ATAD3A / ATPase family AAA domain-containing protein 3A · IHC design guide

Design Immunohistochemistry for ATAD3A

Use the catalog antibody at 1:100–1:200 for paraffin-section IHC (datasheet: A06057-1), with granular cytoplasmic staining as the tissue reference (HPA tissue IHC). Compare high-staining glandular cells with low-staining smooth muscle cells (HPA tissue IHC), and assess specificity because staining may reflect protein from more than one gene (HPA tissue IHC).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for ATAD3A (IHC for ATAD3A): expected localisation Granular cytoplasm (HPA tissue IHC); mitochondrial inner membrane (UniProt), antibody A06057-1, validated IHC image, and IHC protocol steps — Printable **ATAD3A** IHC protocol sheet — expected localisation Granular cytoplasm (HPA tissue IHC); mitochondrial inner membrane (UniProt), antibody A06057-1, controls and protocol steps. Open the full ATAD3A IHC guide →

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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	Granular cytoplasm (HPA tissue IHC); 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	Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific)
	Caveat	Staining may reflect protein from more than one gene (HPA tissue IHC)
	Regulation	Overexpressed in lung adenocarcinoma (UniProt)
	Isoform / epitope	3 isoforms; epitope coverage is unknown (UniProt)

Section 1

Recommended ATAD3A IHC & IF Protocols

The catalog antibody protocol (datasheet) appears alongside published ATAD3A IHC protocols for bladder and lung tissues (PMC10757082; PMC8358594; PMC10808738).

Recommended immunohistochemistry (IHC-P) protocol parameters
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 HIER at 95–98 °C for 20 min (page retrieval rule); the lung adenocarcinoma protocol used 15 min at 95 °C (PMC8358594).

Section 2

What Is the Expected ATAD3A Staining Pattern?

ATAD3A is associated with the mitochondrial inner membrane and matrix nucleoids; its single transmembrane segment spans residues 247–264 (UniProt Q9NVI7 topology). In paraffin section IHC, expect granular cytoplasmic staining across tissues, including strong staining in several glandular and epithelial cell populations (HPA: tissue IHC). Treat tissue intensity as a guide: HPA rates the tissue pattern Approved, with medium consistency against RNA data and a warning that the antibody targets proteins from more than one gene (HPA: tissue reliability).

What am I looking at on my slide?
Granular cytoplasmic staining is prominent in adrenal, appendix, breast, cervical, colonic or duodenal glandular cells, or bronchial respiratory epithelium (HPA: High in these cell populations).	This matches the reported tissue pattern (HPA: granular cytoplasmic expression). Mitochondrial localisation offers a compartment explanation (UniProt Q9NVI7: inner membrane and matrix nucleoids), but chromogenic IHC alone does not resolve the inner membrane.
A predominantly nuclear, membranous or extracellular signal replaces granular cytoplasmic staining (HPA: tissue IHC pattern).	That distribution conflicts with the reported mitochondrial localisation (UniProt Q9NVI7; HPA: subcellular). Check morphology, counterstain and detection artefacts before interpreting it as ATAD3A; the supplied sources do not establish those compartments as the expected IHC result.
The strongest signal falls in smooth muscle cells, chondrocytes or adipocytes while adjacent expected positive cells are weak (HPA: Low in those cell types; High in listed glandular cells).	The relative pattern conflicts with HPA tissue observations. Consider antibody cross-reactivity or endogenous detection activity as possible causes (general IHC practice); HPA specifically cautions that its tissue antibody targets proteins from more than one gene (HPA: tissue reliability).
Diffuse colour covers many cells and extracellular spaces, obscuring discrete cytoplasmic granules (HPA: granular cytoplasmic profile).	This prevents a reliable localisation call. Uneven blocking, excess primary antibody or detection background can produce diffuse staining (general IHC practice); compare a no-primary control before assigning the colour to ATAD3A.
Little or no signal appears in a run that includes an HPA-reported high-staining tissue, such as colon glandular cells (HPA: High in colon glandular cells).	First assess tissue preservation, retrieval, antibody incubation and detection with appropriate run controls (general IHC practice). An absent signal does not by itself prove ATAD3A absence; HPA reports staining levels, not guaranteed positivity under every laboratory condition.

💡Expected ATAD3A appearanceCall a section positive when expected cells show discernible granular cytoplasmic colour, potentially strong in HPA-listed high-staining populations, while dominant nuclear or diffuse extracellular colour warrants an artefact check (HPA: tissue IHC; UniProt Q9NVI7: mitochondrial localisation).

How each factor affects the staining
Mitochondrial topology (UniProt Q9NVI7)	A single transmembrane segment at 247–264 separates an intermembrane region from a matrix region (UniProt Q9NVI7 topology). The payload gives no antibody epitope, so topology cannot predict which region the IHC antibody recognises or which retrieval condition works.
Tissue reference strength (HPA: Approved IHC)	HPA describes granular cytoplasmic staining across tissues, with medium consistency between staining and RNA expression and a multiple-gene targeting caution (HPA: tissue reliability). Use its high and low cell populations as comparisons, without treating their intensity as proof of antibody specificity.
Reported isoforms and processing (UniProt Q9NVI7)	UniProt lists 3 isoforms and an annotated chain spanning residues 2–586, with no signal peptide or propeptide (UniProt Q9NVI7). Without the antibody epitope, these annotations cannot establish isoform coverage or explain a missing IHC signal.
Retrieval choice (general IHC practice)	Optimise antigen retrieval against a known staining reference and assess whether tissue detail remains interpretable (general IHC practice). No ATAD3A-specific retrieval condition or fixation sensitivity is supplied; HPA staining levels and UniProt topology do not establish either effect.
Chromogenic detection controls (general IHC practice)	A no-primary control helps identify background from detection reagents or endogenous enzyme activity (general IHC practice). Keep that signal separate from the granular cytoplasmic reference pattern (HPA: tissue IHC); the payload does not identify a target-specific endogenous activity.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
The expected high-staining cell population is blank (HPA: High in listed cell populations).	Run failure, unsuitable retrieval or an ineffective primary antibody are possibilities (general IHC practice); absence alone cannot identify which step failed.	Review an appropriate positive run control, then check retrieval, primary incubation and detection in sequence (general IHC practice). Reassess the same cell population against the HPA tissue reference.
Most of the section has diffuse chromogenic colour instead of discrete granules (HPA: granular cytoplasmic profile).	Primary antibody excess, insufficient blocking or detection background are possible explanations (general IHC practice).	Compare a no-primary control and optimise blocking, washes and primary concentration (general IHC practice); score localisation only after the cytoplasmic pattern becomes distinguishable.
Colour is strongest in HPA-low cells and weak in HPA-high cells (HPA: tissue IHC levels).	Cross-reactivity or endogenous detection activity may contribute (general IHC practice); HPA also warns of antibody targeting proteins from more than one gene (HPA: tissue reliability).	Check a no-primary control, cell identity and the relative pattern in high and low reference populations (general IHC practice; HPA: tissue IHC). Avoid calling the low-cell signal specific on intensity alone.
The apparent positive result is chiefly nuclear or extracellular (HPA: granular cytoplasmic profile).	Compartment misassignment, counterstain confusion or background may explain the mismatch (general IHC practice).	Recheck morphology and detection controls, then require a granular cytoplasmic pattern before comparing with the mitochondrial localisation reference (HPA: tissue IHC; UniProt Q9NVI7).
Different sections show inconsistent intensity within the same expected cell population (HPA: tissue IHC levels).	Section handling, retrieval or incubation variation can change IHC staining (general IHC practice); the supplied sources do not show ATAD3A-specific fixation sensitivity.	Compare matched run controls and recorded processing conditions (general IHC practice). Interpret intensity only where morphology and the expected cell population remain comparable (HPA: tissue IHC).
IF/ICC question: what localisation should an independent fluorescence check show?	A fluorescence image can address subcellular distribution, while chromogenic tissue IHC provides a coarser localisation readout (general microscopy practice).	Look for a mainly mitochondrial signal (HPA: subcellular ICC-IF; UniProt Q9NVI7), while retaining HPA's multiple-gene antibody caution; consult the separate IF/ICC guide for assay design.

Source: UniProt Q9NVI7 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing ATAD3A staining

Immunohistochemistry of paraffin-embedded human stomach using ATAD3A antibody at dilution of 1:100 .Perform microwave antigen retrieval with 10 mM PBS buffer pH 7.2 before commencing with IHC staining protocol.

A06057-1 · IHCImmunohistochemistry of paraffin-embedded human stomach using ATAD3A antibody at dilution of 1:100 .Perform microwave antigen retrieval with

Immunohistochemistry of paraffin-embedded mouse brain using ATAD3A antibody at dilution of 1:100 .Perform microwave antigen retrieval with 10 mM PBS buffer pH 7.2 before commencing with IHC staining protocol.

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.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for ATAD3A IHC & IF

🧪Run breast first: glandular cells should stain strongly (HPA: High in breast glandular cells). HPA detects ATAD3A in all 45 scored tissues, so use no-primary and isotype controls for the negative comparison; no cell type on the positive slide is established as a true negative, and any unstained cells should lack brown reaction product (HPA: no negative tissue rows).

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 slide and an isotype control matched to the primary antibody’s host species and clonality (standard IHC practice). A validated ATAD3A knockout specimen can provide a biological negative; block endogenous peroxidase and check for pigment or other background before scoring breast staining (standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the selected stomach paraffin-section caption does not state a fixative (selected-SKU tissue-IHC caption: A06057-1). That caption uses microwave retrieval in 10 mM PBS at pH 7.2 and a 1:100 antibody dilution, but it does not establish whether retrieval is required for other tissues (selected-SKU tissue-IHC caption: A06057-1). Frozen sections and IF cannot be ranked as easier from the supplied evidence; assess background in the breast slide with its controls (HPA: High in breast glandular cells; 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 section chromogenic IHC by checking retrieval, compartmental pattern, controls and scoring against the available evidence.

Which retrieval should I use when ATAD3A staining is weak?

Start with microwave antigen retrieval in 10 mM PBS, pH 7.2, the condition used for the catalog antibody’s paraffin section image (IHC caption A06057-1). The caption reports neither heating time nor temperature, so record both during optimization rather than treating an unstated setting as validated (IHC caption A06057-1). If staining remains weak, compare citrate pH 6.0 at 95–98 °C for 20 minutes as this page’s fallback condition (page retrieval rule). Keep the antibody at the caption’s 1:100 starting dilution while comparing retrieval conditions, and assess granular cytoplasmic staining alongside tissue damage and background (IHC caption A06057-1; HPA tissue IHC; standard IHC practice).

Could fixation explain weak or uneven ATAD3A staining?

ATAD3A-specific fixation sensitivity is unknown: the catalog image describes a paraffin section but does not state its fixative (IHC caption A06057-1). For troubleshooting, record the actual fixative and fixation duration for each specimen, then compare sections processed together under the same retrieval and detection conditions (standard IHC practice). Use the caption’s microwave retrieval in 10 mM PBS, pH 7.2, and its 1:100 antibody dilution as documented starting conditions (IHC caption A06057-1). If staining differs across specimens, inspect morphology and processing history before attributing the difference to ATAD3A abundance or to a target-specific fixation effect (standard IHC practice).

What staining pattern is plausible for ATAD3A in tissue sections?

Expect granular cytoplasmic staining at the light microscope level, consistent with the reported tissue pattern and mitochondrial location (HPA tissue IHC; UniProt Q9NVI7 subcellular location). ATAD3A spans the mitochondrial inner membrane at residues 247–264, with its N-terminal region on the intermembrane side and C-terminal region in the matrix (UniProt Q9NVI7 topology). Diffuse nuclear staining alone therefore needs scrutiny against a positive tissue control and a no-primary control (UniProt Q9NVI7 subcellular location; standard IHC practice). Because HPA cautions that its antibodies target proteins from multiple genes, agreement with its granular pattern supports localisation but does not establish antibody specificity (HPA tissue IHC).

How can epitope placement change my interpretation of ATAD3A staining?

ATAD3A has 3 listed isoforms, while the supplied catalog caption does not identify the antibody epitope or establish isoform coverage (UniProt Q9NVI7 isoforms; IHC caption A06057-1). Its transmembrane segment occupies residues 247–264, separating an intermembrane N-terminal region from a matrix-facing C-terminal region (UniProt Q9NVI7 topology). Check the antibody’s documented immunogen or epitope before interpreting an absent signal as loss of every isoform, and compare sections processed under identical retrieval conditions (standard IHC practice). UniProt lists modifications at residues 2, 321 and 491; without epitope mapping, their effect on this IHC result is unknown (UniProt Q9NVI7 modified residues; IHC caption A06057-1).

How should I follow up an IHC pattern with multiplex IF?

Treat IF as a separate validation workflow: the supplied catalog evidence documents paraffin section IHC at 1:100, not an IF protocol (IHC caption A06057-1). Pair ATAD3A with a marker of the expected cell population, such as glandular cells in an HPA high-staining tissue, and assess overlap at the cell level (HPA tissue IHC; standard IF practice). Choose fluorophores after checking the tissue’s autofluorescence in unstained controls, and separate channels enough to limit bleed-through (standard IF practice). Because the epitope is unspecified, optimize permeabilisation empirically; accessibility may differ across the intermembrane N-terminal and matrix-facing C-terminal regions (IHC caption A06057-1; UniProt Q9NVI7 topology; standard IF practice).

How do I reduce diffuse brown background without losing ATAD3A signal?

First compare a no-primary control with the stained section to identify detection-related colour that does not require the ATAD3A antibody (standard IHC practice). In a peroxidase and DAB workflow, block endogenous peroxidase, optimize protein blocking and washes, and monitor DAB development; these are general IHC steps, not ATAD3A-specific validation (standard IHC practice). Keep the documented 1:100 dilution and microwave retrieval in 10 mM PBS, pH 7.2 as starting conditions while changing one variable at a time (IHC caption A06057-1). Retain granular cytoplasmic signal when judging improvement, and flag uniform haze or section-edge colour separately from cellular staining (HPA tissue IHC; standard IHC practice).

What is a defensible way to quantify ATAD3A IHC? ⚠ ANSWER MARKED FOR VERIFICATION

Define the scored cell population and tissue area before reviewing intensity, because HPA reports granular cytoplasmic staining across tissues and low tissue RNA specificity (HPA tissue IHC; HPA RNA specificity; standard IHC practice). Record the percentage of positive cells and cytoplasmic intensity, or calculate an H-score from 0–300 using percentages at intensity grades 0–3 (standard IHC practice). Normalize positive-cell counts to eligible cells, or staining density to analyzed tissue area in mm², excluding folds, necrosis and section edges (standard IHC practice). Apply the same thresholds, retrieval conditions and image settings across groups, and report cell type separately when tissue composition differs (standard IHC practice).

When should I doubt an apparent ATAD3A-positive area?

Give greatest weight to granular cytoplasmic staining in intact cells, matching the HPA tissue pattern and mitochondrial assignment (HPA tissue IHC; UniProt Q9NVI7 subcellular location). Check whether the stained cell population fits the specimen: HPA reports high staining in several glandular cell populations and low staining in adipocytes, chondrocytes and smooth muscle cells (HPA tissue IHC). Isolated nuclear colour, section-edge enhancement, necrotic debris or signal persisting in a no-primary control warrants investigation before scoring; endogenous peroxidase is one possible source in chromogenic workflows (standard IHC practice). HPA explicitly cautions that its tissue antibody evidence may include proteins from more than one gene, so morphology and localisation alone cannot prove ATAD3A specificity (HPA tissue IHC).

Boster reagents

Best ATAD3A / ATPase family AAA domain-containing protein 3A IHC Antibodies

Anti-ATAD3A antibodies have IHC images from paraffin-embedded human stomach and mouse brain (A06057-1 image captions), plus IF/ICC images from Daudi cells (A06057 image captions).

Real IHC data Immunohistochemistry of paraffin-embedded human stomach using ATAD3A antibody at dilution of 1:100 .Perform microwave antigen retrieval with 10 mM PBS buffer pH 7.2 before commencing with IHC staining protocol.

Anti-ATAD3A Antibody

Cat # A06057-1

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Real IF data Immunocytochemistry of ATAD3A in Daudi cells with ATAD3A antibody at 20 μg/mL.

Anti-ATAD3A Antibody

Cat # A06057

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A06057-1 is listed for IHC and has paraffin-section images from human stomach and mouse brain at 1:100 (catalog applications; A06057-1 image captions). A06057 is listed for IF/ICC and has Daudi-cell images at 20 μg/mL (catalog applications; A06057 image captions).

Which to pick: Choose A06057-1 for tissue IHC: its images show paraffin sections with microwave retrieval in 10 mM PBS, pH 7.2; the fixative is unreported (A06057-1 image captions). Choose A06057 for IF/ICC; its images show Daudi cells, while its catalog lists human, mouse and rat reactivity, including rat for cross-species planning (A06057 image captions; catalog reactivity). A06057-1 lists human and mouse reactivity, and clonality is unreported for both antibodies (catalog reactivity; catalog 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.