ACO2 / Aconitate hydratase, mitochondrial · IHC design guide

Design Immunohistochemistry for ACO2

Plan ACO2 paraffin-section IHC around granular cytoplasmic staining (HPA tissue IHC) and mitochondrial localisation (UniProt). This guide covers fixation, retrieval, a catalog antibody concentration of 2–5 μg/ml (datasheet M03096-3), chromogenic detection, and scoring.

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

Immunohistochemistry protocol sheet for ACO2 (IHC for ACO2): expected localisation Granular cytoplasm (HPA tissue IHC); mitochondrion (UniProt), antibody M03096-3, validated IHC image, and IHC protocol steps — Printable **ACO2** IHC protocol sheet — expected localisation Granular cytoplasm (HPA tissue IHC); mitochondrion (UniProt), antibody M03096-3, controls and protocol steps. Open the full ACO2 IHC guide →

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ACO2 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); mitochondrion (UniProt)
	Staining pattern	Widespread granular cytoplasmic staining (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet M03096-3)
	Positive control ⓘ	Adrenal gland+4 more · see all
	Negative control ⓘ	Adipose tissue

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation consistent across sections. (standard IHC practice; not target-specific)
	Caveat	Adipocytes may be unstained (HPA tissue IHC)
	Regulation	RNA enhanced: heart/skeletal/tongue (HPA tissue RNA)
	Isoform / epitope	No isoforms annotated; mature chain spans residues 28–780 (UniProt)

Section 1

Recommended ACO2 IHC & IF Protocols

The catalog antibody’s IHC-P protocol is paired with one published ACO2 IHC protocol using osteosarcoma and paracancerous tissues (PMC9534693 methods).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet M03096-3)
Fixation	Image fixative and duration unreported (datasheet M03096-3); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: EDTA pH 8.0 (datasheet M03096-3); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet M03096-3)
Primary antibody	Mouse monoclonal (clone 4C12D1) anti-ACO2, 2-5 μg/ml (datasheet M03096-3)
Primary incubation	Overnight at 4 °C (datasheet M03096-3)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet M03096-3)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	ACO2-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression in a granular pattern. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA at pH 8.0 (datasheet M03096-3); Tri-EDTA at pH 9 is a published alternative (PMC9534693 methods).

Section 2

What Is the Expected ACO2 Staining Pattern?

ACO2 localizes to mitochondria (UniProt Q99798; HPA subcellular ICC-IF). In paraffin-section IHC, expect granular cytoplasmic staining across many tissues, including glandular cells and glia reported as high (HPA tissue IHC). HPA describes the tissue pattern as ubiquitous and rates its IHC evidence Enhanced, citing agreement between staining and RNA expression (HPA tissue IHC). ACO2 has no transmembrane segment (UniProt Q99798 topology).

What am I looking at on my slide?
Granular cytoplasmic signal in adrenal or appendix glandular cells (HPA tissue IHC: High).	This fits the reported high staining in those cells and ACO2's mitochondrial location (HPA tissue IHC; UniProt Q99798). Judge the granules against tissue structure and controls; a positive control alone does not establish that every stained structure is specific (general IHC practice).
Predominantly nuclear staining or a sharp cell-surface rim.	Neither pattern matches mitochondrial localization or the reported granular cytoplasmic IHC profile (UniProt Q99798; HPA tissue IHC). Check section morphology, counterstain and control slides before interpreting a compartment mismatch as ACO2 (general IHC practice).
Strong signal restricted to adipocytes, with little signal in adjacent cell types.	HPA reports ACO2 as not detected in adipocytes, while describing a broad granular cytoplasmic tissue pattern (HPA tissue IHC). Treat this discordance as possible cross-reactivity or endogenous chromogen activity; compare negative controls before assigning it to ACO2 (general IHC practice).
Uniform color across cells, extracellular areas and section edges.	A uniform deposit obscures the granular cytoplasmic pattern reported by HPA (HPA tissue IHC). Assess background with a primary-antibody omission control, then review blocking, washing and detection conditions (general IHC practice).
No convincing signal in adrenal glandular cells or bone-marrow hematopoietic cells.	Both are reported as high-staining cell populations (HPA tissue IHC). First confirm that the relevant cells are present and preserved; then compare a suitable positive control and review the antibody, retrieval and detection workflow (general IHC practice).

💡Expected ACO2 appearanceCall a section positive when intact glandular cells or glia show clear granular cytoplasmic signal, consistent with reported high staining and mitochondrial location (HPA tissue IHC; UniProt Q99798); isolated nuclear, surface-rim or broad extracellular color is suspect (general IHC practice).

How each factor affects the staining
Compartment and topology	Mitochondrial localization supports granular cytoplasmic interpretation (UniProt Q99798; HPA subcellular ICC-IF). With no transmembrane segment, a crisp cell-surface outline lacks support as the expected ACO2 pattern (UniProt Q99798 topology).
Cell population	HPA reports high staining in several glandular populations, hematopoietic cells and glia, but no detected signal in adipocytes (HPA tissue IHC). Score the named cell population rather than treating a whole tissue as uniformly positive or negative (general IHC practice).
Evidence strength	The reported tissue pattern has Enhanced reliability, and HPA001097 has Enhanced IHC validation (HPA tissue IHC; HPA antibody record). These ratings support the reference pattern; they do not validate an arbitrary reagent or every signal on an individual slide (general IHC practice).
Processing and epitope limits	UniProt annotates the mature ACO2 chain as residues 28–780 and lists no isoforms (UniProt Q99798). The supplied record gives no antibody epitope or target-specific fixation response, so neither differential epitope staining nor fixation sensitivity can be inferred.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Weak or absent staining in an HPA high-staining cell population (HPA tissue IHC).	The slide may lack an interpretable positive cell population, or a general IHC workflow step may have underperformed (general IHC practice).	Verify cell identity and section integrity, then check the positive control, antigen-retrieval run, antibody dilution and detection reagents (general IHC practice).
Nuclear or surface-rim signal dominates the slide.	The compartment conflicts with mitochondrial localization and granular cytoplasmic IHC staining (UniProt Q99798; HPA tissue IHC).	Review morphology and counterstain, compare control slides, and adjust staining or detection conditions if the misplaced signal persists (general IHC practice).
Adipocytes stain strongly despite an otherwise weak section.	HPA reports adipocytes as not detected (HPA tissue IHC). Cross-reactivity or endogenous chromogen activity is possible (general IHC practice).	Check a primary-antibody omission control and the applicable endogenous-activity block; interpret staining by cell type (general IHC practice).
Diffuse deposit hides cytoplasmic granules.	Background from the staining or detection workflow can prevent assessment of the reported granular pattern (general IHC practice; HPA tissue IHC).	Compare omission and positive controls, then review blocking, antibody concentration, washes and chromogen development (general IHC practice).
Two sections show different apparent staining intensity.	The proportion of HPA high-staining cell populations may differ between sections (HPA tissue IHC); staining-run variation is also possible (general IHC practice).	Compare like cell populations and morphology, then evaluate both slides against the same suitable controls before scoring intensity (general IHC practice).
IF/ICC Q&A: What localization should be expected?	HPA approves a mainly mitochondrial ICC-IF location and lists images from A-431, U-251MG and U2OS (HPA subcellular ICC-IF).	Expect mitochondrial localization in that separate application; assess IF/ICC conditions in its own guide, without treating this IHC pattern as an IF/ICC protocol (HPA subcellular ICC-IF).

Source: UniProt Q99798 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing ACO2 staining

IHC analysis of Aconitase 2 using anti-Aconitase 2 antibody (M03096-3). Aconitase 2 was detected in a paraffin-embedded section of human breast cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml mouse anti-Aconitase 2 Antibody (M03096-3) overnight at 4°C. Peroxidase Conjugated Goat Anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Mouse IgG Super Vision Assay Kit (Catalog # SV0001) with DAB as the chromogen.

M03096-3 · IHCIHC analysis of Aconitase 2 using anti-Aconitase 2 antibody (M03096-3). Aconitase 2 was detected in a paraffin-embedded section of human bre

IHC analysis of Aconitase 2 using anti-Aconitase 2 antibody (M03096-3). Aconitase 2 was detected in a paraffin-embedded section of human hepatocellular carcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml mouse anti-Aconitase 2 Antibody (M03096-3) overnight at 4°C. Peroxidase Conjugated Goat Anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Mouse IgG Super Vision Assay Kit (Catalog # SV0001) with DAB as the chromogen.

M03096-3 · IHCIHC analysis of Aconitase 2 using anti-Aconitase 2 antibody (M03096-3). Aconitase 2 was detected in a paraffin-embedded section of human hep

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000100412-ACO2/tissue/cerebral+cortex#imid_364390

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000100412-ACO2/tissue/colon#imid_364300

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000100412-ACO2/tissue/liver#imid_364301

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000100412-ACO2/tissue/kidney#imid_364302

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001097; green = ACO2, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for ACO2 IHC & IF

🧪Run breast tissue first and expect glandular cells to stain (HPA: High in breast glandular cells); the selected IHC caption also documents staining in a paraffin section of human breast cancer (selected-SKU caption: M03096-3). Run adipose tissue as the negative comparator (HPA: Not detected in adipocytes); cells without specific staining within the breast section should show counterstain and background, without granular cytoplasmic DAB signal (standard IHC interpretation).

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 ACO2 in A-431, U-251MG, U2OS, with annotated localisation: Mitochondria (approved) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control; a mouse IgG isotype control matched to the primary antibody; and ACO2-knockout material, if available, as a biological specificity control (selected-SKU caption: mouse primary; standard IHC practice). Block endogenous peroxidase and check for residual DAB signal in the breast section because the selected assay uses HRP/DAB detection (selected-SKU caption: HRP/DAB; standard IHC practice).

⚠️Feasibility: No target-specific fixation window is reported in the supplied evidence, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative unreported). The documented IHC procedure uses heat retrieval in EDTA at pH 8.0, but a requirement for that retrieval condition has not been established (selected-SKU caption: heat retrieval in EDTA, pH 8.0). The supplied evidence does not establish that frozen sections or IF are easier; for IF, assess mitochondrial localization, while in breast IHC check endogenous peroxidase background under HRP/DAB detection (HPA: mitochondria approved; selected-SKU caption: HRP/DAB; standard IHC/IF practice).

HPA tissue IHC evidence for ACO2

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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
Adrenal gland	Glandular cells	High	Protein (IHC)	HPA →
Appendix	Glandular cells	High	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	High	Protein (IHC)	HPA →
Breast	Glandular cells	High	Protein (IHC)	HPA →
Caudate	Glial cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →

Section 3

Advanced ACO2 IHC Tips

Troubleshoot ACO2 staining in paraffin sections by checking retrieval, cellular pattern, controls and scoring against its expected mitochondrial localisation (UniProt Q99798; HPA subcellular).

How should I troubleshoot weak ACO2 staining after antigen retrieval?

Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet M03096-3). The selected tissue image used this retrieval before an overnight incubation at 4°C with 2 μg/ml primary antibody (datasheet M03096-3). If staining remains weak, check that sections were fully deparaffinised, then adjust heating duration while keeping matched control sections together (standard IHC practice). Compare signal in glandular cells with the expected granular cytoplasmic pattern, rather than judging overall brown colour alone (HPA tissue IHC). Excessive retrieval can damage morphology and complicate interpretation, so assess staining alongside tissue preservation (standard IHC practice).

Does fixation explain variable ACO2 staining in paraffin sections?

The selected image identifies a paraffin-embedded section but does not report its fixative, so ACO2-specific fixation sensitivity is unknown (datasheet M03096-3). Record each specimen’s fixative, fixation duration and processing history before comparing staining across batches (standard IHC practice). If signal differs, process a known positive control alongside test sections and hold retrieval and antibody incubation constant (standard IHC practice; HPA tissue IHC). Examine morphology as well as staining, because poor preservation can make granular cytoplasmic signal difficult to score (standard IHC practice; HPA tissue IHC). Do not assign an ACO2-specific fixation effect from its mitochondrial localisation or annotated modifications alone (UniProt Q99798).

What staining pattern should count as correctly localised ACO2?

Expect a granular cytoplasmic IHC pattern consistent with ACO2’s mitochondrial localisation (HPA tissue IHC; HPA subcellular; UniProt Q99798). Evaluate staining within intact cells, using cell boundaries and a light counterstain to separate cytoplasmic granules from nuclear colour (standard IHC practice). High staining in breast glandular cells provides a documented tissue comparison, while staining strength may vary between cell populations (HPA tissue IHC). Diffuse nuclear staining should prompt review of antibody concentration, detection background and section quality before it is scored as ACO2 (HPA subcellular; standard IHC practice). Chromogenic granules alone cannot establish mitochondrial colocalisation; microscopy with a mitochondrial marker can test that assignment (standard IF practice).

Could epitope placement or ACO2 processing change the IHC result?

ACO2 has a reported mature chain spanning residues 28–780, and the supplied record annotates 0 isoforms (UniProt Q99798). Check the antibody’s documented immunogen or epitope before assuming that a processing difference explains absent staining (standard IHC practice; UniProt Q99798). The record lists acetylation and succinylation examples near residues 31, 50 and 138, but provides no evidence that they alter this antibody’s IHC binding (UniProt Q99798). Compare retrieval conditions and a positive control first, with identical detection settings across sections (standard IHC practice). If epitope information is unavailable, report the result as antibody-dependent staining rather than attributing it to a particular ACO2 form (standard IHC practice).

How can IF help resolve an uncertain chromogenic ACO2 pattern?

Use IF as a separate validation experiment, since the selected antibody caption documents tissue IHC conditions rather than an IF protocol (datasheet M03096-3). Multiplex ACO2 with a marker for the expected cell population, such as glandular cells in breast tissue, and with a mitochondrial marker when compartment assignment matters (HPA tissue IHC; HPA subcellular; standard IF practice). Choose spectrally separated fluorophores after checking unstained tissue for autofluorescence; a far-red channel may improve contrast where shorter wavelengths are bright (standard IF practice). Optimise gentle permeabilisation for the intracellular mitochondrial epitope while preserving cellular structure (UniProt Q99798; standard IF practice). Include single-label controls to assess bleed-through before interpreting overlap (standard IF practice).

How do I distinguish ACO2 signal from chromogenic background?

The selected IHC image used 10% goat serum blocking, a peroxidase-linked secondary antibody and DAB development (datasheet M03096-3). Include a primary-antibody omission control and block endogenous peroxidase before chromogen development to assess non-specific brown signal (standard IHC practice). If background persists, inspect whether it tracks tissue edges, damaged areas or all cell compartments instead of the expected granular cytoplasm (HPA tissue IHC; standard IHC practice). Titrate the primary antibody around the documented 2 μg/ml condition while keeping exposure and development comparable (datasheet M03096-3; standard IHC practice). Review wash stringency and secondary-antibody controls before calling widespread diffuse colour increased ACO2 expression (standard IHC practice).

How should I quantify ACO2 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION

Score intact cells within the same defined tissue compartment, because ACO2 staining is reported as granular cytoplasmic and cell populations can differ in intensity (HPA tissue IHC). An H-score combines the percentage of cells at intensity grades 0–3 into a 0–300 scale; record the percentage positive separately when useful (standard IHC practice). For spatial analyses, report positive-cell density per mm² of viable tissue and state the threshold used (standard IHC practice). Normalise comparisons to the same cell type, sampled area, section quality and staining batch, using a shared control section (standard IHC practice). Exclude folds and necrotic regions before scoring, and keep counterstaining and image settings consistent (standard IHC practice).

When is apparent ACO2 positivity more likely to be artefact?

A credible result shows intracellular granular cytoplasmic staining consistent with mitochondrial ACO2, in morphologically intact cells (HPA tissue IHC; HPA subcellular; UniProt Q99798). Breast glandular cells are a documented strongly stained population; adipocytes are listed as not detected in the supplied tissue profile (HPA tissue IHC). Treat nuclear-only colour, staining confined to section edges or necrotic areas, and signal in the primary-antibody omission control as reasons to investigate artefact (HPA subcellular; standard IHC practice). Check endogenous peroxidase activity when brown deposits appear independently of primary antibody (standard IHC practice). Interpret intensity only after comparing appropriate cell populations, tissue preservation and a matched positive control (standard IHC practice).

Boster reagents

Best ACO2 / Aconitate hydratase, mitochondrial IHC Antibodies

M03096-3 has a real ACO2 IHC figure from a human paraffin section (M03096-3 image caption). PB9973 is listed for human ICC/IF (PB9973 catalog).

Real IHC data

Anti-Aconitase 2 Antibody ® (monoclonal, 4C12D1)

Cat # M03096-3

View product →

The rendered M03096-3 card documents IHC on paraffin-embedded human breast cancer; its other captions show human hepatocellular carcinoma, laryngeal squamous cell carcinoma and lung adenocarcinoma (M03096-3 image captions). The catalog lists M03096-3 for IHC with human, mouse and rat reactivity (M03096-3 catalog).

Which to pick: For tissue IHC, choose mouse monoclonal M03096-3: its own figure shows a human paraffin section, and the fixative is unreported (M03096-3 image caption; M03096-3 catalog). For IF/ICC, rabbit polyclonal PB9973 is listed for human ICC/IF at 5 μg/ml (PB9973 catalog). For cross-species planning, rabbit monoclonal M03096-2 lists IHC and ICC/IF applications and human, mouse and rat reactivity, but has no image caption documenting tissue processing (M03096-2 catalog).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry Q99798 (ACON_HUMAN, Aconitate hydratase, mitochondrial).
Human Protein Atlas. ACO2 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. ACO2 subcellular location (ICC-IF): Mainly localized to the mitochondria..
Human Protein Atlas. ACO2 antibody validation summary (1 antibodies).
Pan-Cancer analysis shows that ACO2 is a potential prognostic and immunotherapeutic biomarker for multiple cancer types including hepatocellular carcinoma. Frontiers in oncology 2022 — PMC9748622.
Zinc cooperates with p53 to inhibit the activity of mitochondrial aconitase through reactive oxygen species accumulation. Cancer medicine 2019 — PMC6536939.
Loss of mitochondrial aconitase promotes colorectal cancer progression via SCD1-mediated lipid remodeling. Molecular metabolism 2021 — PMC8042449.
Verification of Ferroptosis Subcluster-Associated Genes Related to Osteosarcoma and Exploration of Immune Targeted Therapy. Oxidative medicine and cellular longevity 2022 — PMC9534693.
PubMed PMID:9630632 — UniProt-cited evidence.
PubMed PMID:15461802 — UniProt-cited evidence.
PubMed PMID:10591208 — UniProt-cited evidence.