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ATAD3A / ATPase family AAA domain-containing protein 3A · IHC design guide
Design Immunohistochemistry for ATAD3A
Plan ATAD3A paraffin-section IHC around the granular cytoplasmic staining observed across tissues (HPA tissue IHC). The IHC-validated antibody has a listed dilution of 1:100–1:200 (datasheet); interpret staining with the reported multi-gene specificity caution (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ATAD3A 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 | Tissue: granular cytoplasm (HPA tissue IHC); molecular: mitochondrial inner membrane (UniProt) | |
| Staining pattern | Granular cytoplasmic staining across 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 | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
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 A06057-1) | |
| Caveat | Tissue staining may include proteins from more than one gene (HPA tissue IHC) | |
| Regulation | Elevated in lung adenocarcinoma (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended ATAD3A IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet) is paired with three published ATAD3A tissue protocols (PMC10757082; PMC8358594; PMC10808738).
| Sample | Paraffin-embedded human stomach tissue; fixative not specified (datasheet A06057-1) |
| Fixation | Image fixative and duration unreported (datasheet A06057-1); 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-ATAD3A, 1:100-1:200 (datasheet A06057-1) |
| 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 | ATAD3A-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Granular cytoplasmic expression in all tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 at 95–98 °C for 20 min (page retrieval); compare microwave retrieval for 15 min if needed (PMC8358594).
Section 2
What Is the Expected ATAD3A Staining Pattern?
ATAD3A is an inner mitochondrial membrane protein, with its N-terminal region facing the intermembrane space and its C-terminal region facing the matrix (UniProt Q9NVI7 topology). In paraffin sections, expect granular cytoplasmic staining, including strong signal in several glandular and epithelial cell populations (HPA tissue IHC). Treat the pattern as provisional evidence for ATAD3A: HPA rates tissue staining Approved, reports medium consistency with RNA, and cautions that the antibody targets proteins from more than one gene (HPA tissue IHC).
| Granular cytoplasmic staining in glandular epithelium. | This fits the reported tissue pattern (HPA tissue IHC). Strong glandular-cell staining is documented in adrenal gland, appendix, breast, cervix, colon, and duodenum (HPA tissue IHC). Read the granules as compatible with mitochondrial localization, while retaining the HPA warning that staining may include proteins from more than one gene (UniProt Q9NVI7; HPA tissue IHC). |
| Predominantly nuclear or continuous cell-surface staining. | This does not match the reported granular cytoplasmic IHC profile or mitochondrial localization (HPA tissue IHC; UniProt Q9NVI7). Check morphology and detection controls before assigning the signal to ATAD3A (standard IHC practice). The UniProt nucleoid association describes a compartment within mitochondria; it does not predict nuclear staining (UniProt Q9NVI7). |
| Strong signal in cells expected to be low, with little signal in adjacent expected-positive cells. | HPA lists smooth muscle cells, chondrocytes, and adipocytes as low, while several glandular populations are high (HPA tissue IHC). Such a reversed distribution warrants investigation of nonspecific binding or endogenous detection activity (standard IHC practice). Low HPA staining is not a validated negative control, and the multigene antibody caution limits target-specific conclusions (HPA tissue IHC). |
| Diffuse chromogen over cells, stroma, or the whole section. | A diffuse field obscures the granular cytoplasmic pattern reported by HPA (HPA tissue IHC). Review blocking, washing, chromogen development, and no-primary controls for background from the detection workflow (standard IHC practice). Uniform color alone cannot establish mitochondrial localization or ATAD3A specificity (UniProt Q9NVI7; standard IHC practice). |
| No signal in an expected-positive tissue compartment. | High staining is reported in bronchial respiratory epithelium and cerebellar Purkinje cells, as well as the listed glandular populations (HPA tissue IHC). First confirm that the expected cells are present and that a positive control developed (standard IHC practice). A negative section alone cannot establish absent ATAD3A, particularly when HPA reports broad granular cytoplasmic expression (HPA tissue IHC). |
💡Expected ATAD3A appearanceCall a result pattern-consistent when the expected cells show clearly resolved granular cytoplasmic staining, with strong signal in an HPA High compartment such as colon glandular cells; diffuse, predominantly nuclear, or cell-surface color is suspect (HPA tissue IHC; UniProt Q9NVI7; standard IHC practice).
| Compartment and topology | ATAD3A spans the inner mitochondrial membrane at residues 247–264; residues 2–246 face the intermembrane space and 265–586 face the matrix (UniProt Q9NVI7 topology). An epitope's position may matter when choosing staining conditions, but no antibody epitope or ATAD3A-specific retrieval response is supplied (UniProt Q9NVI7; supplied evidence). |
| Tissue distribution and comparator choice | HPA reports granular cytoplasmic staining across tissues and low RNA tissue specificity (HPA tissue IHC). Use a documented High cell population to check pattern and detection; smooth muscle cells, chondrocytes, and adipocytes are Low comparators, not proven ATAD3A-negative controls (HPA tissue IHC; standard IHC practice). |
| Antibody interpretation | HPA058968 has Approved IHC status, while HPA cautions that tissue staining targets proteins from more than one gene and has medium consistency with RNA (HPA antibodies; HPA tissue IHC). Interpret matching morphology as supportive rather than uniquely ATAD3A-specific; avoid inferring specificity from staining intensity alone (HPA tissue IHC; standard IHC practice). |
| Isoforms and processing | UniProt lists 3 isoforms and annotates a chain beginning at residue 2, without a signal peptide or propeptide (UniProt Q9NVI7). These annotations do not establish which isoforms an unspecified IHC epitope detects or predict loss of tissue staining through shedding (UniProt Q9NVI7; supplied evidence). |
| IF/ICC Q&A: what should a companion fluorescence image show? | Predominantly mitochondrial localization is reported by HPA ICC-IF; acrosome staining is an additional approved location (HPA subcellular). This supports a mitochondrial localization check in IF/ICC, but HPA cautions that those antibodies target proteins from multiple genes (HPA subcellular). IF/ICC conditions belong to its separate guide (application scope). |
| Situation | Likely cause | Next action |
|---|---|---|
| Granules are faint in a documented High compartment. | The expected cells may be sparse, or the staining workflow may have insufficient detection sensitivity (HPA tissue IHC; standard IHC practice). | Confirm the relevant cell population on the section, then compare a known-positive section processed in the same run; review the catalog antibody's IHC-P protocol for its specified conditions (HPA tissue IHC; standard IHC practice). |
| A known-positive section remains blank. | Failure of primary staining, detection, or chromogen development can produce a blank result (standard IHC practice). | Check reagent steps and a separate positive detection control; assess the supplied tissue before calling ATAD3A absent (standard IHC practice). HPA High labels describe observed staining, not a guarantee for every specimen (HPA tissue IHC). |
| Chromogen appears in the no-primary control. | Endogenous detection activity or background from the detection system may contribute signal (standard IHC practice). | Review the relevant blocking and detection steps, including endogenous enzyme blocking when using enzyme-based chromogenic detection; interpret only signal exceeding control background (standard IHC practice). |
| Staining is diffuse rather than granular. | Background or overdevelopment may conceal the reported cytoplasmic granules (HPA tissue IHC; standard IHC practice). | Compare development time and washing with run controls, and reassess at a magnification that resolves cell-level granules (standard IHC practice). Do not score diffuse color alone as the HPA pattern (HPA tissue IHC). |
| Nuclear or surface staining dominates. | The observed compartment conflicts with mitochondrial inner-membrane localization (UniProt Q9NVI7 topology). | Recheck cellular boundaries and control sections; if the distribution persists, record it as discordant rather than assigning it to ATAD3A (UniProt Q9NVI7; standard IHC practice). |
| Unexpected cells stain more strongly than expected-positive cells. | Nonspecific staining or endogenous activity may alter the distribution; the HPA antibody also carries a multigene-target caution (standard IHC practice; HPA tissue IHC). | Compare the affected cells with HPA High and Low compartments, examine no-primary controls, and qualify any ATAD3A attribution until the distribution is resolved (HPA tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing ATAD3A staining
A06057-1 · IHCImmunohistochemistry of paraffin-embedded human stomach using ATAD3A antibody at dilution of 1:100 .Perform microwave antigen retrieval with
A06057-1 · IHCImmunohistochemistry of paraffin-embedded mouse brain using ATAD3A antibody at dilution of 1:100 .Perform microwave antigen retrieval with 1
A06057 · IFImmunocytochemistry of ATAD3A in Daudi cells with ATAD3A antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000197785-ATAD3A/tissue/cerebral+cortex#imid_16806987
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000197785-ATAD3A/tissue/colon#imid_16806929
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000197785-ATAD3A/tissue/liver#imid_16806914
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000197785-ATAD3A/tissue/kidney#imid_16806931
HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA064867; green = ATAD3A, blue = nucleus, red = microtubules.
HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA064867; green = ATAD3A, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ATAD3A IHC & IF
🧪Run adrenal gland first: glandular cells should show strong staining (HPA: High in adrenal gland glandular cells). HPA detects ATAD3A in all 45 scored tissues, so there is no validated negative tissue; no-primary and isotype controls carry the negative comparison, while cells outside the glandular compartment with weak or absent granular cytoplasmic staining are only relative internal controls (HPA: no negative tissues; UniProt Q9NVI7: mitochondrial localization).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: ATAD3A is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ATAD3A in CACO-2, PC-3, U2OS, Sperm, with annotated localisation: Mitochondria (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only section and an isotype control matched to the primary antibody’s host species and clonality; use ATAD3A knockout material or a validated peptide-block condition as a biological specificity control (standard IHC practice). For adrenal tissue, quench endogenous peroxidase and block endogenous biotin if using biotin-based detection (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected paraffin-section caption does not state a fixative (selected SKU A06057-1 tissue-IHC caption). That caption uses microwave retrieval in 10 mM PBS, pH 7.2, before staining at 1:100; it documents a retrieval method, not that retrieval is required (selected SKU A06057-1 tissue-IHC caption). Frozen-section performance and relative ease are unreported; ICC-IF offers a documented mitochondrial localization check, while endogenous biotin can complicate biotin-based detection in adrenal tissue (HPA: subcellular ICC-IF; standard IHC practice).
HPA tissue IHC evidence for ATAD3A
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene.). 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 → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: ATAD3A is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced ATAD3A IHC Tips
Troubleshoot ATAD3A staining in paraffin sections by checking retrieval, compartmental pattern, controls and scoring before interpreting chromogenic signal.
Which retrieval conditions should I try when ATAD3A staining is weak?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 minutes (page IHC retrieval setting). If staining remains weak, test microwave retrieval in 10 mM PBS, pH 7.2, which was used for paraffin embedded human stomach with antibody A06057-1 (caption: A06057-1). Keep section thickness, antibody dilution and detection conditions constant while comparing retrieval methods (standard IHC practice). Examine tissue preservation as well as signal: excessive heating can damage morphology, while insufficient retrieval can leave epitopes inaccessible (standard IHC practice). Record the chosen method for each staining batch (standard IHC practice).
Could fixation explain inconsistent ATAD3A staining between paraffin sections?
ATAD3A specific sensitivity to fixation is unknown from the supplied evidence (caption: A06057-1, fixative not stated). The human stomach example identifies paraffin sections and retrieval in 10 mM PBS at pH 7.2, but does not report a fixative or fixation duration (caption: A06057-1). Compare sections with documented processing histories using the same retrieval, antibody dilution and detection run, then judge signal alongside preserved morphology (standard IHC practice). If processing histories differ, avoid treating a weaker stain as lower ATAD3A abundance without a matched control (standard IHC interpretation). Record fixation conditions prospectively so later staining differences can be evaluated (standard IHC practice).
What staining pattern supports ATAD3A localisation in tissue sections?
Look for granular cytoplasmic staining rather than a uniform nuclear pattern (HPA: granular cytoplasmic expression in all tissues; UniProt Q9NVI7: mitochondrial inner membrane and matrix localisation). The protein spans the mitochondrial inner membrane at residues 247–264, with residues 2–246 on the intermembrane side and 265–586 on the matrix side (UniProt Q9NVI7 topology). Chromogenic IHC may show cytoplasmic granules without resolving those mitochondrial compartments (standard IHC interpretation). Compare staining with an expected positive tissue region and inspect cell outlines and counterstained nuclei before assigning the signal (HPA: high expression in adrenal glandular cells; standard IHC practice).
How can epitope placement affect ATAD3A IHC results?
Check the antibody immunogen or mapped epitope against the 3 annotated ATAD3A isoforms before interpreting a negative result (UniProt Q9NVI7: isoforms 1, 2 and 3; standard IHC practice). An epitope within residues 2–246 faces the mitochondrial intermembrane space, whereas one within 265–586 faces the matrix (UniProt Q9NVI7 topology). Residue 321 is annotated as phosphoserine and residue 491 as acetyllysine; their effect on this antibody’s staining is unknown (UniProt Q9NVI7 modified residues; supplied antibody evidence). If epitope information is unavailable, report antibody identity and retrieval conditions, and avoid claiming isoform specific staining (standard IHC interpretation).
How should I investigate ATAD3A localisation by IF alongside tissue IHC?
Use IF as a separate localisation check: ATAD3A is mainly mitochondrial in the supplied subcellular evidence (HPA subcellular: mitochondria enhanced; UniProt Q9NVI7 localisation). Multiplex with a marker for the expected cell population, such as glandular cells in a selected positive tissue region, and assess whether ATAD3A signal falls within those cells (HPA tissue IHC: high in adrenal glandular cells; standard IF practice). Choose a fluorophore channel after inspecting unstained tissue autofluorescence and include single stain controls (standard IF practice). For permeabilisation, verify the antibody epitope: residues 2–246 face the intermembrane space and 265–586 face the matrix, so access may differ (UniProt Q9NVI7 topology; standard IF practice).
How can I reduce diffuse brown background without losing ATAD3A signal?
First compare the stained section with a no primary control to locate detection background (standard IHC practice). Apply a peroxidase block before chromogenic detection and use an appropriate protein block; these are general IHC steps, not ATAD3A specific evidence (standard chromogenic IHC practice). Start antibody titration around the reported 1:100 dilution for paraffin embedded human stomach, keeping retrieval and development time fixed during the comparison (caption: A06057-1; standard IHC practice). Shorten DAB development if both tissue and control sections become diffusely brown, and retain a positive region to check that signal remains detectable (standard chromogenic IHC practice; HPA tissue IHC: high in glandular cells).
How should I score ATAD3A staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region of interest before scoring, because HPA reports granular cytoplasmic expression across tissues and differing intensity among cell populations (HPA tissue IHC profile and cell levels; standard IHC practice). Score cytoplasmic intensity as 0–3 and calculate an H score as the sum of each intensity multiplied by its percentage of cells, yielding 0–300 (standard IHC scoring practice). Alternatively, report the percentage of positive cells within the same defined population across sections (standard IHC practice). Normalise counts or stained area to the number of eligible cells or tissue area examined, and apply one threshold across the comparison (standard IHC quantification practice).
When is an apparent ATAD3A positive cell likely to be artefactual?
Give most weight to intracellular granular cytoplasmic staining in preserved cells (HPA tissue IHC: granular cytoplasmic expression; UniProt Q9NVI7: mitochondrial localisation). Be cautious with exclusively nuclear signal or staining confined to cut edges and necrotic regions; compare those areas with intact tissue on the same section (UniProt Q9NVI7 localisation; standard IHC interpretation). Check a no primary control for endogenous enzyme or detection signal, especially when brown precipitate lacks clear cell boundaries (standard chromogenic IHC practice). HPA rates its tissue staining Approved but cautions that its antibodies target proteins from more than one gene, so HPA patterns alone cannot establish ATAD3A specificity (HPA tissue IHC reliability description).
Boster reagents
Best ATAD3A / ATPase family AAA domain-containing protein 3A IHC Antibodies
Real ATAD3A IHC images cover paraffin sections of human stomach and mouse brain (A06057-1 captions); IF/ICC images cover Daudi cells (A06057 captions). Rat reactivity is listed for A06057 (catalog: reactivity).
Real IHC data
Anti-ATAD3A Antibody
Cat # A06057-1
Real IF data
Anti-ATAD3A Antibody
Cat # A06057
A06057-1 lists human and mouse IHC, with paraffin-section images from human stomach and mouse brain (catalog: applications and reactivity; A06057-1 captions). A06057 lists IF/ICC and human, mouse, and rat reactivity, with Daudi-cell images at 20 μg/mL (catalog: applications and reactivity; A06057 captions).
Which to pick: Choose A06057-1 for paraffin tissue IHC: its captions show human stomach and mouse brain at 1:100 after microwave retrieval in 10 mM PBS, pH 7.2; the fixative is unreported (A06057-1 captions). Choose A06057 for IF/ICC; its Daudi-cell images use 20 μg/mL (A06057 captions). For cross-species work, both list human and mouse, while only A06057 lists rat; neither reports a clone, so clonality cannot guide the choice (catalog: reactivity and clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9NVI7 (ATD3A_HUMAN, ATPase family AAA domain-containing protein 3A).
- Human Protein Atlas. ATAD3A tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ATAD3A subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the acrosome. Caution: Based on antibodies targeting proteins from multiple genes..
- Human Protein Atlas. ATAD3A antibody validation summary (3 antibodies).
- The value of ATAD3A as a potential biomarker for bladder cancer. Cancer medicine 2023 — PMC10757082.
- Proteome profiling of formalin-fixed, paraffin-embedded lung adenocarcinoma tissues using a tandem mass tag-based quantitative proteomics approach. Oncology letters 2021 — PMC8358594.
- DEK deficiency suppresses mitophagy to protect against house dust mite-induced asthma. Frontiers in immunology 2023 — PMC10808738.
- Loss of mitochondrial ATPase ATAD3A contributes to nonalcoholic fatty liver disease through accumulation of lipids and damaged mitochondria. The Journal of biological chemistry 2022 — PMC9157002.
- PubMed PMID:20332122 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16710414 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ATAD3A (ATPase family AAA domain-containing protein 3A). Expected localisation Tissue: granular cytoplasm (HPA tissue IHC); molecular: mitochondrial inner membrane (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A06057-1 at 1:100-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human stomach tissue; fixative not specified (datasheet A06057-1); 2. Fixation: Image fixative and duration unreported (datasheet A06057-1); 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-ATAD3A, 1:100-1:200 (datasheet A06057-1); 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 A06057-1); Caveat: Tissue staining may include proteins from more than one gene (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope coverage is unspecified (UniProt); Regulation: Elevated in lung adenocarcinoma (UniProt). Evidence: UniProt Q9NVI7, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the mitochondria. In addition localized to the…, Datasheet A06057-1, PMC10757082, PMC8358594. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ATAD3A/immunohistochemistry-ihc-guide.