This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
HADHB / Trifunctional enzyme subunit beta, mitochondrial · IHC design guide
Design Immunohistochemistry for HADHB
Plan chromogenic HADHB IHC on paraffin sections with the catalog antibody at 2–5 μg/mL (datasheet A04776-2). Compare high staining in colon glandular cells with undetected bone-marrow hematopoietic cells, and score the mitochondrial pattern (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HADHB 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 | Mitochondrial staining in glandular cells (HPA tissue IHC) | |
| Staining pattern | Glandular-cell mitochondrial staining; germinal and non-germinal centers included (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A04776-2) | |
| Positive control | Colon+4 more · see all | |
| Negative control | Bone marrow+2 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Skeletal myocytes stain low despite enhanced tissue RNA (HPA tissue IHC; HPA tissue RNA) | |
| Regulation | RNA enhanced: heart, skeletal muscle, tongue (HPA tissue RNA) | |
| Isoform / epitope | 2 isoforms; chain 34–474; epitope map unreported (UniProt) |
Section 1
Recommended HADHB IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by 3 published HADHB IHC protocols from paraffin sections (datasheet A04776-2; PMC6350214; PMC9743170; PMC12394106).
| Sample | Paraffin-embedded human glioblastoma tissue; fixative not specified (datasheet A04776-2) |
| Fixation | Image fixative and duration unreported (datasheet A04776-2); 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 A04776-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A04776-2) |
| Primary antibody | Rabbit anti-HADHB, 2-5 μg/ml (datasheet A04776-2) |
| Primary incubation | Overnight at 4 °C (datasheet A04776-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A04776-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | HADHB-positive staining in glandular cells of colon (HPA tissue IHC: High). HPA tissue profile: Mitochondrial expression in glandular cells, also including germinal and non-germinal centers. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 for the catalog antibody (datasheet A04776-2); the cited protocols use citrate retrieval (PMC6350214; PMC9743170; PMC12394106).
Section 2
What Is the Expected HADHB Staining Pattern?
HADHB should appear mainly in mitochondria, often as granular cytoplasmic staining (HPA: mitochondrial tissue profile; UniProt P55084: mitochondrial localization). High staining is reported in colon, duodenal, gallbladder, salivary gland and small-intestinal glandular cells, kidney tubular cells, lung alveolar type II cells, and skin endothelial cells (HPA: tissue IHC). HPA rates the tissue profile Enhanced, with medium consistency between staining and RNA data (HPA: reliability). HADHB has no transmembrane segment (UniProt P55084: topology).
| Granular cytoplasmic chromogen in colon glandular cells or kidney tubular cells. | This fits mitochondrial HADHB staining in cell types scored High by tissue IHC (HPA: colon glandular cells; kidney tubular cells). Assess granules within identifiable cells rather than judging overall section color (standard IHC practice). |
| Predominantly nuclear staining, or an exclusively smooth cell-border outline. | These patterns do not match the mainly mitochondrial localization (HPA: subcellular IF; UniProt P55084: localization). Review morphology, antibody specificity and detection controls before calling them HADHB; UniProt also annotates membrane association and endoplasmic reticulum localization (UniProt P55084: localization). |
| Strong staining confined to cells expected to be unstained, such as bone-marrow hematopoietic cells. | HPA reports HADHB not detected in that cell population (HPA: bone marrow IHC). Consider cross-reactivity or endogenous detection activity; neither can be diagnosed from appearance alone (standard IHC practice). |
| Diffuse color across cells and surrounding tissue, obscuring intracellular granules. | The expected mitochondrial pattern cannot be scored confidently (HPA: mitochondrial tissue profile). Check whether background persists in detection controls, then review antibody dilution, blocking, washing and chromogen development (standard IHC practice). |
| No signal in a colon glandular-cell or kidney tubular-cell control. | Both are reported High by HPA, so a blank control calls the staining run into question (HPA: tissue IHC). Verify tissue morphology, retrieval, primary-antibody application and detection before interpreting a test section as negative (standard IHC practice). |
💡Expected HADHB appearanceCall a positive result when identifiable high-expression cells show clear, predominantly granular cytoplasmic staining consistent with mitochondria; uniform nuclear or acellular color is suspect (HPA: colon and kidney tissue IHC; HPA: mitochondrial localization; standard IHC practice).
| Tissue and cell selection | Use the reported cell type, not the organ name alone: colon glandular cells and kidney tubular cells are High, while bone-marrow hematopoietic cells are not detected (HPA: tissue IHC). |
| RNA versus protein readout | Skeletal muscle is RNA-enhanced but its myocytes have Low IHC staining; RNA enrichment alone cannot set an IHC intensity threshold (HPA: RNA specificity; skeletal-muscle tissue IHC). |
| Subcellular interpretation | Mitochondria are the main supported IF location; UniProt also lists mitochondrial inner and outer membranes and endoplasmic reticulum (HPA: subcellular IF; UniProt P55084: localization). Bright-field IHC cannot resolve those membrane assignments (standard IHC practice). |
| Protein context | HADHB complexes with HADHA, which is required for its stability and membrane association (UniProt P55084: subunit; localization note). This biological context does not establish a specific change in paraffin-section staining. |
| Antibody validation | HPA lists HPA037540 as IHC Enhanced and HPA037539 as IHC Supported; HPA066099 has no listed IHC status (HPA: antibody validation). Match the validation record to the antibody actually used. |
| Q: Does the IF/ICC image predict the chromogenic IHC result? | A: It supports a mainly mitochondrial location, but IF/ICC and paraffin IHC have separate antibody-validation records and readouts (HPA: subcellular IF; HPA: antibody validation). Use the IF/ICC guide for that application. |
| Situation | Likely cause | Next action |
|---|---|---|
| The positive-control section is blank. | A staining-run problem is possible because colon glandular cells and kidney tubular cells are reported High (HPA: tissue IHC). The section alone cannot identify the failed step. | Confirm the control contains the stated cells, then check the documented retrieval, primary-antibody application and detection steps (standard IHC practice). |
| Most of the section has a diffuse brown haze. | Diffuse background can obscure the mitochondrial pattern (HPA: tissue profile). Excess detection signal or nonspecific staining are possibilities (standard IHC practice). | Compare an appropriate detection control and review blocking, washing, antibody dilution and chromogen development (standard IHC practice). |
| Strong nuclear color dominates positive-looking cells. | The dominant pattern conflicts with HPA's mainly mitochondrial localization (HPA: subcellular IF). Morphology alone cannot prove which reagent caused it. | Check nuclear counterstain versus chromogen, inspect control sections and reassess antibody specificity before scoring HADHB (standard IHC practice). |
| Bone-marrow hematopoietic cells stain strongly. | That population is reported not detected by HPA; cross-reactivity or endogenous detection activity is possible (HPA: bone-marrow IHC; standard IHC practice). | Compare a primary-antibody omission control and review the detection-system controls before accepting the signal (standard IHC practice). |
| Skeletal-muscle myocytes stain weakly despite RNA enrichment. | This can agree with HPA's Low myocyte IHC score even though skeletal muscle is RNA-enhanced (HPA: tissue IHC; RNA specificity). | Judge the run against a reported high-staining cell population and its morphology, rather than RNA enrichment alone (HPA: colon or kidney tissue IHC; standard IHC practice). |
| A weak region is being called HADHB-negative. | HPA records different staining levels by cell type, including Low and not detected categories (HPA: tissue IHC). Weak color without a working control is inconclusive (standard IHC practice). | Score the specified cell population, compare the same run's positive and detection controls, and report uncertainty if separation remains unclear (standard IHC practice). |
Experiment evidence
IHC & IF images showing HADHB staining
A04776-2 · IHCIHC analysis of HADHB using anti-HADHB antibody (A04776-2). HADHB was detected in a paraffin-embedded section of human glioblastoma tissue.
A04776-2 · IHCIHC analysis of HADHB using anti-HADHB antibody (A04776-2). HADHB was detected in a paraffin-embedded section of human glioblastoma tissue.
A04776-2 · IFIF analysis of HADHB using anti-HADHB antibody (A04776-2). HADHB was detected in an immunocytochemical section of HELA cells. Enzyme antigen
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000138029-HADHB/tissue/cerebral+cortex#imid_9205648
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000138029-HADHB/tissue/colon#imid_9205574
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000138029-HADHB/tissue/liver#imid_9205567
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000138029-HADHB/tissue/kidney#imid_9205568
HPA · ICC-IF SK-MEL-30Human Protein Atlas immunofluorescence, SK-MEL-30, antibody HPA037540; green = HADHB, blue = nucleus, red = microtubules.
HPA · ICC-IF SK-MEL-30Human Protein Atlas immunofluorescence, SK-MEL-30, antibody HPA037540; green = HADHB, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HADHB IHC & IF
🧪Run kidney first: tubular cells should show HADHB staining (HPA: High in kidney cells in tubules). Run bone marrow as the negative tissue (HPA: Not detected in hematopoietic cells); on the kidney slide, use cells outside the tubules as internal background comparators only if they remain unstained, because their HADHB status is not specified by the supplied HPA rows.
Positive control tissue: Colon (Glandular cells, HPA High)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show HADHB in SK-MEL-30, U2OS, Hep-G2, Sperm, NIH 3T3, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an immunoglobulin control matched to the primary antibody’s host and class (selected caption: rabbit anti-HADHB); process HADHB-knockout material in parallel as a biological negative when available. For chromogenic kidney IHC, block endogenous peroxidase and check tubular background from endogenous biotin if using biotin-based detection (HPA: High in kidney cells in tubules; standard IHC practice).
⚠️Feasibility: The selected A04776-2 paraffin-section caption reports heat retrieval in EDTA, pH 8.0, followed by DAB detection, but does not report the fixative or establish that retrieval is required for kidney tissue (selected-SKU tissue-IHC caption). No target-specific fixation window or fixation effect is reported in the supplied evidence; whether frozen sections or IF are easier is also unreported. Assess tubular background carefully when using biotin-based detection (HPA: High in kidney cells in tubules; standard IHC practice).
HPA tissue IHC evidence for HADHB
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 |
|---|---|---|---|---|
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
| Lung | Alveolar cells type II | High | Protein (IHC) | HPA → |
Section 3
Advanced HADHB IHC Tips
HADHB staining in paraffin sections should be assessed against its predominantly mitochondrial localisation and the cell types reported to express it (UniProt P55084; HPA tissue IHC).
How should I adjust antigen retrieval when HADHB staining is weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A04776-2). The selected antibody produced HADHB staining in a paraffin section after that retrieval, followed by 2 μg/ml primary antibody overnight at 4°C (datasheet A04776-2). If staining remains weak, compare retrieval duration on adjacent sections while holding antibody concentration, detection and exposure to chromogen constant (standard IHC practice). Include a section with an HPA-reported high-expressing cell population, such as kidney tubular cells, so a weak result in the test tissue can be distinguished from a failed staining run (HPA tissue IHC; standard IHC practice).
Could fixation explain inconsistent HADHB staining across paraffin blocks?
Target-specific sensitivity of HADHB to fixation is unknown: the selected tissue-IHC caption identifies a paraffin section but does not state its fixative (datasheet A04776-2). Record each block’s fixative, fixation duration and processing history before attributing staining differences to biology (standard IHC practice). Compare blocks using the same EDTA pH 8.0 retrieval and detection conditions, with a shared positive-control section in every run (datasheet A04776-2; standard IHC practice). Review morphology and staining at section edges alongside the control, because uneven processing can confound a comparison; neither HPA expression patterns nor HADHB membrane association establishes a fixation effect (standard IHC practice; HPA tissue IHC; UniProt P55084).
What staining pattern is credible for HADHB in chromogenic sections?
Look for finely granular cytoplasmic staining consistent with mitochondria, assessed within intact cells rather than as diffuse deposit across the section (HPA subcellular; standard IHC practice). UniProt also lists inner and outer mitochondrial membranes and endoplasmic reticulum, and notes that HADHA supports HADHB stability and membrane association (UniProt P55084). Chromogenic light microscopy cannot assign a brown punctum to either mitochondrial membrane, so report the cellular pattern without claiming membrane-side resolution (standard IHC practice; UniProt P55084). Compare the pattern in the test tissue with HPA-reported high staining in kidney tubular or colon glandular cells, while accounting for cell composition in each section (HPA tissue IHC; standard IHC practice).
How do isoforms and epitope uncertainty affect a negative HADHB result?
HADHB has 2 listed isoforms and a processed chain spanning residues 34–474, but the supplied antibody caption does not identify its epitope (UniProt P55084; datasheet A04776-2). Do not assume that this antibody detects both isoforms or that its epitope survives every retrieval condition (UniProt P55084; standard IHC practice). HADHB has no annotated transmembrane segment, while modified residues include alternative acetylation or succinylation at positions 72 and 188; those annotations alone do not establish an effect on staining (UniProt P55084). For an unexpected negative, first verify the positive control and processing, then seek epitope documentation or an independently validated reagent before assigning an isoform-specific explanation (standard IHC practice).
How can IF help check an ambiguous HADHB IHC pattern?
Use IF as a separate localisation check, pairing HADHB with a mitochondrial marker and, where relevant, a marker identifying the expected cell population, such as glandular cells (HPA subcellular; HPA tissue IHC; standard IF practice). Choose fluorophores after checking tissue autofluorescence, and place the weaker signal in a spectrally clear, often far-red channel when the instrument permits (standard IF practice). Because HADHB has no transmembrane segment but associates with mitochondrial membranes, choose permeabilisation to give antibody access to the documented epitope’s side of the membrane; if that side is unknown, compare gentle and stronger conditions empirically (UniProt P55084; standard IF practice). Include single-label and secondary-only controls before comparing IF localisation with the chromogenic IHC pattern (standard IF practice).
How do I reduce diffuse brown background without losing HADHB signal?
Confirm that the background persists in a section lacking primary antibody, then check endogenous peroxidase blocking and the timing of DAB development (standard IHC practice). The selected caption used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and a peroxidase-conjugated secondary for 30 minutes at 37°C (datasheet A04776-2). If control sections are clean but test sections are diffuse, titrate the primary antibody and optimise washing while keeping EDTA pH 8.0 retrieval fixed (datasheet A04776-2; standard IHC practice). Judge improvements against mitochondrial-pattern staining in intact cells, since a uniform brown wash is less consistent with the reported main localisation (HPA subcellular; standard IHC practice).
What should I score when comparing HADHB across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, then record the percentage of positive cells and staining intensity on a 0–3 scale (standard IHC practice). An H-score from 0–300 combines those measures; alternatively, count positive cells per mm² of viable, annotated tissue when cell density is the outcome of interest (standard IHC practice). Normalise to the number or area of the same relevant cell type, rather than whole-section area when tissue composition differs (standard IHC practice). Keep retrieval, antibody incubation, DAB development and image thresholds consistent, and report separate scores for HPA-reported high and low cell populations if both occur in the sampled material (datasheet A04776-2; HPA tissue IHC; standard IHC practice).
How can I distinguish genuine HADHB positivity from staining artefact?
Give greatest weight to staining in intact cells with a granular cytoplasmic pattern compatible with HADHB’s mainly mitochondrial localisation (HPA subcellular; standard IHC practice). Check cell identity: HPA reports high staining in colon glandular cells and kidney tubular cells, but no detectable staining in bone marrow hematopoietic cells in its tissue survey (HPA tissue IHC). Discount staining restricted to cut edges, necrotic regions or deposits outside cells, and use a no-primary control to assess endogenous enzyme activity or detection background (standard IHC practice). A discrepant result merits review of morphology, retrieval and controls before concluding that HADHB expression or localisation has changed (datasheet A04776-2; standard IHC practice).
Boster reagents
Best HADHB / Trifunctional enzyme subunit beta, mitochondrial IHC Antibodies
A04776-2 has IHC data from paraffin-embedded human glioblastoma and liver cancer sections (catalog IHC captions), plus IF data from HeLa cells (catalog IF caption). Listed reactivity includes human, mouse, and rat (catalog reactivity).
Real IHC data
Anti-HADHB Antibody ®
Cat # A04776-2
A04776-2 is listed for IHC and IF/ICC (catalog applications). Its IHC images show human glioblastoma and liver cancer sections (catalog IHC captions), and its IF image shows HeLa cells (catalog IF caption).
Which to pick: For tissue IHC, choose A04776-2: its captions document paraffin-embedded human sections with EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody; the fixative is unreported (catalog IHC captions). For IF/ICC, A04776-2 has an image from HeLa cells using 5 μg/ml primary antibody (catalog IF caption). For mouse or rat samples, A04776-2 lists those species as reactive, but the supplied IHC and IF images document human samples only (catalog reactivity; catalog image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P55084 (ECHB_HUMAN, Trifunctional enzyme subunit beta, mitochondrial).
- Human Protein Atlas. HADHB tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. HADHB subcellular location (ICC-IF): Mainly localized to the mitochondria. In addition localized to the equatorial segment and principal piece..
- Human Protein Atlas. HADHB antibody validation summary (3 antibodies).
- Posttranscriptional Regulation of 14q32 MicroRNAs by the CIRBP and HADHB during Vascular Regeneration after Ischemia. Molecular therapy. Nucleic acids 2019 — PMC6350214.
- Hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta gene as a tumour suppressor in stomach adenocarcinoma. Frontiers in oncology 2022 — PMC9743170.
- HADHB mediates 5-fluorouracil sensitivity in colorectal cancer. Discover oncology 2025 — PMC12394106.
- Mitochondrial protein-enriched artificial nanovesicles: mitochondrial recovery and antioxidation for diabetic wound treatment. Journal of nanobiotechnology 2026 — PMC12964820.
- PubMed PMID:8135828 — UniProt-cited evidence.
- PubMed PMID:9259266 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HADHB (Trifunctional enzyme subunit beta, mitochondrial). Expected localisation Mitochondrial staining in glandular cells (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A04776-2). Validated IHC antibody: A04776-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Colon (Glandular cells); negative Bone marrow. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human glioblastoma tissue; fixative not specified (datasheet A04776-2); 2. Fixation: Image fixative and duration unreported (datasheet A04776-2); 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 A04776-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A04776-2); 8. Primary antibody: Rabbit anti-HADHB, 2-5 μg/ml (datasheet A04776-2); 9. Primary incubation: Overnight at 4 °C (datasheet A04776-2). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Skeletal myocytes stain low despite enhanced tissue RNA (HPA tissue IHC; HPA tissue RNA); Isoform / epitope: 2 isoforms; chain 34–474 ; epitope map unreported (UniProt); Regulation: RNA enhanced: heart, skeletal muscle, tongue (HPA tissue RNA). Evidence: UniProt P55084, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the mitochondria. In addition localized to the…, Datasheet A04776-2, PMC6350214, PMC9743170. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HADHB/immunohistochemistry-ihc-guide.