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ATP5MF / ATP synthase F(0) complex subunit f, mitochondrial · IHC design guide
Design Immunohistochemistry for ATP5MF
Plan ATP5MF paraffin IHC around cytoplasmic staining in most tissues (HPA tissue IHC). The catalog antibody is shown staining paraffin-embedded human lung carcinoma at 1:100 (catalog IHC image caption).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ATP5MF 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 | Cytoplasmic staining (HPA tissue IHC); inner mitochondrial membrane (UniProt) | |
| Staining pattern | Cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A32262) | |
| Caveat | Some cell types lack staining despite the broad tissue profile (HPA tissue IHC) | |
| Regulation | No specific expression regulator reported (UniProt) | |
| Isoform / epitope | 4 isoforms; epitope coverage is not given in the record (UniProt) |
Section 1
Recommended ATP5MF IHC & IF Protocols
The catalog antibody IHC-P protocol (datasheet) is accompanied by a published ATP5MF IHC protocol using clinical breast cancer tissue sections (PMC11472516).
| Sample | Paraffin-embedded human lung carcinoma tissue; fixative not specified (datasheet A32262) |
| Fixation | Image fixative and duration unreported (datasheet A32262); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-ATP5MF, 1:50-1:200 (datasheet A32262) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ATP5MF-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval rule); the article reports retrieval without specifying conditions (PMC11472516).
Section 2
What Is the Expected ATP5MF Staining Pattern?
ATP5MF is a mitochondrial inner-membrane subunit with 1 transmembrane segment at residues 68–85 (UniProt P56134 topology). In paraffin-section IHC, expect cytoplasmic staining in many tissues, especially kidney tubule cells and glandular cells of the adrenal, breast, parathyroid, rectum, salivary gland and stomach, where HPA reports High staining (HPA tissue IHC). HPA rates its tissue profile Approved, with medium consistency against RNA data and external verification pending (HPA tissue IHC).
| Cytoplasmic staining in kidney tubule cells or one of the listed high-staining glandular cell populations (HPA tissue IHC). | This matches the reported IHC distribution (HPA tissue IHC). A granular cytoplasmic appearance is compatible with mitochondrial localisation (UniProt P56134; HPA subcellular), but staining alone does not prove organelle identity (standard IHC practice). |
| Predominantly nuclear or cell-surface chromogen in an otherwise positive tissue (HPA tissue IHC). | This conflicts with the expected mitochondrial inner-membrane location (UniProt P56134). Assess specificity and detection background before interpreting it as ATP5MF (standard IHC practice). HPA's additional nuclear-membrane IF location is uncertain and does not establish a nuclear IHC pattern (HPA subcellular). |
| Strong staining of a cell population reported as Not detected, such as adipocytes or lymph-node germinal-center cells (HPA tissue IHC). | The discrepancy warrants checking cross-reactivity and endogenous detection activity (standard IHC practice). HPA's Not detected category describes its observed specimens; it does not prove that every specimen of that cell type must be negative (HPA tissue IHC). |
| Diffuse chromogen across tissue and surrounding spaces, without a clear cellular pattern (standard IHC practice). | This is difficult to score as ATP5MF: the reported tissue signal is cytoplasmic and the protein is mitochondrial (HPA tissue IHC; UniProt P56134). Review blocking, washing and detection controls for nonspecific background (standard IHC practice). |
| No discernible staining in kidney tubules or the listed high-staining glandular cells (HPA tissue IHC). | An absent signal in these reference-positive populations calls for an assay check, including section quality, retrieval, primary-antibody conditions and detection controls (standard IHC practice). HPA's Approved rating has only medium RNA concordance and awaits external verification (HPA tissue IHC). |
💡Expected ATP5MF appearanceCall positive when cytoplasmic staining is clear in kidney tubule cells or listed High glandular populations (HPA tissue IHC), with a mitochondrial-compatible distribution (UniProt P56134); isolated nuclear, cell-surface or diffuse chromogen is suspect (standard IHC practice).
| Compartment and topology | ATP5MF resides in the mitochondrial inner membrane and has 1 transmembrane segment at residues 68–85 (UniProt P56134). This supports cytoplasmic, mitochondrial-compatible interpretation; the supplied record does not locate the antibody epitope or establish a retrieval condition. |
| Tissue and cell context | HPA reports cytoplasmic expression in most tissues, but levels differ by cell population (HPA tissue IHC). Its High kidney-tubule and listed glandular observations are useful comparators; its Not detected calls are observed results, not universal exclusions (HPA tissue IHC). |
| Antibody evidence | HPA070412 has Approved IHC status; HPA067267 has no supplied IHC status (HPA antibodies). The tissue profile itself has medium antibody–RNA consistency and pending external verification (HPA tissue IHC). Interpret unexpected staining with that evidence limit in mind. |
| IF/ICC Q: What localisation should a separate IF/ICC guide expect? | A: Mainly mitochondria; HPA marks this location supported (HPA subcellular). Its additional nuclear-membrane location is uncertain, so treat that signal cautiously (HPA subcellular). Both listed antibodies have Supported ICC status (HPA antibodies). |
| Protein forms | UniProt lists 4 isoforms and a chain spanning residues 2–94 (UniProt P56134). These annotations do not identify which forms a given IHC antibody detects; avoid assigning cell-level intensity differences to isoforms without antibody-specific evidence. |
| Situation | Likely cause | Next action |
|---|---|---|
| A high-staining reference population gives little or no chromogen (HPA tissue IHC). | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. | Check section integrity, retrieval, primary-antibody conditions and detection with appropriate controls, then reassess the reference population (standard IHC practice). |
| Nuclear staining dominates the section (standard IHC practice). | Nuclear staining conflicts with the mitochondrial inner-membrane assignment (UniProt P56134). HPA labels its additional nuclear-membrane IF finding uncertain (HPA subcellular). | Compare the same run with positive tissue and detection controls; score nuclear-only chromogen separately from the expected cytoplasmic signal (HPA tissue IHC; standard IHC practice). |
| Chromogen appears in HPA Not detected cells, such as adipocytes (HPA tissue IHC). | Cross-reactivity or endogenous detection activity may contribute (standard IHC practice); HPA's category is specimen-based evidence (HPA tissue IHC). | Review the primary-omission and detection controls, then compare cell identity and staining distribution with HPA's observed pattern (standard IHC practice; HPA tissue IHC). |
| Background obscures cell boundaries or intracellular detail (standard IHC practice). | Nonspecific antibody binding or detection background can impair interpretation (standard IHC practice). | Review blocking, washing and primary-antibody conditions using matched controls; interpret ATP5MF only when cellular signal separates from background (standard IHC practice). |
| Signal is present, but mostly on cell surfaces (standard IHC practice). | A surface-dominant pattern does not fit an inner-mitochondrial-membrane protein (UniProt P56134). | Check detection controls and antibody specificity, and compare with the cytoplasmic pattern reported in tissue IHC (standard IHC practice; HPA tissue IHC). |
| Two samples differ markedly in staining intensity (standard IHC practice). | HPA reports variable cell-population levels, from High to Not detected, while its tissue profile has medium RNA concordance (HPA tissue IHC). | Compare like cell populations under matched staining and scoring conditions; document any departure from HPA's observations without treating them as absolute expression limits (standard IHC practice; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing ATP5MF staining
A32262 · IHCImmunohistochemistry (IHC) analyzes of ATP5J2 (G42) pAb in paraffin-embedded human lung carcinoma tissue at 1:100.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000241468-ATP5MF/tissue/cerebral+cortex#imid_19956317
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000241468-ATP5MF/tissue/colon#imid_19956243
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000241468-ATP5MF/tissue/liver#imid_19956252
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000241468-ATP5MF/tissue/kidney#imid_19956237
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA067267; green = ATP5MF, blue = nucleus, red = microtubules.
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA067267; green = ATP5MF, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ATP5MF IHC & IF
🧪Run kidney first: cells in tubules should stain (HPA: High in kidney tubules). Use oral mucosa squamous epithelial cells as the negative tissue (HPA: Not detected); on the kidney slide, treat cells outside tubules as internal negatives only if they remain at background in the observed section, since their ATP5MF staining status is not specified by the supplied HPA row.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ATP5MF in HEK293, MCF-7, U2OS, HeLa, Sperm, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host- and immunoglobulin-matched isotype or nonimmune IgG controls, selected according to the primary antibody’s clonality; use ATP5MF knockout material, if available, as a biological negative (standard IHC practice). For chromogenic kidney IHC, quench endogenous peroxidase and check for endogenous biotin if using avidin–biotin detection (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and the effect of antigen retrieval are unreported in the supplied evidence; optimize retrieval empirically for paraffin sections. The selected catalog antibody caption shows paraffin-section IHC at 1:100, but its fixative is unreported (selected-SKU tissue-IHC caption: A32262). The supplied evidence does not establish that frozen sections or IF are easier; kidney tubules may give background from endogenous biotin with avidin–biotin detection (standard IHC practice).
HPA tissue IHC evidence for ATP5MF
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Pending external verification.). 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 → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | High | Protein (IHC) | HPA → |
| Rectum | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Placenta | Decidual cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ATP5MF IHC Tips
Troubleshoot ATP5MF chromogenic staining in paraffin sections using mitochondrial localisation, tissue IHC patterns, and the selected antibody’s caption as reference points (UniProt P56134; HPA tissue IHC; A32262 caption).
How should I retrieve ATP5MF in paraffin sections when staining is weak?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting). Cool sections consistently, then compare retrieved and unretrieved sections at the same antibody dilution to judge whether retrieval improves specific staining (standard IHC practice). Use 1:100 as a documented starting dilution for A32262 in paraffin-embedded human lung carcinoma tissue, while recognising that its caption does not report retrieval conditions (A32262 caption). Look for cytoplasmic staining consistent with mitochondria, rather than treating stronger staining in every compartment as improvement (HPA tissue IHC; UniProt P56134 localisation). If signal remains weak, vary retrieval duration cautiously and check tissue preservation alongside staining intensity (standard IHC practice).
Could fixation explain weak ATP5MF staining in my paraffin sections?
The selected A32262 image describes paraffin-embedded human lung carcinoma tissue but does not state its fixative (A32262 caption). Target-specific ATP5MF sensitivity to fixative type or fixation duration is therefore unknown from the supplied evidence; assess it experimentally without assigning a cause in advance. Record fixative, fixation duration, processing schedule, and section age for each specimen, and compare matched sections processed under documented conditions (standard IHC practice). Keep the retrieval setting at citrate pH 6.0, 95–98 °C for 20 min while assessing fixation as a variable (page retrieval setting). Judge changes by cytoplasmic staining, preserved morphology, and matched negative controls, since intensity alone cannot establish specificity (HPA tissue IHC; standard IHC practice).
Where should convincing ATP5MF staining appear in chromogenic IHC?
Expect predominantly cytoplasmic staining at the light-microscopy level: ATP5MF is assigned to the mitochondrial inner membrane, and HPA reports cytoplasmic expression in most tissues (UniProt P56134 localisation; HPA tissue IHC). Its annotated transmembrane segment spans residues 68–85, supporting interpretation as a membrane-associated mitochondrial protein rather than a soluble nuclear antigen (UniProt P56134 topology). Compare stained cells with adjacent morphology and a matched negative control before calling fine cytoplasmic signal positive (standard IHC practice). HPA subcellular data also list nuclear membrane localisation as uncertain, so an isolated nuclear rim should prompt validation rather than automatic acceptance (HPA subcellular). Document whether staining is diffuse, granular, or confined to tissue edges, and retain the same criteria across samples (standard IHC practice).
How do isoforms and epitope position affect interpretation of ATP5MF IHC?
ATP5MF has 4 annotated isoforms, but the supplied A32262 caption does not identify its epitope or establish isoform coverage (UniProt P56134 isoforms; A32262 caption). Check the antibody’s documented immunogen or epitope before making isoform-specific claims, and avoid interpreting a negative section as loss of every isoform (standard IHC practice). The protein has one transmembrane segment at residues 68–85 and annotated modifications at residues 2, 3, and 22 (UniProt P56134 topology and modified residues). Those annotations alone do not show whether fixation or retrieval masks this antibody’s epitope. Compare any revised staining condition with the same control tissue, dilution, detection chemistry, and scoring criteria (standard IHC practice).
How can IF help investigate an ambiguous ATP5MF IHC pattern?
Use IF/ICC as a separate validation experiment, since the selected A32262 evidence is a paraffin-section IHC image at 1:100 (A32262 caption). Multiplex ATP5MF with a mitochondrial marker and a marker defining the expected cell type, then ask whether signals coincide within the same cells (UniProt P56134 localisation; standard IF practice). Select fluorophores in channels with low measured tissue autofluorescence and include single-stain and no-primary controls for spectral spillover and background (standard IF practice). Choose and titrate permeabilisation after identifying which side of the inner membrane contains the antibody epitope; that orientation is not provided here (UniProt P56134 topology; standard IF practice). Keep conclusions from IF distinct from chromogenic IHC scoring unless both methods agree on cell distribution (standard IHC/IF practice).
What should I check when ATP5MF IHC shows diffuse brown background?
Inspect no-primary and detection-only controls to distinguish antibody-dependent staining from endogenous peroxidase, nonspecific detection, or pigment (standard chromogenic IHC practice). Apply a peroxidase block before DAB detection and verify that the block reduces control staining without obscuring tissue morphology (standard chromogenic IHC practice). Titrate the antibody around the documented 1:100 use in paraffin-embedded human lung carcinoma tissue, keeping incubation and development times constant (A32262 caption; standard IHC practice). Reassess blocking, washes, and DAB development if colour coats stromal spaces or section edges instead of identifiable cells (standard IHC practice). Evaluate remaining signal against ATP5MF’s expected cytoplasmic mitochondrial distribution and matched tissue controls (HPA tissue IHC; UniProt P56134 localisation).
How should I quantify ATP5MF staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and regions before examining outcomes, and exclude folds, necrosis, and tissue edges consistently (standard IHC practice). For cellular staining, record the percentage of positive cells and an H-score from intensity categories; use positive-cell density per mm² when cell abundance itself matters (standard IHC quantification practice). Normalise counts to viable tissue area or the number of eligible cells, and apply identical thresholds, counterstaining, and DAB development across cases (standard IHC practice). Score cytoplasmic signal separately from nuclear or extracellular colour because mitochondrial localisation is the principal expectation for ATP5MF (UniProt P56134 localisation; HPA tissue IHC). Include a documented control section in each staining run to monitor variation between runs (standard IHC practice).
When is an ATP5MF-positive IHC result likely to be artefactual?
Treat reproducible cytoplasmic staining in intact cells as more plausible than isolated nuclear, extracellular, or edge-restricted colour, given ATP5MF’s mitochondrial localisation (UniProt P56134 localisation; standard IHC practice). Check whether the labelled cell population matches the section: HPA reports high staining in kidney tubule cells but no detected staining in adipocytes (HPA tissue IHC). Avoid calling brown deposits in necrotic areas positive, and compare them with no-primary and peroxidase-block controls before attributing colour to ATP5MF (standard chromogenic IHC practice). HPA’s tissue antibody reliability is Approved with medium agreement between staining and RNA data, pending external verification (HPA tissue IHC). Interpret discordant patterns cautiously and seek an independent antibody or orthogonal assay when the conclusion depends on specificity (standard IHC practice).
Boster reagents
Best ATP5MF / ATP synthase F(0) complex subunit f, mitochondrial IHC Antibodies
The IHC-validated ATP5MF antibody A32262 has a paraffin-embedded human lung carcinoma image (A32262 image caption) and lists Human, Mouse, and Rat reactivity (A32262 catalog).
Real IHC data
Anti-ATP5J2 (G42) ATP5MF Antibody
Cat # A32262
A32262 is listed for IHC (A32262 catalog). Its IHC image shows paraffin-embedded human lung carcinoma tissue at 1:100 (A32262 image caption).
Which to pick: For tissue IHC, choose A32262, a rabbit antibody with a listed IHC dilution of 1:50–1:200 (A32262 catalog); its image supports use on paraffin-embedded human lung carcinoma tissue (A32262 image caption). The caption does not report the fixative (A32262 image caption). For cross-species work, A32262 lists Human, Mouse, and Rat reactivity, but its catalog lists no IF/ICC application or image, so there is no supported IF/ICC pick (A32262 catalog).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P56134 (ATPK_HUMAN, ATP synthase F(0) complex subunit f, mitochondrial).
- Human Protein Atlas. ATP5MF tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ATP5MF subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the nuclear membrane..
- Human Protein Atlas. ATP5MF antibody validation summary (2 antibodies).
- Identification of the metabolic protein ATP5MF as a potential therapeutic target of TNBC. Journal of translational medicine 2024 — PMC11472516.
- PubMed PMID:9653160 — UniProt-cited evidence.
- PubMed PMID:12853948 — UniProt-cited evidence.
- PubMed PMID:12690205 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ATP5MF (ATP synthase F(0) complex subunit f, mitochondrial). Expected localisation Cytoplasmic staining (HPA tissue IHC); inner mitochondrial membrane (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A32262 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung carcinoma tissue; fixative not specified (datasheet A32262); 2. Fixation: Image fixative and duration unreported (datasheet A32262); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-ATP5MF, 1:50-1:200 (datasheet A32262); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A32262); Caveat: Some cell types lack staining despite the broad tissue profile (HPA tissue IHC); Isoform / epitope: 4 isoforms; epitope coverage is not given in the record (UniProt); Regulation: No specific expression regulator reported (UniProt). Evidence: UniProt P56134, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the mitochondria. In addition localized to the…, Datasheet A32262, PMC11472516. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ATP5MF/immunohistochemistry-ihc-guide.