HADHA / Trifunctional enzyme subunit alpha, mitochondrial · IHC design guide

Design Immunohistochemistry for HADHA

Plan chromogenic HADHA IHC in paraffin sections using colon glandular cells, which show high staining, as a positive reference (HPA tissue IHC). The catalog antibody A03666-2 was used at 2 μg/ml on a human colon adenocarcinoma section (datasheet A03666-2).

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

Immunohistochemistry protocol sheet for HADHA (IHC for HADHA): expected localisation Cytoplasmic staining (HPA tissue IHC); mitochondrial inner membrane target (UniProt), antibody A03666-2, validated IHC image, and IHC protocol steps — Printable **HADHA** IHC protocol sheet — expected localisation Cytoplasmic staining (HPA tissue IHC); mitochondrial inner membrane target (UniProt), antibody A03666-2, controls and protocol steps. Open the full HADHA IHC guide →

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HADHA 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 (HPA tissue IHC); mitochondrial inner membrane target (UniProt)
	Staining pattern	Widespread cytoplasmic staining across tissue cell types (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A03666-2)
	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); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A03666-2)
	Caveat	Adipocytes were not detected despite broad tissue staining (HPA tissue IHC)
	Regulation	No staining regulator annotated (UniProt)
	Isoform / epitope	2 isoforms; mature chain starts at residue 37; epitope unknown (UniProt)

Section 1

Recommended HADHA IHC & IF Protocols

The catalog antibody’s IHC-P protocol is followed by three published HADHA IHC protocols for lung, human biopsy, and human myocardial sections (PMC5578913; PMC4636942; PMC13495513).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human bladder cancer tissue; fixative not specified (datasheet A03666-2)
Fixation	Image fixative and duration unreported (datasheet A03666-2); 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 A03666-2); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A03666-2)
Primary antibody	Rabbit anti-HADHA, 2-5 μg/ml (datasheet A03666-2)
Primary incubation	Overnight at 4 °C (datasheet A03666-2)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A03666-2)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	HADHA-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet A03666-2); published biopsy and myocardial protocols used citrate at pH 6.0 (PMC4636942; PMC13495513).

Section 2

What Is the Expected HADHA Staining Pattern?

HADHA is mitochondrial and associated with the inner membrane, with no transmembrane segment (UniProt P40939 topology). In paraffin sections, expect cytoplasmic staining in many cell types (HPA: ubiquitous cytoplasmic expression), including strong staining in several glandular cell populations and Leydig cells (HPA: High). HPA rates its tissue IHC profile Approved, with medium consistency between antibody staining and RNA expression (HPA: reliability description).

What am I looking at on my slide?
Cytoplasmic staining in colon or duodenal glandular cells, with little nuclear signal.	This fits HADHA's mitochondrial location (UniProt P40939; HPA: mitochondria in ICC-IF) and the reported High glandular-cell staining in both tissues (HPA: tissue IHC). Judge the distribution within identified cells rather than expecting every cell in the section to have equal intensity.
Predominantly nuclear, membrane-outline, or extracellular staining.	These compartments do not match the reported mitochondrial location (UniProt P40939; HPA: mitochondria in ICC-IF). Treat a dominant misplaced pattern as suspect; inspect the counterstain and controls, then reassess whether the chromogen is being assigned to the correct compartment (standard IHC practice).
Strong adipocyte staining while expected glandular cells stain weakly.	HPA reports HADHA as Not detected in adipocytes and High in several glandular populations (HPA: tissue IHC). Consider cross-reactivity or endogenous detection activity, especially if the signal also appears in a reagent control; this pattern alone cannot identify which mechanism is responsible (standard IHC practice).
Uniform color across tissue, empty spaces, and section edges.	A field-wide haze obscures the cellular pattern needed to assess mitochondrial HADHA (UniProt P40939 location; standard IHC practice). Compare a matched no-primary control and review blocking, detection reagents, washing, and chromogen development before scoring cells (standard IHC practice).
No signal in an intact colon or duodenum section containing glandular cells.	Both glandular populations are reported High by HPA tissue IHC, so an absent result warrants a run-level check (HPA: tissue IHC). Confirm tissue identity and preservation, then check antibody application, retrieval, and detection against a same-run control (standard IHC practice); absence alone does not prove HADHA loss.

💡Expected HADHA appearanceCall a section positive when identified glandular cells show clear cytoplasmic staining, potentially with granular emphasis, at an intensity consistent with HPA's High category; dominant nuclear staining or strong adipocyte staining is suspect (HPA: tissue IHC; HPA: mitochondrial ICC-IF location; UniProt P40939 topology).

How each factor affects the staining
Cellular location and resolution	HADHA associates with the mitochondrial inner membrane but has no transmembrane segment (UniProt P40939 topology). A chromogenic paraffin section may show an overall cytoplasmic pattern because individual mitochondria are not always resolved (standard IHC practice); HPA describes tissue staining as ubiquitous cytoplasmic.
Tissue and cell selection	Colon, duodenum, small intestine, stomach, and adrenal gland glandular cells, plus testis Leydig cells, are reported High; adipocytes are Not detected (HPA: tissue IHC). Use the named cell population when interpreting a control, since the tissue label alone does not assign staining to every cell.
Antibody evidence	HPA015536 is Approved for IHC; HPA056070 has no IHC validation status in the supplied record (HPA: antibody validation). The overall tissue profile is Approved with medium staining-to-RNA consistency (HPA: reliability description), so assess a new antibody or run against its own controls.
Processing and isoforms	UniProt lists a mature chain spanning residues 37–763 and 2 isoforms (UniProt P40939 processing and isoforms). No antibody epitope or isoform coverage is supplied, so these annotations cannot predict whether a particular IHC antibody detects both isoforms.
IF/ICC Q&A: Where should signal appear?	In IF/ICC, expect mitochondrial signal: HPA reports an enhanced mitochondrial location and Enhanced ICC validation for HPA015536 and HPA056070 (HPA: subcellular ICC-IF; HPA: antibody validation). Those ICC findings do not establish IHC validation for HPA056070.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Glandular cells expected to stain are blank.	A failed detection step, omitted antibody, or unsuitable retrieval may prevent a usable signal (standard IHC practice); HPA reports High staining in colon and duodenal glandular cells (HPA: tissue IHC).	Check a same-run positive section and reagent sequence, then optimize retrieval and antibody conditions for the IHC-validated antibody (standard IHC practice). No HADHA-specific retrieval condition or dilution is supplied.
Signal is mostly nuclear or outlines cell surfaces.	The dominant compartment conflicts with mitochondrial HADHA localisation (UniProt P40939; HPA: mitochondrial ICC-IF location). Misread morphology or nonspecific detection may explain it (standard IHC practice).	Recheck the cells against the counterstain and compare a no-primary control; score only convincing cytoplasmic staining after the misplaced signal is resolved (standard IHC practice).
Adipocytes stain strongly.	HPA reports HADHA as Not detected in adipocytes (HPA: tissue IHC). Cross-reactivity or endogenous activity is possible, but staining alone cannot distinguish them (standard IHC practice).	Compare no-primary and detection controls, inspect which adipose-tissue cells carry the signal, and confirm the result with an independently validated antibody if needed (standard IHC practice).
Diffuse background hides cell boundaries.	Excess detection background, insufficient washing, or overdevelopment can obscure a cellular staining pattern (standard IHC practice). The haze itself provides no evidence of HADHA distribution.	Check the no-primary control; adjust blocking, washing, detection intensity, or development using the established assay workflow, then reassess identifiable cells (standard IHC practice).
A sample differs from the HPA intensity profile.	HPA calls the tissue profile Approved but reports only medium consistency between antibody staining and RNA expression (HPA: reliability description). A difference is therefore a finding to investigate, not proof of altered HADHA.	Verify cell identity and section quality, compare same-run positive and negative controls, and record intensity by the relevant cell population (standard IHC practice).
An ICC-positive antibody gives uncertain paraffin-section staining.	HPA056070 is Enhanced for ICC but has no IHC status in the supplied antibody record (HPA: antibody validation). ICC performance does not establish paraffin IHC performance.	For this IHC interpretation, prioritize an IHC-validated antibody such as HPA015536 and confirm its expected cellular pattern with tissue and reagent controls (HPA: antibody validation; standard IHC practice).

Source: UniProt P40939 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing HADHA staining

IHC analysis of HADHA using anti-HADHA antibody (A03666-2). HADHA was detected in a paraffin-embedded section of human bladder 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 rabbit anti-HADHA Antibody (A03666-2) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A03666-2 · IHCIHC analysis of HADHA using anti-HADHA antibody (A03666-2). HADHA was detected in a paraffin-embedded section of human bladder cancer tissue

IF analysis of HADHA using anti-HADHA antibody (A03666-2). HADHA was detected in an immunocytochemical section of MG63 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-HADHA Antibody (A03666-2) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A03666-2 · IFIF analysis of HADHA using anti-HADHA antibody (A03666-2). HADHA was detected in an immunocytochemical section of MG63 cells. Enzyme antigen

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000084754-HADHA/tissue/cerebral+cortex#imid_4460892

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000084754-HADHA/tissue/colon#imid_4460826

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000084754-HADHA/tissue/liver#imid_4460835

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000084754-HADHA/tissue/kidney#imid_4460828

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA015536; green = HADHA, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for HADHA IHC & IF

🧪Run colon first: glandular cells should stain (HPA: High in colon glandular cells). Run adipose tissue as the negative, where adipocytes are not detected (HPA: Not detected in adipocytes); on the colon slide, use morphologically unstained cells, if present, to assess background rather than assuming every nonglandular cell is negative.

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 HADHA in A-431, U2OS, MCF-7, NIH 3T3, with annotated localisation: Mitochondria (enhanced) (HPA subcellular).

Technical controls: Include a no-primary (secondary-only) control, a rabbit IgG isotype control matched to the primary where possible, and HADHA-knockout tissue or cells as a biological negative (selected-SKU caption: rabbit primary; standard IHC controls). Quench endogenous peroxidase and check any inflammatory cells in colon for background before interpreting DAB staining (standard chromogenic IHC practice).

⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative unreported). The caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required (selected-SKU caption: EDTA retrieval). HPA shows mitochondrial ICC-IF localization, but the evidence does not establish that IF or frozen sections are easier; in colon, inspect luminal material and any pigment before scoring DAB signal (HPA: mitochondria; standard IHC practice).

HPA tissue IHC evidence for HADHA

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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
Adrenal gland	Glandular cells	High	Protein (IHC)	HPA →
Colon	Glandular cells	High	Protein (IHC)	HPA →
Duodenum	Glandular cells	High	Protein (IHC)	HPA →
Small intestine	Glandular cells	High	Protein (IHC)	HPA →
Stomach	Glandular cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

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

Section 3

Advanced HADHA IHC Tips

Troubleshoot HADHA staining in paraffin sections by checking retrieval, controls, mitochondrial localisation and consistent scoring (UniProt P40939; HPA tissue IHC).

Which retrieval conditions should I try when HADHA staining is weak?

Use heat-mediated retrieval in EDTA at pH 8.0 first (datasheet A03666-2). The selected paraffin-section example then used 2 μg/ml catalog antibody overnight at 4°C, so compare retrieval changes while holding antibody conditions constant (datasheet A03666-2). Include a high-staining reference such as colon glandular cells on each run to detect retrieval failure (HPA: High in colon glandular cells; standard IHC practice). If staining remains weak, test an alternative retrieval buffer as a fallback on adjacent sections, with matched detection and exposure to chromogen (standard IHC practice). Reject conditions that increase diffuse background without improving the expected cytoplasmic pattern (HPA: ubiquitous cytoplasmic expression; standard IHC practice).

Could fixation explain weak HADHA staining in paraffin sections?

Target-specific sensitivity to fixation is unknown because the selected paraffin-section caption does not report a fixative (datasheet A03666-2). Record the fixative and processing history for each specimen, then compare sections processed alike before changing antibody concentration or retrieval (standard IHC practice). Keep section thickness and staining batches consistent when assessing whether a weak result follows specimen handling (standard IHC practice). A reference section with high glandular-cell staining can show whether the run worked, but cannot establish HADHA-specific fixation effects (HPA: High in colon glandular cells; standard IHC practice). Do not infer fixation tolerance from mitochondrial localisation or annotated modifications (UniProt P40939 topology and modified residues).

What staining pattern supports mitochondrial HADHA in chromogenic IHC?

Expect a cytoplasmic pattern compatible with mitochondria, potentially granular where resolution permits, because HADHA localises to mitochondria and the inner membrane (UniProt P40939 localisation; HPA: ubiquitous cytoplasmic expression). Its annotated topology has no transmembrane segment, so membrane association does not require a crisp membrane outline in a chromogenic section (UniProt P40939 topology; standard IHC practice). Compare cellular staining with nearby background and the same run’s controls before calling faint cytoplasm positive (standard IHC practice). Colon glandular cells provide a reported high-staining reference, whereas adipocytes are reported as not detected (HPA: High in colon glandular cells; HPA: Not detected in adipocytes). Predominantly nuclear staining warrants specificity checks (UniProt P40939 localisation; standard IHC practice).

How should isoforms and epitope uncertainty affect HADHA IHC interpretation?

HADHA has 2 annotated isoforms, while the supplied product caption does not identify the antibody epitope (UniProt P40939 isoforms; datasheet A03666-2). Its annotated mature chain spans residues 37–763, and modified residues include positions 46, 60 and 129 (UniProt P40939 processing and modified residues). These annotations alone cannot establish whether either isoform or modification changes staining (UniProt P40939 annotations; standard IHC interpretation). If results differ across specimens, check the antibody’s documented immunogen or epitope before assigning an isoform-specific explanation (standard IHC practice). Validate any proposed epitope effect with an independent antibody or orthogonal assay rather than staining intensity alone (standard IHC practice).

How can I assess HADHA localisation by multiplex IF?

For multiplex IF, pair HADHA with a marker identifying the expected cell population, such as a glandular epithelial marker in colon, and assess signal within those cells (HPA: High in colon glandular cells; standard IF practice). Add a mitochondrial marker to test spatial agreement with the reported organelle localisation (UniProt P40939 localisation; HPA subcellular: mitochondria; standard IF practice). Choose spectrally separated fluorophores and place a dim signal in a longer-wavelength channel after checking tissue autofluorescence and single-stain controls (standard IF practice). Optimize permeabilisation for access to the actual epitope: HADHA associates with the inner membrane but lacks an annotated transmembrane segment, and this payload does not locate the antibody epitope on either side (UniProt P40939 topology; standard IF practice). The paraffin IHC conditions do not establish IF conditions for this antibody (datasheet A03666-2).

How do I reduce diffuse or misleading HADHA DAB staining?

Start with a no-primary control and an endogenous peroxidase block to distinguish detection background from antibody-dependent signal (standard IHC practice). The selected paraffin-section example used 10% goat serum before primary antibody and HRP with DAB for detection (datasheet A03666-2). If background persists, titrate the primary antibody below the example’s 2 μg/ml while keeping retrieval and development conditions matched across sections (datasheet A03666-2; standard IHC practice). Examine pigments, tissue edges and damaged areas separately from intact cells because these can mimic positive chromogen (standard IHC practice). Accept staining when cellular distribution agrees with the reported cytoplasmic pattern and appropriate controls (HPA: ubiquitous cytoplasmic expression; standard IHC practice).

How should I score HADHA staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring; HADHA is reported as cytoplasmic in tissue IHC (HPA: ubiquitous cytoplasmic expression; standard IHC practice). For intact cells, record percent positive and intensity, or calculate an H-score from prespecified intensity categories (standard IHC practice). For spatial comparisons, measure positive-cell density per mm² of evaluable tissue and exclude folds, edges and necrosis by the same rule in every section (standard IHC practice). Normalize to the number of eligible cells or evaluable area, rather than whole-section area when tissue content differs (standard IHC practice). Keep retrieval, DAB development and image thresholds constant, with a shared reference section across runs (standard IHC practice).

When is an apparent HADHA-positive result likely to be artefactual?

A credible signal should be cellular and cytoplasmic, consistent with mitochondrial HADHA and the reported tissue IHC pattern (UniProt P40939 localisation; HPA: ubiquitous cytoplasmic expression). Predominantly nuclear or extracellular staining conflicts with that localisation and calls for control review (UniProt P40939 localisation; standard IHC practice). Compare the stained cell type with reported references: colon glandular cells are high, whereas adipocytes are not detected in HPA tissue IHC (HPA: High in colon glandular cells; HPA: Not detected in adipocytes). Treat staining confined to section edges or necrotic regions cautiously, and check the no-primary control for endogenous enzyme or detection signal (standard IHC practice). Interpret intensity alongside morphology and matched controls, since the HPA tissue assessment reports only medium agreement with RNA expression (HPA: Approved, medium consistency; standard IHC practice).

Boster reagents

Best HADHA / Trifunctional enzyme subunit alpha, mitochondrial IHC Antibodies

A03666-2 has IHC image data from human paraffin sections and IF image data from human lung cancer sections and MG63 cells (datasheet: IHC/IF captions).

Real IHC data

Anti-HADHA Antibody ®

Cat # A03666-2

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A03666-2 will render with its human bladder cancer paraffin-section IHC figure; another IHC caption documents human colon adenocarcinoma sections (datasheet: IHC captions). Its IF captions document MG63 cells and human lung cancer paraffin sections, and the catalog lists IHC, IF and ICC applications (datasheet: IF captions; catalog: applications).

Which to pick: Choose A03666-2 for chromogenic tissue IHC: its bladder cancer paraffin-section image used EDTA pH 8.0 heat retrieval, 2 μg/ml primary antibody and DAB detection; the fixative is unreported (datasheet: bladder IHC caption). For IF/ICC, the same rabbit-host SKU has IF images from MG63 cells and human lung cancer sections (catalog: host; datasheet: IF captions). For cross-species work, A03666-2 lists Human, Mouse and Rat reactivity, while the supplied tissue images document human samples (catalog: reactivity; datasheet: IHC/IF captions).

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

References

UniProt Consortium. UniProt entry P40939 (ECHA_HUMAN, Trifunctional enzyme subunit alpha, mitochondrial).
Human Protein Atlas. HADHA tissue IHC expression (reliability: Approved).
Human Protein Atlas. HADHA subcellular location (ICC-IF): Localized to the mitochondria..
Human Protein Atlas. HADHA antibody validation summary (2 antibodies).
Fuelling the mechanisms of asthma: Increased fatty acid oxidation in inflammatory immune cells may represent a novel therapeutic target. Clinical and experimental allergy : journal of the British Society for Allergy and Clinical Immunology 2017 — PMC5578913.
Inhibition of Fatty Acid Oxidation Modulates Immunosuppressive Functions of Myeloid-Derived Suppressor Cells and Enhances Cancer Therapies. Cancer immunology research 2015 — PMC4636942.
Differential expression of metabolic genes distinguishes physiological from pathological cardiac hypertrophy. BMC cardiovascular disorders 2026 — PMC13495513.
Cold exposure-induced β-hydroxybutyrate promotes brown fat mitochondrial lipid droplet contact to ameliorate fatty dysfunction and hepatic steatosis. Acta pharmaceutica Sinica. B 2026 — PMC12891858.
PubMed PMID:8135828 — UniProt-cited evidence.
PubMed PMID:7918661 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.