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COX5A / Cytochrome c oxidase subunit 5A, mitochondrial · IHC design guide
Design Immunohistochemistry for COX5A
Plan COX5A chromogenic IHC in paraffin sections using its granular cytoplasmic tissue pattern (HPA tissue IHC) and mitochondrial inner membrane location (UniProt). Compare cell types with documented high and undetected staining when selecting controls (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
COX5A 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); mitochondrial inner membrane (UniProt) | |
| Staining pattern | Granular cytoplasmic staining in glandular and epithelial cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A07895-2) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue+2 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 | Staining varies by cell type: colon glands high, adipocytes undetected (HPA tissue IHC) | |
| Regulation | No specific regulator annotated (UniProt) | |
| Isoform / epitope | No isoforms; processing removes residues 1–41 from the mature chain (UniProt) |
Section 1
Recommended COX5A IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A07895-2) is accompanied by published COX5A IHC methods for rat brain, renal carcinoma, and gastric tissues (PMC7191708; PMC2783948; PMC12582873).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A07895-2) |
| Fixation | Image fixative and duration unreported (datasheet A07895-2); 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 A07895-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A07895-2) |
| Primary antibody | Rabbit anti-COX5A, 1:50 recommended; image 1:100 (datasheet A07895-2) |
| Primary incubation | Overnight at 4 °C (datasheet A07895-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A07895-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | COX5A-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression with a granular pattern. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet: A07895-2). Microwave retrieval is a published alternative for gastric sections (PMC12582873).
Section 2
What Is the Expected COX5A Staining Pattern?
COX5A is a component of respiratory complex IV in the mitochondrial inner membrane and has no annotated transmembrane segment (UniProt P20674). In paraffin section IHC, expect granular cytoplasmic staining, including in glandular cells of colon and appendix and respiratory epithelial cells of bronchus (HPA tissue IHC). HPA rates the tissue pattern Supported, with medium consistency between antibody staining and RNA expression (HPA tissue IHC).
| Granular cytoplasmic stain in colon glandular cells, with nuclei remaining defined by counterstain. | This matches HPA's general granular cytoplasmic pattern and High staining in colon glandular cells (HPA tissue IHC). It is consistent with mitochondrial localization (UniProt P20674), although a chromogenic pattern alone cannot resolve the inner membrane (standard IHC interpretation). |
| Predominantly nuclear, sharply membranous, or extracellular stain replaces the granular cytoplasmic pattern. | These compartments conflict with the annotated mitochondrial inner membrane location and HPA tissue pattern (UniProt P20674; HPA tissue IHC). Treat the result as suspect; review morphology and detection controls before calling it COX5A signal (standard IHC practice). |
| Strong stain appears in adipocytes, ovarian stroma cells, or soft tissue fibroblasts. | HPA reports COX5A as Not detected in those sampled cell populations (HPA tissue IHC). Consider nonspecific antibody binding or endogenous detection activity; confirm the cell identity and compare controls before interpreting the stain (standard IHC practice). |
| Broad, smooth color covers tissue or surrounding spaces and obscures cell boundaries. | This differs from HPA's granular cytoplasmic profile (HPA tissue IHC). Uneven reagent coverage, residual detection activity, or nonspecific background are possible workflow causes; the appearance alone does not identify which one occurred (standard IHC practice). |
| No stain is visible in colon glandular cells or bronchial respiratory epithelial cells. | Both populations are High in the supplied HPA tissue IHC observations (HPA tissue IHC). A blank result warrants review of section integrity, reagent performance, retrieval conditions, and detection controls; it does not by itself establish biological absence (standard IHC practice). |
💡Expected COX5A appearanceCall a result positive when identifiable cells show granular cytoplasmic chromogen, especially High-staining colon or appendix glandular cells or bronchial respiratory epithelium (HPA tissue IHC); dominant nuclear or extracellular color is inconsistent with the reported pattern and mitochondrial location (HPA tissue IHC; UniProt P20674).
| Compartment and topology | COX5A is annotated in the mitochondrial inner membrane, with no transmembrane segment (UniProt P20674). Evaluate the stain as a cytoplasmic granular pattern at light-microscope resolution (HPA tissue IHC); topology does not identify an antibody epitope. |
| Protein processing | UniProt annotates the mature chain as residues 42–150 of a 150-residue precursor (UniProt P20674). With no epitope location supplied, the record cannot predict whether the antibody recognizes precursor, mature protein, or both. |
| Tissue and cell context | HPA records High stain in several epithelial and glandular populations, but Low stain in skeletal myocytes and Not detected in adipocytes (HPA tissue IHC). Its RNA tissue enhancement in skeletal muscle is a different measurement and does not override the Low protein stain observation (HPA tissue IHC). |
| Strength of IHC evidence | HPA rates the overall tissue IHC profile Supported and lists antibody HPA027526 as IHC Supported (HPA tissue IHC; HPA antibodies). Medium staining–RNA consistency leaves room for discordant samples; use the reported cell pattern as a reference, not an absolute threshold (HPA tissue IHC). |
| IF/ICC evidence: can it independently confirm the compartment? | HPA supplies no main subcellular location or ICC-IF image set for COX5A (HPA subcellular). The IHC pattern therefore has no supplied HPA cell-image confirmation; consult the separate IF/ICC guide for that application. |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive tissue is blank. | The slide may have a failed staining step; HPA reports High signal in colon glandular and bronchial respiratory epithelial cells (HPA tissue IHC). | Check tissue preservation and the run's positive control, then review retrieval, primary antibody, and chromogen steps against the validated IHC workflow (standard IHC practice). No COX5A-specific retrieval condition is supplied. |
| Only nuclei or tissue edges stain. | The distribution conflicts with HPA's granular cytoplasmic profile and UniProt's mitochondrial location (HPA tissue IHC; UniProt P20674). | Compare with a no-primary control, inspect section edges and morphology, and repeat detection if the pattern persists (standard IHC practice). Do not score this distribution as COX5A-positive. |
| Adipocytes or fibroblasts appear strongly positive. | HPA records Not detected in adipocytes and soft tissue fibroblasts; nonspecific binding or detection background is possible (HPA tissue IHC; standard IHC practice). | Verify the cells on the counterstained section and compare a no-primary control with a HPA High-staining reference tissue (standard IHC practice; HPA tissue IHC). |
| Stain is diffuse across cells and empty spaces. | This differs from the HPA granular cytoplasmic profile; background from the staining workflow is possible (HPA tissue IHC; standard IHC practice). | Check reagent coverage and washing, then compare a no-primary control and review blocking of endogenous detection activity where applicable (standard IHC practice). |
| Skeletal myocytes look weaker than expected from the RNA profile. | HPA reports Low IHC staining in skeletal myocytes despite tissue-enhanced RNA in skeletal muscle (HPA tissue IHC). | Score the observed protein stain separately from RNA expression; use cell-resolved IHC references and the same run's controls when judging whether the assay worked (HPA tissue IHC; standard IHC practice). |
| A proposed negative tissue shows scattered granular positive cells. | HPA's Not detected calls apply to named cell populations, such as ovarian stroma cells, rather than every cell in an entire organ (HPA tissue IHC). | Identify the stained cell population before scoring. Record cell-level location and intensity, and compare it with the relevant HPA cell-level observation (HPA tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing COX5A staining
A07895-2 · IHCIHC analysis of COX5A using anti-COX5A antibody (A07895-2). COX5A was detected in a paraffin-embedded section of human breast cancer tissue.
A07895-2 · IHCIHC analysis of COX5A using anti-COX5A antibody (A07895-2). COX5A was detected in a paraffin-embedded section of human colon tissue. Heat me
A07895-1 · IHCImmunohistochemistry (IHC) analyzes of COX5A pAb in paraffin-embedded human colon carcinoma tissue at 1:50.showing Mitochondrion inner membr
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000178741-COX5A/tissue/cerebral+cortex#imid_6908520
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000178741-COX5A/tissue/colon#imid_6908462
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000178741-COX5A/tissue/liver#imid_6908455
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000178741-COX5A/tissue/kidney#imid_6908448
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for COX5A IHC & IF
🧪Run colon first: glandular cells should show COX5A staining (HPA: High in colon glandular cells). Use adipose tissue as the negative tissue because adipocytes are not detected (HPA: Not detected in adipocytes); cells chosen as internal negatives on the colon slide should show counterstain without specific chromogenic signal, although the supplied HPA rows do not identify a verified negative colon cell type.
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for COX5A; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control, a concentration-matched rabbit IgG isotype control, and COX5A knockout material as a biological negative (selected-SKU caption: rabbit primary antibody; standard IHC practice). For DAB detection in colon, quench endogenous peroxidase and assess background in the no-primary control (selected-SKU caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the exact selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative unreported). The caption uses heat retrieval in EDTA at pH 8.0, so test retrieval under those conditions for paraffin IHC; it does not establish that retrieval is required in every preparation (selected-SKU caption: EDTA retrieval). The supplied evidence does not establish that frozen sections or IF/ICC are easier, or identify a COX5A-specific colon artefact; assess endogenous peroxidase background during chromogenic scoring (HPA subcellular: no ICC-IF image cell lines; selected-SKU caption: HRP/DAB detection; standard IHC practice).
HPA tissue IHC evidence for COX5A
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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuropil | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
Section 3
Advanced COX5A IHC Tips
Troubleshoot COX5A staining in paraffin sections by checking retrieval, tissue processing, mitochondrial localisation, controls, and scoring.
What retrieval should I try first for weak COX5A staining?
Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet A07895-2). The documented paraffin-section example used that retrieval before a 1:100 primary incubation overnight at 4°C, so keep those conditions together when establishing a reference run (datasheet A07895-2). If staining remains weak, compare a second retrieval condition on adjacent sections while holding antibody concentration and detection constant (standard IHC practice). Check whether improved granular cytoplasmic signal also appears in a no-primary control; increased diffuse staining alone does not establish better COX5A detection (HPA tissue IHC; standard IHC practice). Avoid judging retrieval from a single field, since section edges can stain differently from the interior (standard IHC practice).
Could fixation explain weak or uneven COX5A staining?
The selected tissue caption describes a paraffin-embedded section but does not report its fixative, so COX5A-specific fixation sensitivity is unknown (datasheet A07895-2). Record the fixative and processing history for each specimen, then compare sections processed alike before changing retrieval or antibody concentration (standard IHC practice). Include a documented positive tissue on the same run to separate a specimen-wide processing problem from an assay failure (standard IHC practice; HPA tissue IHC). If staining varies across a section, inspect morphology and compare central tissue with edges before assigning the difference to COX5A expression (standard IHC practice). Neither the HPA staining profile nor COX5A topology establishes a target-specific response to fixation (HPA tissue IHC; UniProt P20674).
Where should convincing COX5A staining appear in chromogenic IHC?
COX5A resides in the mitochondrial inner membrane, while tissue IHC shows general cytoplasmic expression with a granular pattern (UniProt P20674; HPA tissue IHC). In a chromogenic section, look for cytoplasmic granularity within intact cells and assess it alongside the counterstained nuclei (HPA tissue IHC; standard IHC practice). Predominantly nuclear or extracellular DAB deposition warrants review of the no-primary control, tissue integrity, and detection reagents before scoring (UniProt P20674; standard IHC practice). Compare the same cell type across fields rather than treating every dark area as equivalent signal (standard IHC practice). HPA reports high staining in appendix glandular cells, providing a documented cell-level comparator (HPA tissue IHC).
Could an isoform or inaccessible epitope explain discordant COX5A staining?
The supplied UniProt record lists 0 isoforms and a mature COX5A chain spanning residues 42–150 (UniProt P20674). It also reports no transmembrane segment, but neither that topology nor the supplied caption maps this antibody’s epitope (UniProt P20674; datasheet A07895-2). If staining changes with retrieval, test adjacent sections under controlled conditions before attributing the difference to an isoform (standard IHC practice; UniProt P20674). Acetyllysines at residues 87 and 113 and phosphothreonine at 141 are annotated, but their effect on this antibody is unknown (UniProt P20674). Seek epitope information before making a modification-specific interpretation (standard IHC practice).
How should I adapt COX5A assessment to multiplex IF?
Treat IF as a separate validation exercise: the supplied tissue example documents chromogenic paraffin-section IHC, while HPA supplies no COX5A ICC/IF images (datasheet A07895-2; HPA subcellular). Multiplex COX5A with a marker identifying the expected cell population, such as glandular epithelium when examining HPA-positive glandular cells (HPA tissue IHC; standard IF practice). Select fluorophores after checking tissue autofluorescence and include single-stain controls to assess bleed-through (standard IF practice). COX5A is an inner-membrane protein without a transmembrane segment, but its antibody epitope and membrane-facing side are unspecified (UniProt P20674; datasheet A07895-2). Choose and validate permeabilisation for access to the mapped epitope rather than assuming the IHC retrieval establishes IF access (standard IF practice).
How can I reduce diffuse brown background without losing COX5A signal?
The documented IHC example used 10% goat serum blocking, a 1:100 rabbit primary overnight at 4°C, and DAB development (datasheet A07895-2). Reproduce those documented conditions first, then change one variable at a time while comparing adjacent sections (standard IHC practice). Include a no-primary section to reveal secondary-reagent or chromogen background, and apply a peroxidase block as a general HRP-IHC step (standard IHC practice). If background persists, inspect wash quality and chromogen development while preserving the granular cytoplasmic pattern reported for COX5A (HPA tissue IHC; standard IHC practice). Do not count diffuse staining that also appears in the control as target signal (standard IHC practice).
How should I score COX5A IHC across different cell populations? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and analysis regions before scoring, using intact-cell morphology and granular cytoplasmic staining as inclusion criteria (HPA tissue IHC; standard IHC practice). Report the percentage of positive cells and staining intensity separately, or use an H-score from 0–300 with fixed intensity thresholds (standard IHC practice). Normalise counts to the number of evaluable cells in each population, or report positive-cell density per mm² of evaluable tissue (standard IHC practice). Exclude necrotic and damaged regions consistently, and use identical imaging and threshold settings across compared sections (standard IHC practice). Record cell type because HPA reports different staining levels across tissues and populations (HPA tissue IHC).
What distinguishes true COX5A positivity from IHC artefact?
A credible result shows granular cytoplasmic staining in intact cells, consistent with mitochondrial inner-membrane COX5A and the HPA tissue pattern (UniProt P20674; HPA tissue IHC). Check whether the stained population fits documented observations: appendix glandular cells are high, whereas adipocytes are reported as not detected (HPA tissue IHC). Predominantly nuclear signal, staining confined to section edges, or DAB deposits over necrosis should prompt review of morphology and controls (UniProt P20674; standard IHC practice). A no-primary control helps identify nonspecific detection or endogenous peroxidase contribution to brown staining (standard IHC practice). HPA rates its tissue IHC evidence as Supported with medium agreement between staining and RNA, so interpret unexpected patterns cautiously (HPA tissue IHC).
Boster reagents
Best COX5A / Cytochrome c oxidase subunit 5A, mitochondrial IHC Antibodies
Both antibodies have IHC images from human paraffin sections (catalog IHC captions). Both list Human, Mouse and Rat reactivity; A07895-2 also lists IF/ICC, although no IF image is supplied (catalog).
Real IHC data
Anti-COX5A Antibody
Cat # A07895-2
Real IHC data
Anti-COX5A Antibody
Cat # A07895-1
A07895-1 shows IHC staining in human paraffin-embedded colon carcinoma at 1:50 (A07895-1 IHC caption). A07895-2 shows IHC staining in human paraffin-embedded breast cancer, colon tissue and colon cancer; its application list also includes IF/ICC (A07895-2 IHC captions; catalog applications).
Which to pick: For tissue IHC, A07895-2 has a documented EDTA pH 8.0 retrieval and DAB workflow in human paraffin sections; A07895-1 is another IHC option with a human paraffin-section image (respective IHC captions). For IF/ICC, choose A07895-2 because those applications are listed, while A07895-1 lists IHC without IF/ICC; no IF image is supplied for A07895-2 (catalog applications; catalog IF images). Both are described as polyclonal and list Human, Mouse and Rat reactivity, but their IHC images show human samples only; neither caption reports the fixative (A07895-1 IHC caption; A07895-2 dilution record and IHC captions; catalog reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P20674 (COX5A_HUMAN, Cytochrome c oxidase subunit 5A, mitochondrial).
- Human Protein Atlas. COX5A tissue IHC expression (reliability: Supported).
- Human Protein Atlas. COX5A subcellular location (ICC-IF): Highest expression in HEK293: 849.5 nTPM.
- Human Protein Atlas. COX5A antibody validation summary (1 antibodies).
- COX5A over-expression protects cortical neurons from hypoxic ischemic injury in neonatal rats associated with TPI up-regulation. BMC neuroscience 2020 — PMC7191708.
- Diagnostic biomarkers for renal cell carcinoma: selection using novel bioinformatics systems for microarray data analysis. Human pathology 2009 — PMC2783948.
- Cytochrome c Oxidase Subunit 5A (COX5A) Enhances Gastric Cancer Progression by Augmenting ATP Synthesis and Activating the PI3K/Akt Pathway. Journal of cellular and molecular medicine 2025 — PMC12582873.
- Ulcerative colitis mucosal transcriptomes reveal mitochondriopathy and personalized mechanisms underlying disease severity and treatment response. Nature communications 2019 — PMC6318335.
- PubMed PMID:2853101 — UniProt-cited evidence.
- PubMed PMID:18197981 — UniProt-cited evidence.
- PubMed PMID:16572171 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for COX5A (Cytochrome c oxidase subunit 5A, mitochondrial). Expected localisation Granular cytoplasm (HPA tissue IHC); mitochondrial inner membrane (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A07895-2). Validated IHC antibody: A07895-2 at 1:50 recommended; image 1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A07895-2); 2. Fixation: Image fixative and duration unreported (datasheet A07895-2); 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 A07895-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A07895-2); 8. Primary antibody: Rabbit anti-COX5A, 1:50 recommended; image 1:100 (datasheet A07895-2); 9. Primary incubation: Overnight at 4 °C (datasheet A07895-2). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining varies by cell type: colon glands high, adipocytes undetected (HPA tissue IHC); Isoform / epitope: No isoforms; processing removes residues 1–41 from the mature chain (UniProt); Regulation: No specific regulator annotated (UniProt). Evidence: UniProt P20674, HPA tissue IHC · Supported, HPA cell-line RNA · Highest expression in HEK293: 849.5 nTPM, Datasheet A07895-2, PMC7191708, PMC2783948. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/COX5A/immunohistochemistry-ihc-guide.