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COX5B / Cytochrome c oxidase subunit 5B, mitochondrial · IHC design guide
Design Immunohistochemistry for COX5B
COX5B is a component of mitochondrial complex IV located at the inner membrane (UniProt). This paraffin-section IHC guide helps plan staining and interpret its widespread mitochondrial tissue profile while accounting for medium consistency between antibody staining and RNA expression (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 | Mitochondrial tissue staining (HPA tissue IHC); inner membrane location (UniProt) | |
| Staining pattern | Widespread 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 paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific) | |
| Caveat | Antibody staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Heart and skeletal muscle show tissue-enhanced RNA (HPA RNA) | |
| Isoform / epitope | No annotated isoforms; mature chain 32–129; epitope unspecified (UniProt) |
Section 1
Recommended COX5B IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with one published COX5B staining method for paraffin sections (datasheet A06090-2; PMC12774556).
| 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 at pH 8.0 for the catalog antibody (datasheet A06090-2); the published method used boiling citrate buffer (PMC12774556).
Section 2
What Is the Expected COX5B Staining Pattern?
COX5B is a component of mitochondrial complex IV in the inner membrane and has no annotated transmembrane segment (UniProt P10606). Expect cytoplasmic mitochondrial staining in many cell types: HPA describes ubiquitous mitochondrial expression, with high IHC staining in several sampled tissues (HPA tissue IHC). HPA rates the tissue pattern Supported, meaning medium consistency between antibody staining and RNA expression data (HPA tissue IHC).
| Granular cytoplasmic staining in adrenal glandular cells, with little nuclear staining. | This fits mitochondrial localization (UniProt P10606; HPA subcellular) and the reported high staining of adrenal glandular cells (HPA tissue IHC). Judge the pattern within identifiable cells; a chromogenic IHC image cannot by itself resolve the inner mitochondrial membrane (UniProt P10606 topology; general IHC practice). |
| Predominantly nuclear, surface, or uniformly diffuse staining replaces a mitochondrial pattern. | The dominant compartment conflicts with the reported inner mitochondrial membrane location (UniProt P10606) and mainly mitochondrial ICC/IF localization (HPA subcellular). Treat it as suspect staining; review the negative control, tissue morphology, and detection steps before assigning COX5B expression (general IHC practice). |
| Strong staining appears only in an unexpected cell population while adjacent known-positive cells are blank. | Cell identity matters: HPA reports high staining in breast glandular cells and bronchial respiratory epithelial cells, among others (HPA tissue IHC). This reversal warrants a specificity check for cross-reactivity or endogenous detection activity; HPA provides no universal negative cell type (HPA tissue IHC; general IHC practice). |
| The whole section has diffuse brown haze, including spaces outside cells. | A field-wide deposit cannot establish mitochondrial localization (UniProt P10606; general IHC practice). Compare a no-primary control, check washing and blocking, and assess chromogen development; diffuse background may obscure genuine granular signal (general IHC practice). |
| No staining is visible in an adrenal gland section with recognizable glandular cells. | HPA reports high staining in those cells, so the absence of signal challenges the assay result (HPA tissue IHC). Check section quality, antigen retrieval, antibody and detection performance, and a run control before concluding that the sample lacks COX5B (general IHC practice). |
💡Expected COX5B appearanceCall the IHC result positive when identifiable cells show predominantly granular cytoplasmic staining consistent with mitochondria, particularly high signal in an HPA high-staining population such as adrenal glandular cells; dominant nuclear staining or section-wide haze is suspect (HPA tissue IHC; HPA subcellular; UniProt P10606; general IHC practice).
| Compartment and topology | COX5B is assigned to the mitochondrial inner membrane, with no annotated transmembrane segment; evaluate the visible mitochondrial pattern rather than expecting a cell-surface outline (UniProt P10606). |
| Tissue and cell selection | HPA reports high IHC staining in adrenal and breast glandular cells, bronchial respiratory epithelial cells, and several other listed populations; chondrocytes in soft tissue are listed as low (HPA tissue IHC). Low is not an established negative control (HPA tissue IHC). |
| Strength of IHC evidence | The tissue pattern is Supported, with medium antibody–RNA consistency; HPA034517 is IHC Supported and ICC Enhanced. The ICC rating does not upgrade its IHC validation (HPA tissue IHC; HPA antibodies). |
| RNA versus observed protein | Heart and skeletal muscle are tissue-enhanced in HPA RNA data, but the supplied IHC examples do not assign either an observed staining level; do not use that RNA label as an IHC intensity prediction (HPA tissue IHC). |
| Processing and epitope information | UniProt annotates the mature chain as residues 32–129; the supplied evidence does not locate the antibody epitope. Processing-dependent staining differences therefore cannot be predicted here (UniProt P10606; HPA antibodies). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in an HPA high-staining cell population. | The assay may have failed; absence alone cannot establish lack of protein where high IHC staining is reported (HPA tissue IHC; general IHC practice). | Verify cell identity and section quality, then review retrieval, antibody incubation, detection reagents, and a positive run control (general IHC practice). |
| Diffuse brown staining across cells and surrounding tissue. | Background from detection chemistry or insufficient washing can overwhelm the expected mitochondrial pattern (UniProt P10606; general IHC practice). | Inspect a no-primary control; optimize blocking, washing, antibody concentration, and chromogen development for the assay (general IHC practice). |
| Strong nuclear or cell-surface staining dominates. | This distribution conflicts with the reported mitochondrial location; nonspecific binding or interpretation of counterstain may be involved (UniProt P10606; HPA subcellular; general IHC practice). | Compare with the counterstained negative control and recognizable mitochondrial-like cytoplasmic signal; reassess antibody specificity before scoring (general IHC practice). |
| An unexpected cell type stains strongly while expected cells do not. | Cross-reactivity or endogenous detection activity is possible; HPA cell-specific examples do not validate every cell population (HPA tissue IHC; general IHC practice). | Check morphology and cell identity, then compare no-primary and detection controls before assigning the signal to COX5B (general IHC practice). |
| Staining is weak in soft-tissue chondrocytes. | HPA lists chondrocytes as low, so weak signal there can fit the reported pattern (HPA tissue IHC). | Assess assay performance in an HPA high-staining population; do not treat chondrocytes as a proven COX5B-negative control (HPA tissue IHC; general IHC practice). |
| What should ICC/IF show? | HPA reports mainly mitochondrial localization, plus approved acrosome localization; its listed antibody has ICC Enhanced status (HPA subcellular; HPA antibodies). | Look for a mitochondrial fluorescence pattern and assess acrosome signal only in appropriate sperm images; use the separate IF/ICC guide for the method (HPA subcellular; general IF 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: glandular cells should stain (HPA: High in 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 any unstained cells on the positive slide as background references rather than confirmed COX5B-negative cells (HPA: no negative tissue rows).
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: Include a no-primary, secondary-only slide and a rabbit immunoglobulin control matched to the primary antibody’s clonality; a COX5B knockout specimen, if available, provides a biological negative (caption: rabbit anti-COX5B primary; standard IHC controls). Block endogenous peroxidase in breast sections before HRP/DAB detection (caption: peroxidase-conjugated secondary and DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A06090-2 caption does not state the fixative (caption: fixative not stated). That paraffin-section example used heat retrieval in EDTA at pH 8.0, but it does not establish that retrieval is required (caption: EDTA retrieval); the supplied evidence does not establish that frozen sections or IF are easier. For IF, mitochondrial signal is an expected localization pattern (HPA: mitochondria enhanced; UniProt P10606: inner mitochondrial membrane); assess breast IHC against the no-primary background when interpreting DAB staining (HPA: High in breast glandular cells; standard IHC practice).
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 staining in paraffin sections by checking retrieval, tissue integrity, mitochondrial localisation, detection background, and cell-level scoring.
How should I optimise retrieval when COX5B staining is weak in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for COX5B paraffin-section IHC (datasheet A06090-2). Keep heating and cooling consistent across slides, then compare the suspect section with a positive-control section processed in the same run (standard IHC practice). The documented staining used 2 μg/ml primary antibody overnight at 4°C, so check that exposure before changing retrieval (datasheet A06090-2). If staining remains weak, test a shorter or longer heating interval on serial sections while watching for tissue damage; record the interval and cooling conditions because the caption specifies neither (datasheet A06090-2; standard IHC practice).
Could fixation explain weak COX5B staining in my paraffin sections?
Target-specific COX5B sensitivity to fixative type or fixation duration is unknown from the supplied evidence (datasheet A06090-2: fixative not stated). The documented image shows staining in a paraffin-embedded human breast cancer section, but its caption does not identify the fixative (datasheet A06090-2). Compare sections with known processing histories under identical EDTA pH 8.0 retrieval and antibody conditions, and assess morphology alongside signal (datasheet A06090-2; standard IHC practice). For future specimens, record fixative, time to fixation, fixation duration, and processing schedule; these variables can affect paraffin-section antigen accessibility in routine IHC (standard IHC practice).
Where should convincing COX5B chromogenic staining appear?
Expect predominantly cytoplasmic staining with a granular mitochondrial distribution, interpreted at the resolution available in chromogenic sections (UniProt P10606: inner mitochondrial membrane; HPA: ubiquitous mitochondrial expression). COX5B is a component of respiratory complex IV in the mitochondrial inner membrane and has no annotated transmembrane segment (UniProt P10606). Assess the pattern within intact cells and compare it with tissue morphology, since dense DAB deposits can obscure individual organelles (standard IHC practice). Strong isolated nuclear or extracellular deposits warrant review of counterstain, section damage, and detection controls before being called COX5B positive (UniProt P10606 localisation; standard IHC practice).
How do processing and epitope location affect COX5B staining?
The COX5B record annotates a 129-amino-acid precursor and a processed chain spanning residues 32–129, with no annotated isoforms (UniProt P10606). It also lists lysine acetylation at residues 68, 86, and 121, but the supplied caption does not identify the antibody epitope (UniProt P10606; datasheet A06090-2). Therefore, do not attribute weak staining to cleavage or acetylation without epitope information and direct comparison on matched sections (standard IHC interpretation). If antibody documentation supplies an immunogen sequence, map it against the processed chain before interpreting discrepant retrieval results; continue to score only morphologically credible mitochondrial staining (UniProt P10606; standard IHC practice).
How can I assess COX5B localisation in a multiplex IF follow-up?
For a follow-up IF experiment, pair COX5B with a marker identifying the cell population under study; glandular cells are a documented high-staining population in breast tissue (HPA: High in breast glandular cells). Include a mitochondrial reference marker in a separate channel if the question is subcellular colocalisation (UniProt P10606 localisation; standard IF practice). Inspect unstained tissue in each channel, then choose fluorophores whose emission is separable from tissue autofluorescence and from one another (standard IF practice). Because the antibody epitope and its accessible side of the inner mitochondrial membrane are unreported, titrate permeabilisation and interpret loss of signal cautiously (datasheet A06090-2; UniProt P10606 topology; standard IF practice).
What should I check when COX5B DAB staining is diffuse?
The documented chromogenic workflow used a peroxidase-conjugated secondary and DAB, making enzyme-related and secondary-related background practical checks (datasheet A06090-2; standard IHC practice). Run a no-primary control, apply a routine endogenous peroxidase block, and compare background in intact tissue with deposits at folds or damaged edges (standard IHC practice). The caption specifies 10% goat-serum block and 2 μg/ml primary antibody overnight at 4°C; use those conditions as the documented starting point (datasheet A06090-2). If diffuse staining persists, titrate primary concentration and detection development on serial sections while preserving visible granular cytoplasmic signal and readable counterstain (UniProt P10606 localisation; standard IHC practice).
How should I quantify COX5B across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Score COX5B within a predefined cell population and region, recording both the percentage of positive cells and cytoplasmic intensity to calculate an H-score (standard IHC practice). Use the same staining threshold, DAB development conditions, and counterstain across comparison groups; exclude folds, necrosis, and section edges from the analysis mask (standard IHC practice). Normalise the positive-cell count to the number of eligible cells, or report positive-cell density per mm² of eligible tissue when cell counting is impractical (standard IHC practice). Record cell composition because COX5B expression is broadly mitochondrial and HPA reports varying staining levels across cell types (HPA: ubiquitous mitochondrial expression; HPA: tissue IHC profile).
Which COX5B staining patterns suggest artefact rather than true signal?
A credible positive call combines staining in intact cells with a cytoplasmic mitochondrial pattern, consistent with COX5B's inner-membrane localisation and the HPA tissue profile (UniProt P10606; HPA: ubiquitous mitochondrial expression). Compare the stained cell type with adjacent morphology: high staining is reported in breast glandular cells, while chondrocytes are listed as low (HPA: breast glandular cells High; HPA: chondrocytes Low). Treat nuclear-only staining, abrupt edge enhancement, necrotic deposits, and signal in a no-primary control as reasons to investigate artefact (UniProt P10606 localisation; standard IHC practice). Check endogenous peroxidase blocking before interpreting widespread DAB colour as target signal, since the documented assay uses peroxidase detection (datasheet A06090-2; standard IHC practice).
Boster reagents
Best COX5B / Cytochrome c oxidase subunit 5B, mitochondrial IHC Antibodies
The IHC-validated anti-COX5B antibody has human paraffin-section IHC images and IF images from A549 cells and human paraffin sections; catalog reactivity includes mouse and rat (A06090-2 image captions; catalog reactivity).
Real IHC data
Anti-COX5B Antibody ®
Cat # A06090-2
A06090-2 is listed for IHC, IF and ICC, with paraffin-section IHC images from human breast, endometrial, colorectal and lung cancers (catalog applications; A06090-2 IHC image captions). Its IF images cover A549 cells and paraffin-embedded human ovary and breast cancer sections (A06090-2 IF image captions).
Which to pick: For chromogenic tissue IHC, choose A06090-2: its own breast cancer IHC caption documents a paraffin section, EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody; the fixative is unreported (A06090-2 IHC image caption). For IF/ICC, the same SKU is listed for both applications and has the IF images described above; clonality is unreported (catalog applications; A06090-2 IF image captions; catalog clone field). For mouse or rat samples, A06090-2 is listed as reactive in both species, although its supplied IHC images show human tissue (catalog reactivity; A06090-2 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 Mitochondrial tissue staining (HPA tissue IHC); inner membrane location (UniProt). 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 paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific); Caveat: Antibody staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: No annotated isoforms; mature chain 32–129 ; epitope unspecified (UniProt); Regulation: Heart and skeletal muscle show tissue-enhanced RNA (HPA 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. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/COX5B/immunohistochemistry-ihc-guide.