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AADAT / Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial · IHC design guide
Design Immunohistochemistry for AADAT
Plan chromogenic AADAT IHC in paraffin sections using hepatocytes and renal proximal tubules as positive references (HPA tissue IHC). Assess cytoplasmic staining (HPA tissue IHC) and start with the catalog antibody’s 1:50–1:200 IHC dilution range (datasheet A09111).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
AADAT 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 in tissue; mitochondrial protein (HPA tissue IHC; UniProt) | |
| Staining pattern | Hepatocyte and proximal-tubule cytoplasm; low GI gland staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, heat-mediated (datasheet A09111) | |
| Positive control | Kidney+1 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 paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | RNA and antibody staining have medium agreement (HPA tissue IHC) | |
| Regulation | Liver-enhanced RNA expression (HPA tissue RNA) | |
| Isoform / epitope | 2 isoforms; mature chain spans residues 30–425; check epitope coverage (UniProt) |
Section 1
Recommended AADAT IHC & IF Protocols
Compare the catalog antibody’s citrate pH 6.0 HIER protocol (datasheet A09111) with the published TNBC IHC protocol (PMC12874059).
| Sample | Paraffin-embedded human liver tissue; fixative not specified (datasheet A09111) |
| Fixation | Image fixative and duration unreported (datasheet A09111); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6.0 (datasheet A09111); 20 min, 95–100 °C (standard) |
| 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-AADAT, 1:50-1:200 (datasheet A09111) |
| 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 | AADAT-positive staining in proximal tubules (cell body) of kidney (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in liver, renal tubules and low expression in the gastrointestinal tract. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER for the catalog antibody (datasheet A09111); the paper reports pressure cooking for 10 minutes without specifying a retrieval buffer (PMC12874059).
Section 2
What Is the Expected AADAT Staining Pattern?
In paraffin-section IHC, expect cytoplasmic AADAT staining in hepatocytes and renal proximal tubules, with weaker staining in some gastrointestinal glandular cells (HPA tissue IHC). UniProt places AADAT in mitochondria and annotates no transmembrane segment (UniProt Q8N5Z0). HPA rates its tissue IHC reliability Enhanced, while reporting medium consistency between antibody staining and RNA expression (HPA tissue IHC).
| Hepatocytes and renal proximal tubule cell bodies show moderate cytoplasmic chromogen; gastrointestinal glandular cells show weaker staining. | This matches the reported tissue pattern: hepatocytes and proximal tubules stain at Medium level, while the gastrointestinal tract has low expression (HPA tissue IHC). Score the relevant cells and their cytoplasm, rather than treating every colored structure in the section as target signal (general IHC practice). |
| Strong nuclear staining appears in otherwise positive cells, or a membrane rim dominates the chromogenic IHC result. | Nuclear dominance conflicts with cytoplasmic tissue IHC and mitochondrial annotation; investigate artefact (HPA tissue IHC; UniProt Q8N5Z0). A membrane rim needs separate evaluation: HPA reports approved plasma-membrane localisation in ICC-IF, so its IF observation cannot simply be dismissed or assumed to validate membrane staining in paraffin IHC (HPA subcellular ICC-IF). |
| Adipocytes, respiratory epithelial cells or bone-marrow hematopoietic cells stain prominently. | HPA reports AADAT as Not detected in those cells (HPA tissue IHC). A strong result there raises concern for cross-reactivity, endogenous chromogenic activity or nonspecific binding (general IHC practice). Review the negative control and compare the suspect cells with hepatocytes or proximal tubules before assigning target positivity. |
| Color spreads across stroma, lumina and many unrelated cells without clear cell-body localisation. | This is less persuasive than the reported cytoplasmic staining of selected cell types (HPA tissue IHC). Diffuse color can reflect background from detection chemistry, antibody concentration or incomplete blocking (general IHC practice); it does not establish a new AADAT-positive tissue pattern. |
| Hepatocytes or renal proximal tubules lack visible signal in the same run. | Both are reported at Medium staining level and provide useful positive reference cells (HPA tissue IHC). Check section quality, retrieval, antibody application and detection with run controls (general IHC practice). Absence of staining alone does not prove biological absence, particularly given HPA's reported medium staining–RNA consistency (HPA tissue IHC). |
💡Expected AADAT appearanceCall the IHC result consistent when moderate cytoplasmic staining marks hepatocytes or renal proximal tubule cell bodies, with only low gastrointestinal glandular staining expected; prominent staining of HPA Not detected cell types is a suspect positive (HPA tissue IHC).
| Tissue and cell selection | Liver hepatocytes and kidney proximal tubules are reported at Medium level; gastrointestinal glandular cells are low (HPA tissue IHC). Use the first two as positive references and interpret a faint gastrointestinal result in that context, without imposing a single intensity threshold across tissues. |
| Compartment evidence | UniProt assigns AADAT to mitochondria and annotates no transmembrane segment; tissue IHC describes cytoplasmic expression (UniProt Q8N5Z0; HPA tissue IHC). Conventional chromogenic IHC may show cell-body cytoplasm without resolving individual mitochondria (general microscopy practice). |
| IHC versus IF/ICC localisation | Q: Should an IF/ICC plasma-membrane image redefine the paraffin IHC pattern? A: No. HPA's approved ICC-IF main location is plasma membrane, with vesicles additional, whereas its tissue IHC profile is cytoplasmic (HPA subcellular ICC-IF; HPA tissue IHC). Interpret each application against its own evidence. |
| Protein forms and epitope information | UniProt lists two isoforms and a chain spanning residues 30–425 (UniProt Q8N5Z0). No antibody epitope or isoform-specific IHC result is supplied, so neither feature predicts which form the IHC-validated antibody detects or explains a missing tissue signal. |
| Validation strength | HPA lists antibody HPA037502 as IHC Enhanced and ICC Approved; its tissue profile notes medium consistency with RNA expression (HPA antibodies; HPA tissue IHC). These labels support using the reported pattern as a reference, while leaving discrepant cells and compartments to be checked with controls. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected hepatocyte and proximal-tubule staining is absent. | A failed retrieval, primary-antibody or detection step is possible (general IHC practice); both cell types have Medium staining in HPA (HPA tissue IHC). | Review the run's positive control and each IHC step, then repeat under the established paraffin IHC conditions (general IHC practice). Do not infer AADAT loss from one blank section. |
| The whole section has diffuse brown background. | Nonspecific antibody binding, excess primary antibody or detection background can obscure a cell-specific pattern (general IHC practice). | Compare a no-primary control, check blocking and detection, and optimize primary-antibody concentration within a validated IHC workflow (general IHC practice). Reassess hepatocytes and proximal tubules against the HPA pattern (HPA tissue IHC). |
| Unexpected cell types stain strongly. | Cross-reactivity or endogenous chromogenic activity is possible (general IHC practice), especially in cells reported Not detected by HPA (HPA tissue IHC). | Inspect tissue-matched negative controls and the detection-only control; confirm that expected positive cells stain in the same run (general IHC practice; HPA tissue IHC). Treat the unexpected cells as unresolved until those checks agree. |
| Nuclear color dominates the section. | That compartment conflicts with HPA's cytoplasmic tissue profile and UniProt's mitochondrial annotation (HPA tissue IHC; UniProt Q8N5Z0). | Check counterstain, chromogen deposition and no-primary control before scoring nuclei as positive (general IHC practice). Re-evaluate whether cytoplasmic cell-body staining remains in the reported positive cells (HPA tissue IHC). |
| Gastrointestinal glands look nearly negative while liver and kidney controls stain. | Gastrointestinal glandular staining is reported as low, compared with Medium hepatocyte and proximal-tubule staining (HPA tissue IHC). | Interpret the faint result against the stronger controls and local background (general IHC practice). Avoid increasing detection solely to force gastrointestinal glands to match liver or kidney intensity (HPA tissue IHC). |
| A membrane rim appears in paraffin IHC and resembles the ICC-IF report. | HPA approves plasma-membrane localisation in ICC-IF, while its tissue IHC profile describes cytoplasm; the supplied evidence does not resolve this application difference (HPA subcellular ICC-IF; HPA tissue IHC). | Score the IHC slide against its tissue controls and cytoplasmic reference pattern; inspect negative controls for edge staining (HPA tissue IHC; general IHC practice). Record persistent membrane staining as a discrepancy, rather than assigning it automatically to AADAT. |
Experiment evidence
IHC & IF images showing AADAT staining
A09111 · IHCImmunohistochemistry of paraffin-embedded human liver using AADAT Rabbit pAb at dilution of 1:200 .Perform high pressure antigen retrieval w
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000109576-AADAT/tissue/cerebral+cortex#imid_19774857
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000109576-AADAT/tissue/colon#imid_19791119
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000109576-AADAT/tissue/liver#imid_19791136
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000109576-AADAT/tissue/kidney#imid_19791129
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA037502; green = AADAT, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA037502; green = AADAT, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for AADAT IHC & IF
🧪Run liver first: hepatocytes should show staining (HPA: Medium in hepatocytes; UniProt Q8N5Z0: higher expression in liver). Use adipose tissue as the negative comparison: adipocytes are listed as not detected (HPA: Not detected in adipocytes). On the liver slide, morphologically intact cells without specific chromogen deposition can serve as internal background comparators; the HPA row does not establish that every non-hepatocyte is AADAT-negative (HPA: Medium in hepatocytes).
Positive control tissue: Kidney (Proximal tubules (cell body), HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show AADAT in A-431, U-251MG, U2OS, with annotated localisation: Plasma membrane (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) slide, a concentration-matched rabbit polyclonal isotype control, and an AADAT knockout specimen if available (catalog caption: rabbit pAb; standard IHC control practice). Quench endogenous peroxidase and assess pigment-related background in liver before interpreting chromogenic signal (standard liver IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A09111 liver caption does not report a fixative (selected-SKU caption). That caption specifies high-pressure retrieval in 10 mM citrate, pH 6.0, before paraffin-section IHC at 1:200; it does not establish whether retrieval is essential under other conditions (selected-SKU caption). The supplied evidence does not establish whether frozen sections or IF are easier; IF localisation needs independent validation because HPA reports plasma membrane and vesicles while UniProt assigns AADAT to mitochondria (HPA subcellular; UniProt Q8N5Z0: Mitochondrion).
HPA tissue IHC evidence for AADAT
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Kidney | Proximal tubules (cell body) | Medium | Protein (IHC) | HPA → |
| Liver | Hepatocytes | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced AADAT IHC Tips
Troubleshoot AADAT staining in paraffin sections by checking retrieval, compartment, cell identity, and controls before interpreting intensity.
Which retrieval should I start with for weak AADAT staining?
Use heat-mediated retrieval in 10 mM citrate at pH 6.0 for paraffin AADAT IHC (datasheet A09111). The selected liver image used high-pressure retrieval and a 1:200 antibody dilution, so compare weak runs against those documented conditions (caption A09111). Include liver hepatocytes or kidney proximal tubules as reference cells when assessing recovery of signal (HPA tissue IHC). If staining remains weak, compare a second heat-retrieval condition on adjacent sections while keeping detection and imaging settings fixed (general IHC practice). Check tissue preservation and the no-primary control before treating stronger staining as improved AADAT detection (general IHC practice).
Could fixation explain variable AADAT staining between sections?
Target-specific sensitivity of AADAT to fixation is unknown from the supplied evidence; the selected paraffin liver caption does not state its fixative (caption A09111). Record each specimen’s fixative, fixation duration, processing history, and section age before comparing staining intensity (general IHC practice). Process matched sections together, apply the documented 10 mM citrate retrieval at pH 6.0, and include a reference tissue in every run (datasheet A09111; general IHC practice). If specimens differ in fixation history, interpret an intensity difference cautiously until matched processing or an independent assay supports it (general IHC practice).
How should I assess mitochondrial versus membrane-like AADAT staining?
AADAT is annotated as mitochondrial and has no transmembrane segment, making a predominantly cytoplasmic pattern biologically plausible in tissue IHC (UniProt Q8N5Z0). HPA tissue IHC reports cytoplasmic staining in liver and renal tubules, while its cell-based IF record reports mainly plasma membrane and additional vesicle localisation (HPA tissue IHC; HPA subcellular). Compare the pattern within hepatocytes and proximal tubules against neighboring negative or low-staining cells on the same slide (HPA tissue IHC; general IHC practice). Treat a crisp membrane-only rim as unresolved until controls or an orthogonal localisation assay explain the difference between datasets (UniProt Q8N5Z0; HPA subcellular; general IHC practice).
Could the antibody epitope miss an AADAT isoform?
AADAT has 2 annotated isoforms, but the supplied evidence does not identify the catalog antibody’s epitope or establish isoform-specific recognition (UniProt Q8N5Z0; caption A09111). The annotated mature chain spans residues 30–425, and residue 263 has alternative modification annotations, which makes epitope mapping relevant when investigating discordant stains (UniProt Q8N5Z0). Obtain the immunogen or epitope sequence and compare it with both isoform sequences before claiming shared or selective detection (general IHC practice). If available, use an antibody against a separate region or an independent assay on matched samples to test whether the observed cell pattern is reproducible (general IHC practice).
How can IF help check an ambiguous AADAT IHC pattern?
For a separate IF experiment, multiplex AADAT with a marker for hepatocytes or proximal tubules, the cells reported positive in tissue IHC (HPA tissue IHC; general IF practice). Choose spectrally separated fluorophores and place the weaker signal in a channel with low tissue autofluorescence, checking unstained sections first (general IF practice). Because AADAT is annotated as mitochondrial without a transmembrane segment, intracellular epitope access generally calls for permeabilisation after fixation; adjust detergent strength once the antibody’s epitope is known (UniProt Q8N5Z0; general IF practice). Compare compartment patterns cautiously: HPA cell-based IF reports membrane and vesicle localisation, whereas tissue IHC reports cytoplasmic staining (HPA subcellular; HPA tissue IHC).
What controls distinguish AADAT signal from chromogenic background?
Run a no-primary control beside the catalog antibody to identify detection-system staining and residual endogenous peroxidase activity (general chromogenic IHC practice). Apply a peroxidase block before the enzyme detection step, then develop DAB for a consistent, monitored interval across comparison slides (general chromogenic IHC practice). Compare staining in hepatocytes and proximal tubules with cells reported negative or low for AADAT, while recognizing that expression varies among tissue compartments (HPA tissue IHC). Diffuse precipitate, section-edge staining, or color in the no-primary control should prompt review of blocking, washing, and DAB development before biological interpretation (general chromogenic IHC practice).
How should I score AADAT IHC across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell compartment and region of interest before scoring; hepatocytes and kidney proximal tubules are documented reference populations for AADAT (HPA tissue IHC; general IHC practice). For cellular staining, report the percentage of positive cells and an H-score on a 0–300 scale, using the same intensity thresholds across slides (general IHC practice). Normalize positive counts to the number of eligible cells, or report positive-cell density per mm² of evaluable tissue when cell counts are impractical (general IHC practice). Exclude folds, necrosis, and slide edges by a prespecified rule, and compare batches through a shared reference section (general IHC practice).
When is an apparent AADAT-positive cell likely an artefact?
A convincing result should show reproducible cellular staining in an expected population, such as hepatocytes or proximal tubules, with appropriate control behavior (HPA tissue IHC; general IHC practice). A mitochondrial annotation and cytoplasmic tissue pattern support intracellular signal, but the HPA cell-based membrane and vesicle pattern makes compartment disagreement worth independent follow-up (UniProt Q8N5Z0; HPA tissue IHC; HPA subcellular). Question isolated staining in a cell population reported as not detected, especially when it tracks section edges, damaged areas, or necrosis (HPA tissue IHC; general IHC practice). If color persists without primary antibody, investigate endogenous enzyme activity or detection background before calling the cells AADAT-positive (general chromogenic IHC practice).
Boster reagents
Best AADAT / Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial IHC Antibodies
A09111 has real IHC data from paraffin-embedded human liver (A09111 IHC image caption). IF/ICC is listed without an IF image (catalog applications; catalog IF image alts). Human, mouse, and rat reactivity is listed (catalog reactivity).
Real IHC data
Anti-AADAT Antibody
Cat # A09111
A09111 is listed for IHC, IF, and ICC and for human, mouse, and rat reactivity (catalog applications; catalog reactivity). Its IHC image shows paraffin-embedded human liver stained at 1:200 after high-pressure retrieval in 10 mM citrate, pH 6.0 (A09111 IHC image caption); no IF image is supplied (catalog IF image alts).
Which to pick: Choose A09111 for paraffin-section tissue IHC based on its human liver image (A09111 IHC image caption); the fixative is unreported (A09111 IHC image caption). For IF/ICC, A09111 is listed with a 1:50–1:200 dilution range, though no IF image is supplied (catalog applications; catalog IF dilution; catalog IF image alts). A09111 is the listed option for human, mouse, and rat, and its IHC caption identifies it as a rabbit polyclonal antibody (catalog reactivity; A09111 IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8N5Z0 (AADAT_HUMAN, Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial).
- Human Protein Atlas. AADAT tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. AADAT subcellular location (ICC-IF): Mainly localized to the plasma membrane. In addition localized to vesicles..
- Human Protein Atlas. AADAT antibody validation summary (1 antibodies).
- AADAT-Driven Metabolic Control of Malate and CoQ(10) Shapes Immune Evasion in Triple-Negative Breast Cancer. bioRxiv : the preprint server for biology 2026 — PMC12874059.
- PubMed PMID:12126930 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for AADAT (Kynurenine/alpha-aminoadipate aminotransferase, mitochondrial). Expected localisation Cytoplasmic staining in tissue; mitochondrial protein (HPA tissue IHC; UniProt). Antigen retrieval: Citrate pH 6.0 HIER, heat-mediated (datasheet A09111). Validated IHC antibody: A09111 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Kidney (Proximal tubules (cell body)); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human liver tissue; fixative not specified (datasheet A09111); 2. Fixation: Image fixative and duration unreported (datasheet A09111); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: Citrate pH 6.0 (datasheet A09111); 20 min, 95–100 °C (standard); 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-AADAT, 1:50-1:200 (datasheet A09111); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: RNA and antibody staining have medium agreement (HPA tissue IHC); Isoform / epitope: 2 isoforms; mature chain spans residues 30–425 ; check epitope coverage (UniProt); Regulation: Liver-enhanced RNA expression (HPA tissue RNA). Evidence: UniProt Q8N5Z0, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the plasma membrane. In addition localized to…, Datasheet A09111, PMC12874059. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/AADAT/immunohistochemistry-ihc-guide.