary concentration and shorten chromogen development one variable at a time while preserving a positive control (standard IHC practice). Distinguish granular cytoplasmic signal from uniform haze, edge deposits and precipitate by inspecting cell boundaries and adjacent sections (UniProt P10606 topology; standard IHC practice). The caption does not specify a peroxidase blocking step (datasheet A06090-2).
How should I quantify COX5B staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region before scoring, and exclude folds, necrosis, section edges and obvious DAB precipitate (standard IHC practice). Within comparable regions, record the percentage of positive cells and staining intensity on a 0–3 scale, then calculate an H score from 0–300 if intensity differences matter (standard IHC practice). Alternatively, measure positive cell density per mm² when cell abundance is the biological question (standard IHC practice). Normalise to the number of evaluable cells or viable tissue area, as appropriate, and keep imaging and threshold settings fixed (standard IHC practice). Stratify by cell type because HPA describes widespread expression with cell dependent staining levels (HPA: tissue IHC profile).
How can I separate true COX5B staining from tissue artefact?
Favour staining within intact cells that has a cytoplasmic, mitochondria compatible distribution, because COX5B belongs to the mitochondrial inner membrane (UniProt P10606 topology). HPA reports high staining in breast glandular cells and a broadly mitochondrial tissue profile, so judge intensity against the cell type and morphology present rather than expecting a universally negative tissue (HPA: High in breast glandular cells; HPA: tissue IHC profile). Treat isolated nuclear or extracellular signal, sharp section edge enhancement and staining confined to necrotic regions as suspect (UniProt P10606 topology; standard IHC practice). Check no primary antibody and peroxidase controls for endogenous enzyme or detection background, then compare with a concurrently stained positive section (standard IHC practice).
Boster reagents
Best COX5B / Cytochrome c oxidase subunit 5B, mitochondrial IHC Antibodies
A06090-2 has IHC and IF images of human paraffin sections and an IF/ICC image of A549 cells (image captions). Human, mouse, and rat reactivity is listed (catalog).
Real IHC data
Anti-COX5B Antibody ®
Cat # A06090-2
A06090-2 has IHC images of paraffin sections from human breast, endometrial, colorectal, and lung cancers (IHC image captions). Its IF images show A549 cells and paraffin sections from human ovary and breast cancers (IF image captions).
Which to pick: Choose A06090-2 for chromogenic IHC of paraffin sections: its breast cancer caption reports EDTA pH 8 retrieval, 2 μg/ml primary antibody, and HRP/DAB detection (IHC image caption). The same SKU has IF/ICC examples and a listed IF dilution of 5 μg/ml (IF image captions; catalog). A06090-2 lists mouse and rat reactivity, although its shown IHC sections are human; the fixative used for those paraffin sections is unreported (catalog reactivity; IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P10606 (COX5B_HUMAN, Cytochrome c oxidase subunit 5B, mitochondrial).
- Human Protein Atlas. COX5B tissue IHC expression (reliability: Supported).
- Human Protein Atlas. COX5B subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the acrosome..
- Human Protein Atlas. COX5B antibody validation summary (1 antibodies).
- Transcriptome analysis reveals the sex-switching mechanism of juvenile hermaphroditism in silver pomfret (Pampus argenteus). Biology of sex differences 2025 — PMC12261592.
- Integrative Multi-Omics Analysis and Experiments Validation Identify COX5B as a Novel Therapeutic Target for Lung Adenocarcinoma. Oncology research 2025 — PMC12774556.
- Loss of COX5B inhibits proliferation and promotes senescence via mitochondrial dysfunction in breast cancer. Oncotarget 2015 — PMC4791237.
- Abnormal Expression of COX5B Gene and Disorder of Mitochondrial Function in Cryptorchid Rats. Journal of cellular and molecular medicine 2024 — PMC11588429.
- PubMed PMID:2840351 — UniProt-cited evidence.
- PubMed PMID:1646156 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for COX5B (Cytochrome c oxidase subunit 5B, mitochondrial). Expected localisation Inner mitochondrial membrane; tissue signal is widespread (UniProt; HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A06090-2). Validated IHC antibody: A06090-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A06090-2); 2. Fixation: Image fixative and duration unreported (datasheet A06090-2); 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 A06090-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A06090-2); 8. Primary antibody: Rabbit anti-COX5B, 2-5 μg/ml (datasheet A06090-2); 9. Primary incubation: Overnight at 4 °C (datasheet A06090-2). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Chondrocytes show low staining despite broad expression (HPA tissue IHC); Isoform / epitope: No isoforms annotated; mature chain spans residues 32–129 (UniProt); Regulation: Heart and skeletal muscle have enhanced RNA (HPA tissue RNA). Evidence: UniProt P10606, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the mitochondria. In addition localized to the…, Datasheet A06090-2, PMC12774556, PMC11588429. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/COX5B/immunohistochemistry-ihc-guide.