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OPA1 / Dynamin-like GTPase OPA1, mitochondrial · IHC design guide
Design Immunohistochemistry for OPA1
Plan chromogenic OPA1 IHC in paraffin sections around the granular cytoplasmic tissue pattern (HPA tissue IHC). Kidney tubules stain highly, while adipocytes are undetected (HPA tissue IHC); OPA1 processing makes antibody epitope position relevant (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
OPA1 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 in tissue sections (HPA tissue IHC); inner mitochondrial membrane at the molecular level (UniProt) | |
| Staining pattern | Granular cytoplasmic staining in most tissues; high in kidney tubules (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB9731) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation conditions consistent between sections. (standard IHC practice; not target-specific) | |
| Caveat | An unknown epitope may favor a long or short processed form (UniProt) | |
| Regulation | Loss of membrane potential drives OMA1 cleavage (UniProt) | |
| Isoform / epitope | Six isoforms; long and short forms make epitope position relevant (UniProt) |
Section 1
Recommended OPA1 IHC & IF Protocols
The catalog antibody’s IHC-P protocol uses EDTA retrieval (datasheet PB9731). Published IHC protocols cover heart tissue (PMC9110818) and an ischemic brain injury study (PMC5555107).
| Sample | Paraffin-embedded Mouse Liver tissue; fixative not specified (datasheet PB9731) |
| Fixation | Image fixative and duration unreported (datasheet PB9731); 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 PB9731); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9731) |
| Primary antibody | Rabbit anti-OPA1, 0.5-1μg/ml (datasheet PB9731) |
| Primary incubation | Overnight at 4 °C (datasheet PB9731) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9731) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | OPA1-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression with a granular pattern in most tissues. No signal in the no-primary control. |
💡Decision noteTry heat-mediated EDTA pH 8.0 first (datasheet PB9731). Citrate pH 6.0 is a published alternative (PMC5555107).
Section 2
What Is the Expected OPA1 Staining Pattern?
OPA1 is associated with the mitochondrial inner membrane and intermembrane space (UniProt O60313 topology). In paraffin section IHC, expect granular cytoplasmic staining in many cell types, including strong staining in cardiomyocytes and kidney tubule cells (HPA tissue IHC: High). HPA rates the tissue staining profile Supported, with medium consistency between antibody staining and RNA expression (HPA tissue IHC: reliability).
| Granular cytoplasmic staining in cardiomyocytes or kidney tubule cells, with preserved cell boundaries. | This matches the reported tissue pattern and high staining levels in those cells (HPA tissue IHC: granular cytoplasm; High in cardiomyocytes and kidney tubules). The mitochondrial location supports a granular cytoplasmic interpretation (UniProt O60313 topology). Judge the pattern in identifiable cells, rather than from overall slide darkness (general IHC practice). |
| Predominantly nuclear IHC staining, with little or no granular cytoplasmic signal. | This is discordant with the reported tissue IHC pattern and warrants an artefact check (HPA tissue IHC: granular cytoplasm). It is not an absolute exclusion of OPA1: HPA reports nucleoplasm as an approved ICC-IF location, alongside supported mitochondrial localization (HPA subcellular ICC-IF). Interpret the nuclear result in the context of the assay used. |
| Strong staining in adipocytes while a known high-staining cell population is weak. | HPA reports OPA1 as not detected in adipocytes, but High in several glandular cell populations, cardiomyocytes and kidney tubule cells (HPA tissue IHC). This contrast raises concern for cross-reactivity or endogenous detection activity (general IHC practice). A single unexpected cell population cannot establish the cause; compare cell morphology and detection controls (general IHC practice). |
| Broad, smooth color over cells and surrounding tissue that obscures intracellular granules. | The distribution does not resolve the granular cytoplasmic profile reported for tissue IHC (HPA tissue IHC: profile). Diffuse deposition can arise from nonspecific antibody binding or detection background (general IHC practice). Assess whether a no-primary control carries the same color and whether recognizable high-staining cells retain a distinct pattern (general IHC practice). |
| No visible signal in cardiomyocytes or kidney tubule cells. | These are reported as High by HPA, so a blank result calls for a technical check before concluding that OPA1 is absent (HPA tissue IHC: High). Confirm tissue preservation and the detection run, then review the antibody's documented IHC conditions (general IHC practice). HPA's Supported rating does not guarantee staining in every preparation (HPA tissue IHC: reliability). |
💡Expected OPA1 appearanceA convincing IHC positive shows distinct granular cytoplasmic color in identifiable cardiomyocytes or kidney tubule cells, which HPA rates High; smooth background or nuclear-only color without that pattern is questionable (HPA tissue IHC: profile and levels; UniProt O60313 topology).
| Subcellular location and epitope context | OPA1 occupies the mitochondrial inner membrane and intermembrane space; its recorded transmembrane segment spans residues 97–113 (UniProt O60313 topology). These facts support a mitochondrial interpretation of granular cytoplasm, but they do not identify the catalog antibody's epitope or establish which retrieval condition will work. |
| Cell population and tissue context | HPA describes granular cytoplasmic expression in most tissues and low tissue RNA specificity, while individual cell populations range from High to Not detected (HPA tissue IHC). Score the relevant cells: cardiomyocytes are High, skeletal myocytes Low, and adipocytes Not detected in the supplied profile (HPA tissue IHC). |
| Long and short OPA1 forms | OPA1 has a membrane-bound long form and a soluble short form produced by proteolytic cleavage; both relate to mitochondrial inner membrane biology (UniProt O60313 processing and function). Six isoforms are listed (UniProt O60313). Without an antibody epitope or form-specific validation, IHC color cannot assign a particular form or cleavage state. |
| Strength of staining evidence | The HPA tissue profile is Supported, with medium consistency against RNA expression, and both listed antibodies have Supported IHC status (HPA tissue IHC; HPA antibodies: HPA036926 and HPA036927). Treat the reported cell pattern as a useful reference while checking each staining run against its own controls (general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known high-staining cells are blank. | The result conflicts with HPA's High calls for cardiomyocytes and kidney tubule cells; the cause is undetermined (HPA tissue IHC). | Check tissue integrity, staining-run controls, detection reagents and the catalog antibody's documented IHC conditions (general IHC practice). Do not infer OPA1 absence from a failed control. |
| The whole section has diffuse color. | Nonspecific binding or detection background may obscure the reported granular cytoplasmic pattern (general IHC practice; HPA tissue IHC: profile). | Review a no-primary control, blocking, antibody dilution and washes; compare whether high-staining cells retain discrete granules (general IHC practice; HPA tissue IHC: High cell populations). |
| Nuclei dominate the IHC signal. | Nuclear-only IHC is discordant with HPA's granular cytoplasmic tissue profile, although HPA ICC-IF also reports nucleoplasm (HPA tissue IHC; HPA subcellular ICC-IF). | Inspect the counterstain and no-primary control, then compare cytoplasmic signal in a reported high-staining cell population (general IHC practice; HPA tissue IHC). |
| Adipocytes stain strongly. | That differs from HPA's Not detected call for adipocytes; cross-reactivity or endogenous detection activity is possible (HPA tissue IHC; general IHC practice). | Check cell identification and a no-primary control. Review detection blocking before interpreting this signal as OPA1 (general IHC practice). |
| Signal is unexpectedly weak after retrieval. | The supplied HPA and UniProt records do not establish OPA1-specific retrieval requirements or fixation sensitivity. | Use the catalog antibody's documented IHC conditions as the starting point; compare controlled retrieval changes only if needed (general IHC practice). |
| How should IF/ICC nuclear and mitochondrial staining be interpreted? | HPA reports nucleoplasm as approved and mitochondria as supported in ICC-IF images (HPA subcellular ICC-IF). | Interpret that observation within the IF/ICC assay and its controls; use the granular cytoplasmic tissue profile when judging paraffin IHC (HPA subcellular ICC-IF; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing OPA1 staining
PB9731 · IHCIHC analysis of OPA1 using anti-OPA1 antibody (PB9731). OPA1 was detected in paraffin-embedded section of Mouse Liver Tissue. Heat mediated
PB9731 · IHCIHC analysis of OPA1 using anti-OPA1 antibody (PB9731). OPA1 was detected in paraffin-embedded section of Rat Kidney Tissue. Heat mediated a
PB9731 · IFIF analysis of OPA1 using anti-OPA1 antibody (PB9731). OPA1 was detected in immunocytochemical section of U20S cells. Enzyme antigen retriev
A00508-1 · IFIF analysis of OPA1 using anti-OPA1 antibody (A00508-1). OPA1 was detected in an immunocytochemical section of U2OS cells. Enzyme antigen re
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000198836-OPA1/tissue/cerebral+cortex#imid_8978114
HPA · Salivary glandHuman Protein Atlas IHC, Salivary gland. Source: https://www.proteinatlas.org/ENSG00000198836-OPA1/tissue/salivary+gland#imid_8978062
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000198836-OPA1/tissue/colon#imid_8958080
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000198836-OPA1/tissue/liver#imid_8958081
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA036927; green = OPA1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA036927; green = OPA1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for OPA1 IHC & IF
🧪Run heart muscle first: cardiomyocytes should stain (HPA: High in cardiomyocytes). Use adipose tissue as the negative tissue; adipocytes are not detected (HPA: Not detected in adipocytes), and mature erythrocytes within the heart section, if present, should lack specific OPA1 staining because they lack mitochondria (standard IHC practice).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show OPA1 in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (approved), Mitochondria (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a nonimmune IgG control matched to the primary antibody’s host species and isotype; assess specificity in matched OPA1 knockout material or by immunizing-peptide competition if available (standard IHC practice). For heart sections, quench endogenous peroxidase and check endogenous biotin background because the selected protocol uses biotin-based DAB detection (PB9731 caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the fixative for the selected paraffin-section example is unreported (PB9731 caption: fixative not stated). That example uses heat retrieval in EDTA, pH 8.0, but does not establish that retrieval is required or optimal (PB9731 caption: EDTA retrieval). The supplied evidence does not establish whether frozen sections or IF are easier; in heart tissue, account for endogenous biotin in chromogenic IHC and lipofuscin autofluorescence if evaluating IF (standard IHC/IF practice).
HPA tissue IHC evidence for OPA1
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 → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
| Ovary | Follicle cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced OPA1 IHC Tips
Troubleshoot chromogenic OPA1 staining in paraffin sections by checking retrieval, compartmental pattern, and controls alongside staining intensity (datasheet PB9731; HPA tissue IHC).
How should I retrieve OPA1 when staining is weak in paraffin sections?
Start with heat-mediated retrieval in EDTA pH 8.0 for the catalog antibody, matching its tissue-section example (datasheet PB9731). Keep heating and cooling conditions consistent across sections, then compare staining with a known positive tissue and a no-primary control (standard IHC practice; HPA: high in heart cardiomyocytes). The example used 1 μg/ml primary antibody overnight at 4°C, so check those conditions before changing retrieval chemistry (datasheet PB9731). If staining remains weak, test a different retrieval buffer only on matched sections, because altered retrieval can also increase background or damage morphology (standard IHC practice).
Could fixation explain uneven OPA1 staining across my paraffin sections?
Target-specific fixation sensitivity is unknown: the catalog tissue-section caption does not state a fixative, so its staining cannot establish how OPA1 responds to fixation (datasheet PB9731). Record the fixation method and duration for each specimen, and compare sections processed together before attributing uneven signal to biology (standard IHC practice). Check tissue preservation, section adhesion, and retrieval uniformity when staining varies across a slide, especially near folded or damaged areas (standard IHC practice). Use the same positive control and EDTA pH 8.0 retrieval in each run to separate processing variation from changes in OPA1 staining (datasheet PB9731; standard IHC practice).
What staining pattern is credible for OPA1 in tissue sections?
Expect predominantly granular cytoplasmic staining in chromogenic sections, consistent with the tissue profile and OPA1’s mitochondrial inner-membrane and intermembrane-space locations (HPA tissue IHC; UniProt O60313 localisation). Examine signal within the expected cell population rather than treating uniform brown color across every compartment as specific (standard IHC practice). HPA reports high staining in heart cardiomyocytes and kidney tubule cells, which can serve as reference patterns when those tissues are available (HPA tissue IHC). HPA also reports nucleoplasmic signal in cell-based imaging, so investigate a nuclear pattern with mitochondrial colocalisation or another specificity control before accepting or rejecting it in tissue IHC (HPA subcellular; standard IHC practice).
Can this stain distinguish long and short OPA1 isoforms?
Do not assign a chromogenic signal to one OPA1 form without knowing the antibody’s epitope and testing form-specific recognition (UniProt O60313 processing; standard IHC practice). The long chain spans residues 88–960, whereas the short chain spans 195–960; an epitope in their shared sequence would not distinguish them by tissue staining alone (UniProt O60313 processing). The long form is membrane tethered and the short form resides in the intermembrane space, but both can contribute to a granular cytoplasmic pattern (UniProt O60313 function; HPA tissue IHC). If processing is the research question, pair IHC with an independently validated assay that resolves OPA1 forms (standard IHC practice).
How can IF help investigate an ambiguous OPA1 IHC pattern?
Use IF as a separate validation experiment and multiplex OPA1 with a marker for the expected cell population, plus a mitochondrial marker to assess compartmental overlap (HPA tissue IHC; UniProt O60313 localisation; standard IF practice). Choose spectrally separated fluorophores and inspect unstained tissue in each channel, because tissue autofluorescence can obscure a granular signal (standard IF practice). OPA1 has an inner-membrane segment at residues 97–113 and extensive intermembrane-space sequence, but the catalog antibody’s epitope is unspecified (UniProt O60313 topology; datasheet PB9731). Optimise permeabilisation for access to the verified epitope’s side of the membrane, while checking that mitochondrial morphology remains interpretable (UniProt O60313 topology; standard IF practice).
What should I check when DAB obscures granular OPA1 staining?
Run a no-primary control to assess detection-system background, and inspect whether brown signal tracks tissue edges, damaged regions, or endogenous pigment rather than cells (standard IHC practice). The catalog example used 10% goat serum blocking, biotinylated secondary antibody, an avidin-biotin complex, and DAB, so evaluate each relevant reagent step when reproducing that workflow (datasheet PB9731). Include an endogenous peroxidase block and consider endogenous biotin when using this detection system (standard IHC practice). If controls are clean but staining is diffuse, compare matched sections while adjusting antibody concentration or wash stringency, preserving the documented EDTA pH 8.0 retrieval as the starting condition (datasheet PB9731; standard IHC practice).
How should I quantify OPA1 staining across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue area before scoring, then record either percent positive cells and intensity as an H-score or the density of positive cells per mm² (standard IHC practice). Apply one threshold and the same acquisition settings to every section, using a no-primary control to help set the background boundary (standard IHC practice). Normalise positive-cell counts to the number of eligible cells, or staining density to evaluable tissue area, and exclude folds and necrotic regions by a preset rule (standard IHC practice). Report intensity and distribution separately, because the expected granular cytoplasmic pattern can change without a simple change in positive-cell fraction (HPA tissue IHC; standard IHC practice).
How do I distinguish genuine OPA1 signal from artefact?
Prioritise staining with a granular cytoplasmic pattern in intact cells, consistent with OPA1’s mitochondrial location and the reported tissue-IHC profile (UniProt O60313 localisation; HPA tissue IHC). Compare cell identity with reference patterns such as high staining in heart cardiomyocytes or kidney tubule cells, while recognising that OPA1 is expressed across tissues (HPA tissue IHC; UniProt O60313 tissue specificity). Edge-only staining, necrotic-area signal, or color reproduced in the no-primary control warrants an artefact check; endogenous peroxidase can also generate chromogenic signal (standard IHC practice). Evaluate isolated nuclear staining cautiously because cell-based imaging reports nucleoplasmic OPA1, but that observation alone does not validate nuclear staining in paraffin tissue (HPA subcellular; standard IHC practice).
Boster reagents
Best OPA1 / Dynamin-like GTPase OPA1, mitochondrial IHC Antibodies
Anti-OPA1 antibodies have IHC images from paraffin sections of mouse liver, rat kidney and human lung cancer tissue (PB9731 captions), plus an IF image from U2OS cells (A00508-1 caption).
Real IHC data
Anti-OPA1 Antibody ®
Cat # PB9731
Real IF data
Anti-OPA1 Antibody ®
Cat # A00508-1
PB9731 will render with its own mouse liver paraffin-section IHC image; its other IHC captions show rat kidney and human lung cancer tissue (PB9731 IHC captions). A00508-1 will render with its own U2OS cell IF image (A00508-1 IF caption).
Which to pick: Choose PB9731 for tissue IHC: its application list includes IHC, and its captions document paraffin sections across human, mouse and rat samples (PB9731 applications and IHC captions). Choose A00508-1 for IF/ICC when its U2OS cell example fits the experiment (A00508-1 applications and IF caption); PB9731 is the better documented cross-species IHC choice because it lists human, mouse and rat reactivity and has an IHC caption for each species (PB9731 reactivity and IHC captions). The PB9731 captions report paraffin embedding but do not report the fixative (PB9731 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O60313 (OPA1_HUMAN, Dynamin-like GTPase OPA1, mitochondrial).
- Human Protein Atlas. OPA1 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. OPA1 subcellular location (ICC-IF): Mainly localized to the nucleoplasm and mitochondria..
- Human Protein Atlas. OPA1 antibody validation summary (2 antibodies).
- Fusion or Fission: The Destiny of Mitochondria In Traumatic Brain Injury of Different Severities. Scientific reports 2017 — PMC5569027.
- SS31 Alleviates Pressure Overload-Induced Heart Failure Caused by Sirt3-Mediated Mitochondrial Fusion. Frontiers in cardiovascular medicine 2022 — PMC9110818.
- Lycium Barbarum Polysaccharides Decrease Hyperglycemia-Aggravated Ischemic Brain Injury through Maintaining Mitochondrial Fission and Fusion Balance. International journal of biological sciences 2017 — PMC5555107.
- Mitochondrial role in adaptive response to stress conditions in preeclampsia. Scientific reports 2016 — PMC5004102.
- PubMed PMID:9628581 — UniProt-cited evidence.
- PubMed PMID:20843780 — UniProt-cited evidence.
- PubMed PMID:16641997 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for OPA1 (Dynamin-like GTPase OPA1, mitochondrial). Expected localisation Granular cytoplasm in tissue sections (HPA tissue IHC); inner mitochondrial membrane at the molecular level (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9731). Validated IHC antibody: PB9731 at 0.5-1μg/ml (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 Mouse Liver tissue; fixative not specified (datasheet PB9731); 2. Fixation: Image fixative and duration unreported (datasheet PB9731); 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 PB9731); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9731); 8. Primary antibody: Rabbit anti-OPA1, 0.5-1μg/ml (datasheet PB9731); 9. Primary incubation: Overnight at 4 °C (datasheet PB9731). Watch for: Fixation: Keep fixation conditions consistent between sections. (standard IHC practice; not target-specific); Caveat: An unknown epitope may favor a long or short processed form (UniProt); Isoform / epitope: Six isoforms; long and short forms make epitope position relevant (UniProt); Regulation: Loss of membrane potential drives OMA1 cleavage (UniProt). Evidence: UniProt O60313, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the nucleoplasm and mitochondria., Datasheet PB9731, PMC9110818, PMC5555107. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/OPA1/immunohistochemistry-ihc-guide.