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COX5A / Cytochrome c oxidase subunit 5A, mitochondrial · IHC design guide
Design Immunohistochemistry for COX5A
Plan COX5A staining in paraffin sections using its granular cytoplasmic tissue pattern and cell-specific staining levels (HPA tissue IHC). This guide covers fixation, antibody use, controls and scoring for a protein of the mitochondrial inner membrane (UniProt).
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); inner mitochondrial membrane (UniProt) | |
| Staining pattern | General granular cytoplasmic staining (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 paraffin-section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A07895-2) | |
| Caveat | Skeletal myocytes show low staining despite muscle-enhanced RNA (HPA tissue IHC) | |
| Regulation | Muscle-enhanced RNA expression (HPA tissue RNA) | |
| Isoform / epitope | No isoforms annotated; mature chain spans 42–150 (UniProt) |
Section 1
Recommended COX5A IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with published COX5A staining in rat brain, renal tumors, and gastric tumors (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 EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A07895-2); the gastric study reports microwave retrieval without a buffer specification (PMC12582873).
Section 2
What Is the Expected COX5A Staining Pattern?
COX5A is a complex IV component associated with the mitochondrial inner membrane; UniProt annotates no transmembrane segment (UniProt P20674). In paraffin sections, expect granular cytoplasmic staining, including strong staining in colon glandular cells and bronchial respiratory epithelial cells (HPA: tissue IHC). HPA rates the tissue pattern Supported, with medium consistency between antibody staining and RNA expression (HPA: tissue IHC).
| Granular cytoplasmic signal in colon glands, with nuclei largely unstained. | This fits the reported general cytoplasmic granular pattern and High staining in colon glandular cells (HPA: tissue IHC). The granularity is consistent with a mitochondrial protein (UniProt P20674 location), but a brightfield IHC image alone cannot resolve the inner mitochondrial membrane. |
| Predominantly nuclear, cell-surface, or continuous membrane-edge staining. | That distribution conflicts with the reported granular cytoplasmic IHC pattern (HPA: tissue IHC) and mitochondrial inner-membrane location (UniProt P20674). Treat it as suspect; compare the tissue morphology and negative controls before calling it COX5A. |
| Dominant staining in adipocytes or ovarian stromal cells while nearby expected-positive cells are weak. | HPA reports COX5A as Not detected in adipocytes and ovarian stromal cells (HPA: tissue IHC). Strong signal there warrants checks for cross-reactivity or endogenous detection activity (general IHC practice); the HPA observations are comparisons, not absolute exclusions for every specimen. |
| Broad, even brown haze across cells and extracellular areas. | A diffuse field obscures the reported granular cytoplasmic pattern (HPA: tissue IHC). Consider nonspecific background, endogenous detection activity, or excess chromogen development (general IHC practice); haze alone cannot establish COX5A expression. |
| No staining in a well-preserved colon gland or bronchial respiratory epithelium. | Both cell populations are reported High by HPA (HPA: tissue IHC). First suspect an assay or specimen problem and check controls (general IHC practice). A negative result in one section does not overturn the HPA tissue-level pattern. |
💡Expected COX5A appearanceCall positive a granular cytoplasmic signal in the expected cells, especially High-staining colon glands or bronchial respiratory epithelium (HPA: tissue IHC); dominant nuclear or diffuse background signal is suspect against that pattern (HPA: tissue IHC; UniProt P20674 location).
| Location and topology | COX5A is assigned to the mitochondrial inner membrane without an annotated transmembrane segment (UniProt P20674). Expect a cytoplasmic mitochondrial pattern at slide scale; topology does not justify cell-surface staining. |
| Tissue choice | Colon glandular cells and bronchial respiratory epithelial cells are High, while adipocytes are Not detected (HPA: tissue IHC). These provide a useful within-study contrast, subject to tissue preservation and assay controls (general IHC practice). |
| RNA versus protein pattern | RNA is tissue enhanced in heart muscle, skeletal muscle, and tongue, yet skeletal myocytes have Low IHC staining (HPA: tissue IHC). Do not use RNA enrichment alone to demand strong chromogenic signal in a given cell type. |
| Antibody evidence | The listed rabbit polyclonal HPA027526 has Supported IHC status, and the tissue profile has medium antibody–RNA consistency (HPA: antibodies; HPA: tissue IHC). Interpret unexpected patterns with controls rather than treating the reference image as universal. |
| Processing and epitope limits | UniProt annotates the mature COX5A chain as residues 42–150 and lists modified residues, but the supplied sources do not map the IHC antibody epitope (UniProt P20674; HPA: antibodies). They do not establish a retrieval requirement or fixation sensitivity. |
| IF/ICC Q&A: what should I expect? | A mitochondrial distribution is biologically plausible (UniProt P20674 location), but HPA provides no ICC-IF images or main-location call for COX5A (HPA: subcellular). Its IHC tissue pattern cannot serve as an IF validation result. |
| Situation | Likely cause | Next action |
|---|---|---|
| Colon glands expected to be positive are blank. | The reference cell type is High by HPA (HPA: tissue IHC); an empty section may reflect an assay or specimen issue (general IHC practice). | Confirm that glands are present and intact, review the run's positive control, then check antibody dilution, retrieval, and detection steps against the chosen assay instructions (general IHC practice). |
| Most of the section shows uniform brown background. | Uniform haze differs from the reported granular cytoplasmic pattern (HPA: tissue IHC). Nonspecific binding or excess chromogen development are general possibilities (general IHC practice). | Inspect a no-primary control, review blocking and washes, and assess development time (general IHC practice). Re-evaluate only the cell-associated granular signal after background is controlled. |
| Brown signal remains in a no-primary control. | Endogenous detection activity or a reagent-dependent background can produce chromogen without primary antibody (general IHC practice). | Check the detection system's blocking and control steps; for HRP-based detection, review the endogenous-peroxidase block (general IHC practice). Do not score control-matched deposits as COX5A. |
| Nuclei or cell borders stain more strongly than cytoplasm. | The distribution conflicts with HPA's granular cytoplasmic profile (HPA: tissue IHC) and UniProt's mitochondrial location (UniProt P20674). | Compare a positive tissue and no-primary control, review counterstain and morphology, and reassess specificity before scoring the signal (general IHC practice). |
| Adipocytes stain strongly and consistently. | Adipocytes are reported Not detected in the HPA tissue IHC profile (HPA: tissue IHC). Unexpected staining may reflect background or cross-reactivity (general IHC practice). | Compare adjacent expected-positive cells and a no-primary control; document the discrepancy rather than using adipocyte staining as the sole positive readout (HPA: tissue IHC; general IHC practice). |
| Skeletal myocytes look weak despite RNA enrichment. | HPA calls skeletal-muscle RNA tissue enhanced but skeletal myocyte IHC Low (HPA: tissue IHC). Weak IHC there is therefore compatible with the supplied observations. | Judge the run using morphology and a reported High IHC cell population, such as colon glandular cells, before changing assay conditions (HPA: tissue IHC; general 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 appendix first: glandular cells should stain (HPA: High in appendix glandular cells). Use adipose tissue as the negative comparator (HPA: Not detected in adipocytes); on the appendix slide, treat non-glandular cells as internal negatives only if they show background staining without granular cytoplasmic signal, since no internal-negative cell type is identified in the supplied HPA rows (HPA: appendix glandular-cell row).
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 appropriate to the primary’s clonality; and COX5A knockout material processed alongside the test section (caption: rabbit primary; standard IHC practice). For appendix DAB staining, block endogenous peroxidase and check background in inflammatory or blood-rich areas (standard IHC practice).
⚠️Feasibility: The selected-SKU caption reports paraffin-section IHC with heat retrieval in EDTA at pH 8.0, but its fixative is unreported; a COX5A-specific fixation window or fixation effect is also unreported (A07895-2 caption). That caption supports testing the stated retrieval condition but does not establish retrieval dependence; the supplied evidence does not establish whether frozen sections or IF are easier (A07895-2 caption; HPA subcellular: no ICC-IF images). In appendix, endogenous peroxidase in inflammatory or blood-rich areas can complicate DAB interpretation (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, cellular pattern, controls and scoring before interpreting differences in chromogenic signal.
Which retrieval conditions should I start with for COX5A staining in paraffin sections?
Start with heat-mediated antigen retrieval in EDTA buffer at pH 8.0 (datasheet A07895-2). The selected tissue image used that retrieval before overnight incubation at 4°C with antibody diluted 1:100 (caption A07895-2). If staining is weak, first check heating consistency, section adhesion and whether the positive control developed before changing retrieval conditions (general IHC practice). Compare any fallback buffer on adjacent sections with the same detection and exposure conditions, because a stronger DAB signal can also reflect increased background (general IHC practice). The caption does not report a retrieval duration (caption A07895-2).
How should I investigate weak COX5A staining when fixation varies between specimens?
Target-specific fixation sensitivity is unknown: the selected image identifies a paraffin-embedded section but does not state its fixative (caption A07895-2). Record the fixative, time before fixation and fixation duration for each specimen, then compare sections processed and stained together (general IHC practice). Include a tissue control that stained reliably in the same run, and inspect morphology for poor preservation before attributing low signal to COX5A abundance (general IHC practice). Keep retrieval, the 1:100 primary dilution and overnight 4°C incubation consistent during that comparison (caption A07895-2). Neither tissue staining patterns nor protein topology establish a COX5A-specific fixation effect.
What COX5A staining pattern is plausible in chromogenic tissue sections?
Expect a granular cytoplasmic pattern at light-microscope resolution (HPA: general cytoplasmic expression with a granular pattern), consistent with COX5A residing in the mitochondrial inner membrane (UniProt P20674: subcellular location). Evaluate granules within intact cells alongside tissue morphology; diffuse nuclear staining alone conflicts with that expected location (UniProt P20674: subcellular location; general IHC interpretation). Check a positive reference compartment such as colon glandular cells before calling a test section negative (HPA: High in colon glandular cells). Because DAB does not resolve individual mitochondrial membranes, describe the observed cytoplasmic pattern rather than claiming that each brown granule proves inner-membrane localisation (general IHC practice).
Could isoforms or epitope accessibility explain discordant COX5A staining?
The supplied record lists 0 isoforms and a processed chain spanning residues 42–150 (UniProt P20674: isoforms and processing). COX5A has no annotated transmembrane segment, while acetylated residues 87 and 113 and phosphorylated residue 141 are recorded (UniProt P20674: topology and modified residues). Without a mapped antibody epitope, these annotations cannot identify which modification or processing event affects staining (record and caption A07895-2: epitope not supplied). If two antibodies disagree, compare their documented epitopes when available and test both on adjacent sections under matched retrieval and detection conditions (general IHC practice). Avoid assigning an isoform-specific explanation from staining alone.
How should I assess COX5A by IF alongside the IHC result?
Treat IF/ICC as a separate validation question: the selected antibody evidence describes chromogenic staining of a paraffin section, with no IF/ICC result supplied (caption A07895-2). For multiplexing, pair COX5A with a marker identifying the expected cell population, such as a glandular-cell marker when assessing colon glands (HPA: High in colon glandular cells; general IF practice). Choose fluorophores after checking unstained tissue autofluorescence and reserve the cleaner channel for the weaker signal (general IF practice). COX5A resides in the mitochondrial inner membrane without an annotated transmembrane segment, but its antibody epitope and membrane-facing side are unspecified (UniProt P20674: location and topology; caption A07895-2). Validate permeabilisation against epitope accessibility rather than assuming which side it exposes (general IF practice).
How can I distinguish COX5A signal from nonspecific brown staining?
Include a no-primary control and inspect whether brown deposits persist after the primary antibody is omitted (general IHC practice). Block endogenous peroxidase before HRP detection, particularly when background appears across unrelated cell compartments; peroxidase blocking is a general workflow step, not evidence of COX5A localisation (general IHC practice). The selected image used goat-serum blocking, a peroxidase-conjugated secondary and DAB development (caption A07895-2). If background rises with signal, compare adjacent sections using the documented 1:100 primary dilution against a more dilute condition while holding detection time constant (caption A07895-2; general IHC practice). Judge residual staining against the expected granular cytoplasmic pattern (HPA: staining profile).
What should I score when comparing COX5A IHC across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell compartment and region before scoring, then record the percentage of positive cells and staining intensity to calculate an H-score (general IHC practice). An H-score sums each intensity category’s percentage multiplied by its category value, yielding 0–300 when intensity runs from 0–3 (general IHC practice). Normalise comparisons to viable cells of the same type, sampled area and staining batch; record density per mm² when cell counts per area are more useful (general IHC practice). Score glandular cells separately from surrounding tissue when using a colon reference, because HPA reports High staining specifically in colon glandular cells (HPA: colon). Exclude necrotic and folded regions before comparing scores (general IHC practice).
When is apparent COX5A positivity more likely to be artefact?
A credible positive result shows cytoplasmic granularity in preserved cells, matching the reported tissue pattern and mitochondrial location (HPA: staining profile; UniProt P20674: subcellular location). Treat isolated nuclear staining, staining confined to section edges, or dense deposits over necrosis as suspect until controls and morphology support them (general IHC interpretation). Check a no-primary control for endogenous enzyme signal and nonspecific detection, and compare a known positive compartment such as appendix glandular cells (general IHC practice; HPA: High in appendix glandular cells). Cell identity matters: HPA reports no detected staining in adipocytes, so a strong adipocyte signal deserves closer review (HPA: Not detected in adipocytes). Chromogenic intensity alone cannot establish respiratory activity (UniProt P20674: complex IV component; general IHC interpretation).
Boster reagents
Best COX5A / Cytochrome c oxidase subunit 5A, mitochondrial IHC Antibodies
Both antibodies have human paraffin-section IHC images (IHC captions). A07895-2 also lists IF/ICC, without a supplied IF image (catalog applications; IF image list). Both list Human, Mouse, and Rat reactivity (catalog reactivity).
Real IHC data
Anti-COX5A Antibody
Cat # A07895-2
Real IHC data
Anti-COX5A Antibody
Cat # A07895-1
A07895-2 will render with an IHC image from human breast cancer tissue in a paraffin section; its other supplied IHC captions cover human colon and colon cancer tissue (A07895-2 IHC captions). A07895-1 will render with an IHC image from paraffin-embedded human colon carcinoma tissue (A07895-1 IHC caption).
Which to pick: For paraffin-section IHC, either SKU has its own human tissue image; A07895-2 documents EDTA retrieval and overnight primary incubation, while A07895-1 shows a primary-omission control (respective IHC captions). Choose A07895-2 for IF/ICC because those applications are listed for it; no IF image is supplied (A07895-2 catalog applications; IF image list). For Mouse or Rat samples, both list reactivity, but the supplied tissue images are human only; fixation is unreported for both (catalog reactivity; respective IHC captions).
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); inner mitochondrial 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 paraffin-section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A07895-2); Caveat: Skeletal myocytes show low staining despite muscle-enhanced RNA (HPA tissue IHC); Isoform / epitope: No isoforms annotated; mature chain spans 42–150 (UniProt); Regulation: Muscle-enhanced RNA expression (HPA tissue RNA). 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.