This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
ATP5MF / ATP synthase F(0) complex subunit f, mitochondrial · IHC design guide
Design Immunohistochemistry for ATP5MF
This guide uses the cytoplasmic tissue pattern to plan ATP5MF paraffin IHC (HPA tissue IHC). Use kidney tubules as a high-staining reference (HPA tissue IHC), adipocytes as a no-stain comparator (HPA tissue IHC), and titrate the catalog antibody at 1:50–1:200 (datasheet).
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 | IHC cytoplasmic; protein at mitochondrial inner membrane (HPA tissue IHC; UniProt) | |
| Staining pattern | Cytoplasmic stain in most tissues; high in glandular cells (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 | Keep fixation consistent across sections (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 A32262) | |
| Caveat | Adipocytes lack detectable stain despite the broad tissue pattern (HPA tissue IHC) | |
| Regulation | No specific regulator reported (UniProt) | |
| Isoform / epitope | 4 isoforms; epitope impact is unknown (UniProt) |
Section 1
Recommended ATP5MF IHC & IF Protocols
The catalog antibody's IHC-P protocol is paired with one published 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 HIER at 95–98 °C for 20 min (page retrieval rule); the published protocol does not specify retrieval conditions (PMC11472516).
Section 2
What Is the Expected ATP5MF Staining Pattern?
ATP5MF is an inner mitochondrial membrane subunit with a transmembrane segment at residues 68–85 (UniProt P56134 topology). In paraffin section IHC, expect cytoplasmic staining in many tissues, especially glandular cells and kidney tubular cells scored High by HPA (HPA tissue IHC). HPA rates its tissue pattern Approved, with medium agreement between staining and RNA data, pending external verification (HPA tissue IHC).
| Cytoplasmic signal in kidney tubular cells or adrenal, breast, parathyroid, rectal, salivary, or stomach glandular cells. | This fits the listed High staining cell populations (HPA tissue IHC). ATP5MF resides in the mitochondrial inner membrane (UniProt P56134); chromogenic IHC should be read as cytoplasmic staining, without assuming that individual mitochondria will resolve. |
| Predominantly nuclear filling, extracellular deposits, or a surface outline replaces cytoplasmic staining in a reference positive cell population. | That distribution does not fit the principal mitochondrial location (UniProt P56134; HPA subcellular ICC-IF). Review morphology, counterstain, and controls before calling it ATP5MF. An uncertain nuclear membrane location in ICC-IF does not establish diffuse nuclear IHC staining (HPA subcellular ICC-IF). |
| Strong color appears in a cell population HPA scored Not detected, while reference positive cells stain weakly or not at all. | Consider cross-reactivity or endogenous detection activity (general IHC practice). HPA lists adipocytes, prostate glandular cells, and skeletal myocytes among its Not detected examples; that designation describes the reported staining, not a universal absence of ATP5MF (HPA tissue IHC). |
| Uniform haze spans cells and surrounding tissue, obscuring cell boundaries and the expected cytoplasmic distribution. | Treat this as background until controls show otherwise (general IHC practice). A broadly expressed cytoplasmic profile is reported (HPA tissue IHC), but it does not justify featureless color across compartments or a signal of similar strength in negative controls. |
| A reference positive tissue has no convincing cytoplasmic signal. | First compare it with the stated High cell population and an appropriate run control (HPA tissue IHC; general IHC practice). An absent result alone cannot establish biological absence: antibody performance, tissue handling, retrieval, or detection may need investigation (general IHC practice). |
💡Expected ATP5MF appearanceCall a result positive when interpretable cytoplasmic chromogen appears in the expected cells, including HPA High kidney tubules or listed glandular populations (HPA tissue IHC), consistent with mitochondrial ATP5MF (UniProt P56134); dominant nuclear filling, extracellular color, or equally strong negative controls argue against a specific call (general IHC practice).
| Compartment and resolution | ATP5MF is an inner mitochondrial membrane protein with one annotated transmembrane segment at 68–85 (UniProt P56134 topology). Its mitochondrial address supports a cytoplasmic IHC readout; section thickness and chromogenic resolution limit how precisely that pattern can be assigned to individual organelles (general IHC practice). |
| Reference tissue and cell selection | HPA reports High staining in kidney tubular cells and several glandular populations, Medium in appendix glandular cells, and Not detected in selected cell populations (HPA tissue IHC). Score the named cells within a tissue rather than treating an entire organ as uniformly positive or negative. |
| Strength of IHC evidence | The tissue profile is Approved but has medium staining–RNA consistency and awaits external verification (HPA tissue IHC). HPA070412 has an Approved IHC status; HPA067267 has no listed IHC status (HPA antibodies). These labels support cautious interpretation, not proof that every positive cell is specific. |
| Isoforms and epitope uncertainty | UniProt lists 4 ATP5MF isoforms and a chain spanning residues 2–94 (UniProt P56134). The supplied sources do not map an IHC antibody epitope or establish isoform recognition. Do not attribute staining differences to a particular isoform from these records. |
| IF/ICC Q&A: what pattern is expected? | Mainly mitochondrial signal is supported in ICC-IF; an additional nuclear membrane location is marked uncertain (HPA subcellular ICC-IF). That observation can inform compartment checks, but it does not supply an IF/ICC protocol or validate a nuclear membrane call in chromogenic tissue IHC. |
| Situation | Likely cause | Next action |
|---|---|---|
| Kidney tubular cells show no signal in an otherwise readable section. | The selected cells are reported High, so a completely absent result warrants a run-level check (HPA tissue IHC). Failed detection or a poorly performing IHC assay is possible (general IHC practice). | Confirm the tubular cells are present, inspect the positive run control, and check primary antibody, retrieval, and detection records (general IHC practice). Avoid calling the tissue negative from this section alone. |
| Only a weak stain appears in a population expected to be High. | The HPA level is an observed category, not a calibrated intensity standard for every specimen or run (HPA tissue IHC). Variation in the assay or specimen may affect the visible result (general IHC practice). | Compare cells and controls stained in the same run; verify antibody dilution and chromogen development against the validated IHC method (general IHC practice). Do not infer an ATP5MF isoform change without epitope evidence (UniProt P56134). |
| Color is concentrated in nuclei instead of the cytoplasm. | Diffuse nuclear filling conflicts with the principal mitochondrial localization (UniProt P56134; HPA subcellular ICC-IF). Nuclear membrane labeling in ICC-IF is only an uncertain additional observation (HPA subcellular ICC-IF). | Recheck focus and counterstain, then compare primary antibody omission and reference positive sections (general IHC practice). Do not score diffuse nuclear color as a specific ATP5MF result. |
| Strong staining appears in cells HPA scored Not detected. | Examples include prostate glandular cells and adipocytes (HPA tissue IHC). Cross-reactivity or endogenous detection activity can mimic target staining (general IHC practice); an HPA Not detected score is not proof that all specimens must be blank. | Compare the cell type and compartment with a reference positive section, then inspect omission and detection controls (general IHC practice). Record the unexpected pattern separately from a supported ATP5MF call. |
| Broad brown haze obscures cell boundaries. | Nonspecific antibody binding or endogenous detection activity may add background in chromogenic IHC (general IHC practice). The HPA cytoplasmic profile does not establish specificity for featureless staining (HPA tissue IHC). | Review primary antibody omission and detection controls; adjust blocking, washes, or detection conditions within the validated IHC workflow (general IHC practice). Score only interpretable cellular signal. |
| Two antibodies give different tissue patterns. | Their listed IHC statuses differ: HPA070412 is Approved, while HPA067267 has no IHC status in the supplied list (HPA antibodies). Neither the tissue profile nor the status labels identify the cause of discordance (HPA tissue IHC; HPA antibodies). | Compare the same named cell populations and controls across matched sections, then report the discordance (general IHC practice). Prefer an antibody with documented IHC validation for an IHC interpretation (HPA antibodies). |
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 cells in tubules). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the kidney slide, use non-tubular cells as an internal background comparison only where they are visibly unstained, because HPA does not identify a negative kidney cell type (HPA: High in kidney cells in tubules).
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 a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species, clonality and concentration; use ATP5MF knockout tissue or peptide blocking, if a suitable blocking peptide is available, to test target specificity (standard IHC practice). Block endogenous peroxidase for chromogenic detection, and check kidney sections for endogenous biotin if using avidin–biotin detection (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and retrieval dependence are unreported; the A32262 paraffin-section lung carcinoma caption gives 1:100 but does not report a fixative (selected IHC caption). Whether frozen sections or IF/ICC are easier is unreported, although mitochondrial localization is supported by ICC-IF images (HPA: mitochondria, supported). Kidney tubules can show endogenous biotin signal with avidin–biotin detection, so inspect the negative controls before scoring tubular staining (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 staining in paraffin sections using the selected antibody’s tissue image, cellular localisation evidence, and matched controls.
Which retrieval conditions should I try first for weak ATP5MF staining?
Use citrate pH 6.0 heat-induced retrieval at 95–98 °C for 20 min (page retrieval rule: cytoplasmic / membrane antigen). Allow sections to cool in retrieval buffer, then wash consistently before applying the primary antibody (standard IHC practice). The selected antibody has been shown in paraffin-embedded human lung carcinoma at 1:100, but its image caption does not specify retrieval conditions (A32262 caption). If signal remains weak, compare a longer citrate retrieval with a small, matched test of an alternative buffer, while checking tissue integrity (standard IHC practice). Judge improvement against kidney tubular cells, reported as high staining, rather than background alone (HPA: High in kidney tubules).
Could fixation explain inconsistent ATP5MF staining between paraffin blocks?
Target-specific fixation sensitivity is unknown: the selected antibody’s paraffin-section caption does not report a fixative or fixation time (A32262 caption). Record the fixative, time in fixative, processing history, and section age for each block before comparing staining (standard IHC practice). If blocks differ, stain adjacent sections together with the same citrate pH 6.0, 95–98 °C, 20 min retrieval and detection workflow (page retrieval rule; standard IHC practice). Compare preserved, viable regions and a common positive control, because tissue damage can complicate interpretation of weak chromogenic signal (standard IHC practice). Neither the tissue staining profile nor ATP5MF’s membrane topology establishes a fixation effect (HPA tissue IHC; UniProt P56134 topology).
What staining pattern should count as correctly localised ATP5MF?
Expect predominantly cytoplasmic staining at light-microscope resolution: ATP5MF is an inner mitochondrial membrane component, while tissue IHC reports cytoplasmic expression in most tissues (UniProt P56134 localisation; HPA tissue IHC). A fine granular cytoplasmic pattern is compatible with mitochondria, but chromogenic IHC cannot resolve the inner membrane itself (standard IHC interpretation). Use kidney tubular cells as a strong reference and assess whether staining remains cell-associated in intact regions (HPA: High in kidney tubules; standard IHC practice). HPA reports mitochondrial localisation as supported and an additional nuclear-membrane location as uncertain (HPA subcellular). Predominantly nuclear or diffuse extracellular DAB should prompt review of controls, counterstain, and tissue preservation before attribution to ATP5MF (standard IHC practice).
How could isoforms or epitope accessibility affect the IHC result?
ATP5MF has 4 annotated isoforms and one transmembrane segment at residues 68–85 (UniProt P56134 isoforms and topology). The supplied antibody caption does not identify its epitope, so it cannot establish which isoforms the observed paraffin-section signal represents (A32262 caption). Check the antibody’s documented immunogen or epitope information before interpreting absent staining as loss of every isoform (standard antibody-validation practice). Acetylation at residues 2 and 22, and phosphorylation at residue 3, are annotated, but their effects on this antibody’s IHC staining are unknown (UniProt P56134 modified residues). Compare matched positive and negative-control sections under the same retrieval conditions when evaluating apparent epitope loss (standard IHC practice).
How should I assess ATP5MF by IF alongside chromogenic IHC?
Treat IF as a separate assay and compare its cellular pattern with the chromogenic IHC result; HPA supports mitochondrial localisation in ICC/IF images (HPA subcellular). Multiplex with a validated marker for the expected cell type in the chosen specimen, so cell identity can be checked alongside ATP5MF signal (standard IF practice). Choose a far-red fluorophore when the tissue shows strong shorter-wavelength autofluorescence, and include an unstained section to assess that background (standard IF practice). Plan permeabilisation around the antibody epitope’s side of the mitochondrial inner membrane; that side is not specified in the supplied antibody caption (UniProt P56134 topology; A32262 caption). The IF comparison does not establish fixation or permeabilisation conditions for this antibody (A32262 caption).
How can I separate ATP5MF signal from chromogenic background?
Run a no-primary control and inspect pigment, damaged areas, and section edges before assigning brown deposits to ATP5MF (standard IHC practice). Block endogenous peroxidase before DAB detection, then compare a polymer-based detection system if an avidin–biotin method produces tissue background (standard chromogenic IHC practice). The selected antibody’s paraffin-section image uses 1:100; use that as a documented starting dilution when checking whether antibody concentration contributes to diffuse staining (A32262 caption). Keep counterstain and DAB development comparable across control and test sections (standard IHC practice). True signal should follow intact cells and a plausible cytoplasmic distribution, consistent with tissue IHC and mitochondrial localisation (HPA tissue IHC; UniProt P56134 localisation).
How should I score ATP5MF staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Score ATP5MF within defined, viable cell populations and exclude folds, necrosis, and section edges before measuring chromogenic signal (standard IHC scoring practice). An H-score combines the percentages of cells at intensity 0–3 into a 0–300 score; report percent positive cells separately if intensity is unreliable (standard IHC scoring practice). For spatial comparisons, report positive-cell density per mm² of viable tissue and state the positivity threshold (standard IHC scoring practice). Normalise comparisons to the same cell population, sampled viable area, staining run, and reference control, since tissue composition and processing affect measured signal (standard IHC practice). Kidney tubular cells are a documented high-staining reference, while HPA’s tissue calls are descriptive rather than assay-specific thresholds (HPA: High in kidney tubules; HPA tissue IHC).
Which findings support genuine ATP5MF staining rather than artefact?
Look for reproducible, cell-associated cytoplasmic staining in preserved tissue, consistent with ATP5MF’s mitochondrial inner-membrane localisation and HPA tissue IHC (UniProt P56134 localisation; HPA tissue IHC). Kidney tubular cells provide a documented high-staining comparison; lung macrophages are reported low, so cell identity matters when interpreting a mixed section (HPA: High in kidney tubules; HPA: Low in lung macrophages). The selected 1:100 image documents staining in paraffin-embedded human lung carcinoma, without establishing that all carcinoma cells should stain (A32262 caption). Signal confined to cut edges, necrotic material, or a no-primary control favors artefact, including residual endogenous enzyme activity (standard IHC practice). Treat prominent nuclear-membrane staining cautiously because that additional location remains uncertain (HPA subcellular).
Boster reagents
Best ATP5MF / ATP synthase F(0) complex subunit f, mitochondrial IHC Antibodies
One anti-ATP5MF antibody has real IHC data from paraffin-embedded human lung carcinoma tissue (A32262 IHC image caption); Human, Mouse, and Rat reactivity is listed (A32262 catalog).
Real IHC data
Anti-ATP5J2 (G42) ATP5MF Antibody
Cat # A32262
A32262 lists IHC for Human, Mouse, and Rat (A32262 catalog). Its IHC figure shows paraffin-embedded human lung carcinoma tissue at 1:100; no IF figure is supplied (A32262 IHC image caption; A32262 catalog).
Which to pick: For tissue IHC, choose A32262: IHC is listed, and its own figure shows a paraffin-embedded human lung carcinoma section (A32262 catalog; A32262 IHC image caption). For IF/ICC, no SKU in this payload has a listed IF application or figure (A32262 catalog). For cross-species IHC, A32262 lists Human, Mouse, and Rat reactivity, though the pictured example is human; the caption does not report the fixative (A32262 catalog; A32262 IHC image caption).
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 IHC cytoplasmic; protein at mitochondrial inner membrane (HPA tissue IHC; 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: Keep fixation consistent across sections (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 A32262); Caveat: Adipocytes lack detectable stain despite the broad tissue pattern (HPA tissue IHC); Isoform / epitope: 4 isoforms; epitope impact is unknown (UniProt); Regulation: No specific 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.