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HADH / Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial · IHC design guide
Design Immunohistochemistry for HADH
Plan HADH staining in paraffin sections using the catalog antibody’s IHC protocol. The guide covers granular cytoplasmic staining (HPA tissue IHC), mitochondrial matrix localisation (UniProt), and interpretation across broadly positive tissues (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HADH 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 matrix (UniProt) | |
| Staining pattern | Ubiquitous granular cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03650-1) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | None in HPA (detected in all 44 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 | Broad expression limits tissue-specific interpretation (HPA tissue IHC) | |
| Regulation | No specific expression regulator reported (UniProt) | |
| Isoform / epitope | 3 isoforms; mature chain starts at residue 13; epitope map unknown (UniProt) |
Section 1
Recommended HADH IHC & IF Protocols
The catalog antibody’s IHC-P protocol uses EDTA pH 8.0 retrieval (datasheet A03650-1). Three published HADH IHC protocols below provide tissue-specific comparisons (PMC12860641; PMC11402447; PMC13326317).
| Sample | Paraffin-embedded human colon adenocarcinoma tissue; fixative not specified (datasheet A03650-1) |
| Fixation | Image fixative and duration unreported (datasheet A03650-1); 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 A03650-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03650-1) |
| Primary antibody | Rabbit anti-HADH, 2-5 μg/ml (datasheet A03650-1) |
| Primary incubation | Overnight at 4 °C (datasheet A03650-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A03650-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | HADH-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression with a granular expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet A03650-1); citrate retrieval is documented for colorectal and renal tissue (PMC12860641; PMC11402447).
Section 2
What Is the Expected HADH Staining Pattern?
HADH is a mitochondrial matrix protein with no transmembrane segment (UniProt Q16836 localisation and topology). In paraffin-section IHC, expect granular cytoplasmic staining across multiple cell types (HPA: ubiquitous granular cytoplasmic expression; Enhanced tissue reliability). HPA reports high staining in adipocytes, glandular cells, endocrine cells, hematopoietic cells and glial cells in the listed tissues (HPA: tissue IHC).
| Granular cytoplasmic staining in adipocytes, with nuclei relatively clear. | This fits the reported IHC pattern and a mitochondrial matrix location (HPA: High in adipocytes; HPA: granular cytoplasmic expression; UniProt Q16836 localisation). Routine chromogenic IHC can support this compartment-level interpretation without resolving individual mitochondria (standard IHC practice). Compare staining with nearby tissue and detection controls before judging intensity (standard IHC practice). |
| Strong nuclear or sharply outlined surface staining dominates the section. | That distribution conflicts with the expected mitochondrial, granular cytoplasmic pattern (UniProt Q16836 localisation and topology; HPA: tissue IHC profile). Treat it as a localisation mismatch and investigate background or antibody specificity; the image alone cannot establish which mechanism caused it (standard IHC interpretation). |
| An unexpected cell population stains strongly while an HPA-listed high population is blank. | Cross-reactivity or endogenous detection activity is possible (standard IHC practice). HPA describes broad HADH expression and supplies no negative cell list, so an unlisted positive cell type is not, by itself, evidence of a false positive (HPA: ubiquitous expression; negative list empty). Confirm cell identity and inspect detection controls (standard IHC practice). |
| Uniform colour obscures cell boundaries and granular cytoplasmic detail. | Diffuse deposition prevents a confident localisation call, even where the tissue contains an HPA-listed positive population (HPA: tissue IHC; standard IHC interpretation). Background from detection chemistry or insufficient washing is a general possibility; neither HPA nor UniProt identifies a HADH-specific cause here (standard IHC practice). |
| No staining appears in adipocytes in adipose tissue. | This conflicts with an HPA high-staining example, but one blank section does not establish absence of HADH (HPA: High in adipose-tissue adipocytes). Review tissue quality, retrieval, antibody application and detection controls as general IHC checks (standard IHC practice). The supplied sources give no HADH-specific fixation sensitivity or retrieval condition. |
💡Expected HADH appearanceCall a positive result when cell-type-associated granular cytoplasmic staining is visible, especially in HPA-listed high cells; dominant nuclear or surface signal is suspect (HPA: tissue IHC; UniProt Q16836 localisation and topology).
| Compartment and topology | HADH resides in the mitochondrial matrix and has no transmembrane segment (UniProt Q16836). Score granular cytoplasmic staining against that expectation; do not require visible mitochondrial outlines in routine chromogenic sections (HPA: granular cytoplasmic IHC; standard IHC interpretation). |
| Tissue breadth | HPA calls expression ubiquitous with low tissue RNA specificity and reports high IHC staining in several cell populations (HPA: tissue IHC). Use its named examples as positive references, while allowing other cell types to stain; the supplied HPA record lists no negative tissue or cell population. |
| Evidence strength | The tissue profile has Enhanced reliability because antibody staining is highly consistent with RNA expression data (HPA: tissue IHC reliability). HPA039588 is Supported for IHC and HPA043888 is Enhanced for IHC (HPA: antibody validation); these grades support interpretation but do not validate an unspecified catalog antibody. |
| Processing and isoforms | UniProt lists a mature chain at residues 13–314 and 3 isoforms (UniProt Q16836 processing and isoforms). The supplied record does not locate the antibody epitope or establish isoform coverage, so a staining difference cannot be assigned to processing or a particular isoform. |
| IF/ICC Q: what should fluorescence show? | A: Mainly mitochondrial localisation, supported by HPA ICC-IF images; this agrees with the UniProt matrix annotation (HPA: mitochondria, supported; UniProt Q16836 localisation). IF/ICC images are a localisation cross-check for IHC interpretation, not a paraffin-IHC intensity standard (standard assay interpretation). |
| Situation | Likely cause | Next action |
|---|---|---|
| The expected granular pattern is absent in an HPA-listed high population. | The result conflicts with the reference pattern, but the source data do not identify a HADH-specific technical cause (HPA: tissue IHC). | Check tissue integrity, retrieval execution, antibody application and detection controls; repeat with a documented positive tissue if needed (standard IHC practice). |
| Nuclei stain more strongly than cytoplasm. | Predominantly nuclear signal conflicts with the mitochondrial matrix annotation and granular cytoplasmic IHC profile (UniProt Q16836; HPA: tissue IHC). | Review primary-omission and detection controls, then compare the pattern with an HPA-listed positive cell population (standard IHC practice; HPA: tissue IHC). |
| A crisp cell-surface rim dominates. | Surface localisation conflicts with HADH's matrix location and lack of a transmembrane segment (UniProt Q16836 localisation and topology). | Inspect background controls and whether the signal tracks tissue edges or other nonspecific deposition patterns (standard IHC practice). |
| Colour is diffuse across cells and extracellular areas. | The background masks the granular cytoplasmic pattern described by HPA; excess detection signal is one general possibility (HPA: tissue IHC; standard IHC practice). | Review blocking, washes, detection development and a primary-omission control before scoring positive cells (standard IHC practice). |
| Only an unlisted cell population stains. | Specificity or endogenous activity may need review, but HPA's ubiquitous profile does not exclude that cell type (HPA: tissue IHC; standard IHC practice). | Confirm cell identification, check detection controls and compare an HPA-listed high population on a suitable section (standard IHC practice; HPA: tissue IHC). |
| Two sections differ markedly in staining intensity. | The supplied sources establish a qualitative pattern, not a HADH-specific fixation effect or a quantitative intensity threshold (HPA: tissue IHC; UniProt Q16836). | Review section quality and keep retrieval, detection and scoring conditions comparable before interpreting the difference (standard IHC practice). |
Experiment evidence
IHC & IF images showing HADH staining
A03650-1 · IHCIHC analysis of HADH using anti-HADH antibody (A03650-1). HADH was detected in a paraffin-embedded section of human colon adenocarcinoma tis
A03650-1 · IHCIHC analysis of HADH using anti-HADH antibody (A03650-1). HADH was detected in a paraffin-embedded section of human colon adenocarcinoma tis
A03650-1 · IFIF analysis of HADH using anti-HADH antibody (A03650-1). HADH was detected in an immunocytochemical section of HELA cells. Enzyme antigen re
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000138796-HADH/tissue/cerebral+cortex#imid_10356977
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000138796-HADH/tissue/colon#imid_10356919
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000138796-HADH/tissue/liver#imid_10356912
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000138796-HADH/tissue/kidney#imid_10356913
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA043888; green = HADH, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA043888; green = HADH, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HADH IHC & IF
🧪Run adipose tissue first and look for HADH staining in adipocytes (HPA: High in adipocytes); a cytoplasmic mitochondrial pattern is expected (HPA subcellular: mitochondria supported). HPA detects HADH in all 44 scored tissues, so there is no supported negative tissue; no-primary and isotype controls carry the negative, while unstained nonadipocyte cells on the positive slide are only local background references, not validated HADH-negative cells (HPA: no negative tissue rows; standard IHC practice).
Positive control tissue: Adipose tissue (Adipocytes, HPA High)
Negative control tissue: None in HPA: HADH is detected in all 44 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 HADH in A-431, A-549, U2OS, NIH 3T3, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and an isotype control matched to the rabbit primary’s class and clonality (caption: rabbit anti-HADH; standard IHC practice); a HADH knockout specimen provides a biological negative if available (standard IHC practice). Block endogenous peroxidase before HRP–DAB detection and check that signal is confined to adipocyte cytoplasm rather than the lipid vacuoles (caption: HRP–DAB detection; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A03650-1 paraffin-section caption does not state the fixative (caption: fixative unreported). That caption demonstrates staining after heat retrieval in EDTA at pH 8.0, but does not establish whether retrieval is required (caption: heat-mediated EDTA retrieval). Paraffin IHC is demonstrated; the supplied evidence does not establish that frozen sections or IF are easier, and adipocyte lipid vacuoles leave a thin cytoplasmic rim that can complicate scoring (caption: paraffin-section IHC; standard histology practice).
HPA tissue IHC evidence for HADH
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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Endocrine cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Adipocytes | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: HADH is detected in all 44 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced HADH IHC Tips
Troubleshoot HADH staining in paraffin sections by checking retrieval, compartment, cell type and background before comparing chromogenic signal across samples.
What retrieval should I try first for weak HADH staining in paraffin sections?
Start with heat-mediated retrieval in EDTA buffer at pH 8.0 (datasheet A03650-1). The selected paraffin-section example used that retrieval before incubation with 2 μg/ml primary antibody overnight at 4°C (datasheet A03650-1). If staining remains weak, compare retrieval duration on matched sections while holding antibody concentration and DAB development constant (standard IHC practice). Check that any gain appears as granular cytoplasmic staining, consistent with the reported tissue pattern and mitochondrial location (HPA: tissue IHC; UniProt Q16836: mitochondrion matrix). Include a section processed without primary antibody so stronger retrieval is not mistaken for improved HADH detection when background rises (standard IHC practice).
How should I assess whether fixation is masking HADH in my sections?
The selected HADH image shows a paraffin-embedded section, but its caption does not state the fixative, so target-specific fixation sensitivity is unknown (datasheet A03650-1). Do not infer a preferred fixative or fixation duration from the granular tissue staining or mitochondrial topology (HPA: tissue IHC; UniProt Q16836: topology). If fixation histories differ, compare sections with documented processing using the same EDTA pH 8.0 retrieval and primary-antibody conditions (datasheet A03650-1; standard IHC practice). Evaluate tissue preservation and background alongside staining intensity, because fixation differences can affect both morphology and antigen accessibility (standard IHC practice). Record the fixative and duration before attributing a signal difference to HADH expression (standard IHC practice).
Where should convincing HADH signal appear in chromogenic IHC?
Expect granular cytoplasmic staining rather than a nuclear or cell-surface pattern: HADH resides in the mitochondrial matrix, and its protein has no annotated transmembrane segment (UniProt Q16836: subcellular location and topology). Tissue IHC reports ubiquitous cytoplasmic expression with a granular appearance (HPA: tissue IHC). Compare the granules with cell boundaries and the counterstain at high magnification, and check whether the pattern repeats across intact cells (standard IHC practice). Strong diffuse nuclear signal warrants a background and detection-control review before interpretation (UniProt Q16836: mitochondrion matrix; standard IHC practice). Cell-type intensity may vary; HPA reports high staining in adipocytes and several glandular or endocrine cell populations (HPA: tissue IHC).
Could isoforms or epitope accessibility explain uneven HADH staining?
HADH has 3 annotated isoforms, and the supplied product caption does not identify the antibody epitope or establish isoform coverage (UniProt Q16836: isoforms; datasheet A03650-1). The annotated mature chain spans residues 13–314, while modified residues include acetylation and succinylation sites near residues 80–87 (UniProt Q16836: processing and modified residues). Those annotations alone cannot show whether an isoform or modification changes binding in paraffin sections (UniProt Q16836: isoforms and modified residues). Compare retrieval and antibody conditions on matched sections, then consult epitope documentation if available before assigning uneven signal to an isoform (standard IHC practice). Score reproducible cellular patterns separately from section edges and damaged areas (standard IHC practice).
How can I check a HADH IHC pattern with multiplex IF?
Use IF as an independent localisation check: HADH is mainly mitochondrial, while tissue IHC describes granular cytoplasmic staining (HPA: subcellular location and tissue IHC). In an adipose tissue experiment, multiplex HADH with an adipocyte marker and assess whether signal lies within the expected cells (HPA: high in adipocytes; standard IF practice). Choose a far-red fluorophore when tissue autofluorescence compromises shorter-wavelength channels, and include single-label and unstained controls (standard IF practice). Because HADH is in the mitochondrial matrix, assess permeabilisation after fixation so antibody can access an intracellular epitope (UniProt Q16836: subcellular location; standard IF practice). Validate IF staining independently; the selected product example documents chromogenic IHC in a paraffin section (datasheet A03650-1).
How do I distinguish HADH staining from DAB or tissue background?
The selected assay used an HRP-linked secondary antibody and DAB, making peroxidase blocking and development controls relevant to interpretation (datasheet A03650-1; standard IHC practice). Compare a no-primary section and, where appropriate, an antibody-diluent control to locate detection-system or tissue background (standard IHC practice). Keep the 10% goat-serum blocking step and 2 μg/ml primary concentration as documented starting conditions while optimizing one variable at a time (datasheet A03650-1). Assess whether brown signal forms intracellular granules consistent with mitochondrial HADH rather than coating tissue edges or pooling in damaged regions (UniProt Q16836: mitochondrion matrix; HPA: tissue IHC; standard IHC practice). Apply the same DAB development endpoint to comparison sections (standard IHC practice).
How should I quantify HADH IHC across samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, because HADH staining is reported across many cell types with differing intensities (HPA: tissue IHC). For intact cells, record percent positive and an H-score based on predefined intensity categories; retain representative images for review (standard IHC practice). For spatial comparisons, measure positive-cell density per mm² of viable, analysable tissue and report that denominator explicitly (standard IHC practice). Keep section thickness, retrieval, primary incubation and DAB development consistent; the selected example used EDTA pH 8.0 and 2 μg/ml antibody (datasheet A03650-1; standard IHC practice). Exclude necrosis and section edges using the same rules for every sample (standard IHC practice).
When is an apparent HADH-positive result likely to be artefactual?
Treat reproducible granular cytoplasmic signal in intact cells as more credible than staining confined to nuclei, surfaces or section edges (UniProt Q16836: mitochondrion matrix and topology; HPA: tissue IHC; standard IHC practice). HADH expression is broadly distributed, so a positive cell is not identified by staining alone; use morphology and a cell-type marker when identity matters (HPA: low tissue specificity; standard IHC practice). Exclude necrotic regions and check no-primary controls before interpreting diffuse brown deposits as protein expression (standard IHC practice). With HRP and DAB detection, review peroxidase-block controls if tissue-associated brown signal persists without primary antibody (datasheet A03650-1; standard IHC practice). Compare matched sections under the same retrieval and development conditions before calling an intensity difference biological (standard IHC practice).
Boster reagents
Best HADH / Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial IHC Antibodies
A03650-1 has real IHC images from human paraffin sections and IF images from human cells and a paraffin section (catalog image captions). Its listed reactivity is human, mouse and rat (catalog applications and reactivity).
Real IHC data
Anti-HADH Antibody ®
Cat # A03650-1
A03650-1 has IHC images from human colon adenocarcinoma and glioblastoma paraffin sections (catalog IHC image captions). The same SKU has IF images from HeLa cells and a human intestinal cancer paraffin section, and lists IHC, ICC and IF applications (catalog IF image captions; catalog applications).
Which to pick: Choose A03650-1 for tissue IHC: its own image captions show paraffin sections with EDTA retrieval at pH 8.0 and primary antibody at 2 μg/ml; the fixative is unreported (catalog IHC image captions). Choose the same SKU for IF/ICC because both applications are listed and its IF captions show human cells and a paraffin section (catalog applications; catalog IF image captions). For cross-species work, the catalog lists human, mouse and rat reactivity, but its IHC-specific dilution listing names human and rat, so mouse IHC requires separate validation (catalog reactivity; catalog dilution listing).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q16836 (HCDH_HUMAN, Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial).
- Human Protein Atlas. HADH tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. HADH subcellular location (ICC-IF): Mainly localized to the mitochondria..
- Human Protein Atlas. HADH antibody validation summary (2 antibodies).
- A nuclear isoform of hydroxyacyl-COA dehydrogenase inhibits tumor progression in colorectal cancer. iScience 2026 — PMC12860641.
- HADH suppresses clear cell renal cell carcinoma progression through reduced NRF2-dependent glutathione synthesis. Translational oncology 2024 — PMC11402447.
- Role of medium- and short-chain L-3-hydroxyacyl-CoA dehydrogenase in the regulation of body weight and thermogenesis. Endocrinology 2011 — PMC3359510.
- Intratumoral microbiota promote pancreatic cancer progression via NaAc-mediated activation of the GPR43/AMPK/HADH pathway. Journal of translational medicine 2026 — PMC13326317.
- PubMed PMID:8687463 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HADH (Hydroxyacyl-coenzyme A dehydrogenase, mitochondrial). Expected localisation Granular cytoplasm (HPA tissue IHC); mitochondrial matrix (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03650-1). Validated IHC antibody: A03650-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative None in HPA (detected in all 44 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon adenocarcinoma tissue; fixative not specified (datasheet A03650-1); 2. Fixation: Image fixative and duration unreported (datasheet A03650-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet A03650-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03650-1); 8. Primary antibody: Rabbit anti-HADH, 2-5 μg/ml (datasheet A03650-1); 9. Primary incubation: Overnight at 4 °C (datasheet A03650-1). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Broad expression limits tissue-specific interpretation (HPA tissue IHC); Isoform / epitope: 3 isoforms; mature chain starts at residue 13; epitope map unknown (UniProt); Regulation: No specific expression regulator reported (UniProt). Evidence: UniProt Q16836, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the mitochondria., Datasheet A03650-1, PMC12860641, PMC11402447. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HADH/immunohistochemistry-ihc-guide.