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BCKDHA / 2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial · IHC design guide
Design Immunohistochemistry for BCKDHA
Plan paraffin IHC for BCKDHA using the catalog antibody’s documented workflow (datasheet A04561-1). Expect granular cytoplasmic tissue staining and account for reported off-target binding when interpreting it (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
BCKDHA 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 in tissue (HPA tissue IHC); mitochondrial matrix expected (UniProt) | |
| Staining pattern | Granular cytoplasmic staining in glandular and other cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A04561-1) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Presumed off-target staining may complicate interpretation (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | 2 isoforms; mature chain spans aa 46–445; epitope map unknown (UniProt) |
Section 1
Recommended BCKDHA IHC & IF Protocols
The catalog antibody’s IHC-P protocol uses EDTA retrieval (datasheet A04561-1). One published CRC tissue microarray protocol provides additional staining conditions (PMC13324575).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A04561-1) |
| Fixation | Image fixative and duration unreported (datasheet A04561-1); 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 A04561-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A04561-1) |
| Primary antibody | Rabbit anti-BCKDHA, 2-5 μg/ml (datasheet A04561-1) |
| Primary incubation | Overnight at 4 °C (datasheet A04561-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A04561-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | BCKDHA-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression with a granular, mitochondrial pattern. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet A04561-1); optimize staining on your sections.
Section 2
What Is the Expected BCKDHA Staining Pattern?
BCKDHA is a mitochondrial matrix protein with no transmembrane segment (UniProt P12694: location and topology). In paraffin sections, expect granular cytoplasmic staining across many cell types (HPA tissue IHC: ubiquitous granular, mitochondrial pattern; reliability: Supported). HPA reports high staining in adrenal and appendix glandular cells, breast adipocytes, bronchial respiratory epithelial cells, and bone marrow hematopoietic cells (HPA tissue IHC: High).
| Granular cytoplasmic staining appears in cells of a tissue with an HPA high-staining example. | This fits the reported mitochondrial pattern (HPA tissue IHC: ubiquitous granular pattern). Compare cells within the section: a high rating describes the listed cell population, not every structure in that tissue (HPA tissue IHC: cell-specific High entries). |
| Signal is predominantly nuclear or sharply outlines plasma membranes. | That compartment conflicts with a matrix protein lacking a transmembrane segment (UniProt P12694: location and topology). Treat it as suspect staining; review controls and the staining procedure before assigning it to BCKDHA (general IHC practice). |
| An unexpected cell population stains more strongly than the documented high-staining population. | Consider cross-reactivity or endogenous chromogenic activity (general IHC practice). HPA reports ubiquitous expression and lists no negative cell population, so an unlisted positive cell alone does not establish an off-target result (HPA tissue IHC: profile and positive/negative lists). |
| Broad, smooth color obscures cells and lacks discernible cytoplasmic granules. | This does not match the reported granular pattern (HPA tissue IHC: profile). Check background and detection controls, then judge signal only where individual cells and their cytoplasm remain interpretable (general IHC practice). |
| No signal appears in a tissue and cell population reported as High. | First consider a failed stain or missed positive cells, then review tissue integrity and detection controls (general IHC practice). An HPA High entry is a useful comparison, but does not guarantee every section or antibody will stain identically (HPA tissue IHC: High entries). |
💡Expected BCKDHA appearanceCall a result consistent when identifiable cells show granular cytoplasmic signal in the mitochondrial distribution, especially an HPA High population; predominant nuclear, membranous, or smooth diffuse color is suspect (HPA tissue IHC: profile and High entries; UniProt P12694: matrix location and topology; general IHC practice).
| Subcellular location and topology (UniProt P12694) | BCKDHA resides in the mitochondrial matrix and has no transmembrane segment (UniProt P12694). Assess cytoplasmic granularity, rather than a membrane outline, when interpreting a positive stain (HPA tissue IHC: granular mitochondrial profile). |
| Maturation (UniProt P12694: chains 46–445) | The annotated mature chain spans residues 46–445 (UniProt P12694: processing). The supplied sources do not locate the antibody epitope, so processing cannot predict whether this antibody recognizes precursor, mature protein, or both. |
| Isoforms and modifications (UniProt P12694) | Two isoforms and several modified residues are annotated, including phosphorylation at residues 337–339 and 347 (UniProt P12694). No supplied antibody epitope or staining comparison links these features to an IHC intensity change. |
| Validation and interpretation (HPA antibodies and tissue IHC) | The listed antibody, HPA036640, has Supported IHC status (HPA antibodies). HPA also cautions that the assay targets protein from more than one gene and reports disregarded presumed off-target binding; interpret unexpected patterns cautiously (HPA tissue IHC: reliability description). |
| IF/ICC Q: Is its observed cellular pattern established here? | A: HPA provides no ICC-IF images or assigned main location (HPA subcellular). Mitochondrial matrix localization is a UniProt annotation, not an ICC-IF validation result (UniProt P12694: location). No IF/ICC protocol is supplied here. |
| Situation | Likely cause | Next action |
|---|---|---|
| Predominant nuclear or membrane-edge color. | The pattern conflicts with matrix localization and the reported granular cytoplasm (UniProt P12694: location/topology; HPA tissue IHC: profile). | Inspect a positive control and detection control, and reassess localization in intact cells before scoring (general IHC practice). |
| Unexpected cells stain while documented High cells do not. | Possible nonspecific binding, endogenous chromogenic activity, or failed staining of the expected cells (general IHC practice); HPA warns of presumed off-target binding (HPA tissue IHC). | Compare cell populations in the same section and inspect appropriate detection controls (general IHC practice). Do not classify every unlisted cell as negative: HPA reports ubiquitous expression (HPA tissue IHC). |
| Smooth color covers much of the section. | Background or excessive detection signal may obscure the granular pattern (general IHC practice; HPA tissue IHC: profile). | Check background controls and review antibody concentration, blocking, washes, and detection conditions using the applicable IHC procedure (general IHC practice). |
| No signal in an HPA High population. | The expected cells may be absent from the section, or the staining run may have failed (general IHC practice; HPA tissue IHC: cell-specific High entries). | Confirm the cell population and tissue morphology, then check the run's positive control and detection steps (general IHC practice). Do not infer target-specific fixation sensitivity from this result. |
| Only faint, poorly resolved cytoplasmic color. | Granules may be hard to distinguish from background at the current staining and imaging conditions (general IHC practice; HPA tissue IHC: granular profile). | Compare with a documented High population and its controls; reassess signal against background before calling the compartment (HPA tissue IHC: High entries; general IHC practice). |
| A strong stain is called BCKDHA solely because it is cytoplasmic. | Cytoplasmic location alone is less specific than the reported granular pattern, and HPA notes presumed off-target binding (HPA tissue IHC: profile and reliability description). | Check granularity, cell context, and controls together; record atypical signal separately from the expected pattern (HPA tissue IHC: profile; general IHC practice). |
Experiment evidence
IHC & IF images showing BCKDHA staining
A04561-1 · IHCIHC analysis of BCKDHA using anti-BCKDHA antibody (A04561-1). BCKDHA was detected in a paraffin-embedded section of human breast cancer tiss
A04561-1 · IHCIHC analysis of BCKDHA using anti-BCKDHA antibody (A04561-1). BCKDHA was detected in a paraffin-embedded section of human colon cancer tissu
A04561-1 · IFIF analysis of BCKDHA using anti-BCKDHA antibody (A04561-1). BCKDHA was detected in an immunocytochemical section of A549 cells. Enzyme anti
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000248098-BCKDHA/tissue/cerebral+cortex#imid_9377777
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000248098-BCKDHA/tissue/colon#imid_9332930
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000248098-BCKDHA/tissue/liver#imid_9332931
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000248098-BCKDHA/tissue/kidney#imid_9332932
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for BCKDHA IHC & IF
🧪Start with breast tissue: adipocytes should stain strongly (HPA: High in breast adipocytes). HPA detects BCKDHA in all 45 scored tissues, so there is no validated negative tissue; use no-primary and isotype controls, and treat cells without visible signal on the positive slide as background references rather than confirmed negative cells (HPA: no negative tissue rows).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: BCKDHA is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for BCKDHA; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Run a no-primary, secondary-only slide and a matched rabbit isotype control; use BCKDHA-knockout material as a biological specificity control if available (selected-SKU caption: rabbit primary antibody; standard IHC control practice). Check endogenous peroxidase and biotin background because the caption uses biotin-based detection with DAB (selected-SKU caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative unreported). Heat retrieval in EDTA at pH 8.0 is a documented starting condition, but the caption does not establish that retrieval is required (selected-SKU caption: heat-mediated EDTA retrieval). Score staining in the thin cytoplasmic rim of adipocytes rather than their lipid vacuoles (HPA: High in breast adipocytes; standard tissue morphology); the supplied evidence does not establish whether frozen sections or IF are easier.
HPA tissue IHC evidence for BCKDHA
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Caution, targets protein from more than one gene. Presumed off target binding observed and disregarded.). 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 | Adipocytes | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: BCKDHA is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced BCKDHA IHC Tips
Troubleshoot BCKDHA staining by checking retrieval, compartment, antibody specificity and cell type before comparing signal across sections.
What retrieval should I use when BCKDHA staining is weak in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A04561-1). The selected tissue image used this retrieval, followed by 10% goat serum blocking and 2 μg/ml primary antibody overnight at 4°C (datasheet A04561-1). If signal is weak, check heating consistency and section adhesion before changing retrieval conditions (standard IHC practice). Compare any adjusted condition with the documented EDTA condition on adjacent sections, keeping antibody concentration and detection constant (standard IHC practice). Score granular cytoplasmic staining in intact cells; diffuse staining alone does not match the reported tissue pattern (HPA: tissue IHC profile).
Can fixation explain weak or uneven BCKDHA staining?
The selected image describes a paraffin section but does not state its fixative, so BCKDHA specific sensitivity to fixation is unknown (datasheet A04561-1). Record each specimen’s fixative, fixation duration and processing history before comparing staining across blocks (standard IHC practice). Uneven staining can reflect differences in processing, retrieval or section thickness; test matched sections with the same EDTA pH 8.0 retrieval and detection conditions (standard IHC practice; datasheet A04561-1). Include intact tissue away from cut edges when judging signal, and document any tissue damage that limits comparison (standard IHC practice). Do not assign a BCKDHA specific fixation effect without a controlled comparison (datasheet A04561-1: fixative unreported).
Where should convincing BCKDHA signal appear in tissue sections?
Expect a granular cytoplasmic pattern because BCKDHA is a mitochondrial matrix protein and tissue IHC describes granular mitochondrial staining (UniProt P12694 localisation; HPA: tissue IHC profile). The protein has no annotated transmembrane segment, so a crisp cell-surface rim would be unexpected and merits investigation (UniProt P12694 topology). Check signal within morphologically intact cells at high magnification, alongside a no-primary control for detection background (standard IHC practice). Do not equate every cytoplasmic deposit with mitochondria: the HPA tissue assay is marked Supported but notes presumed off-target binding (HPA: tissue IHC reliability). Compare repeated sections and the expected intracellular pattern before calling a compartment-specific result (standard IHC practice).
Could isoforms or modified residues change what this antibody detects?
BCKDHA has 2 annotated isoforms, but the supplied evidence does not map the catalog antibody’s epitope or establish which isoforms it recognizes (UniProt P12694 isoforms; datasheet A04561-1). The record also lists phosphorylation near residues 337–347 and alternative modification at 356; their effect on this antibody’s staining is untested here (UniProt P12694 modified residues; datasheet A04561-1). Avoid explaining a staining difference as isoform loss or phosphorylation without epitope information and an independent assay (standard IHC practice). Keep retrieval and detection fixed when comparing specimens, and document the antibody identifier in each run (standard IHC practice). Interpret discordant staining cautiously given the HPA off-target caveat (HPA: tissue IHC reliability).
How should I investigate BCKDHA localisation with multiplex IF?
Treat IF as a separate validation exercise: the supplied product evidence documents chromogenic tissue IHC, while HPA supplies no ICC/IF images for this target (datasheet A04561-1; HPA: subcellular record). Pair BCKDHA with a marker that identifies the expected cell population, such as the adipocytes reported positive in breast tissue, and assess overlap within individual cells (HPA: breast adipocytes High; standard IF practice). Choose fluorophores in channels with low tissue autofluorescence and inspect single-stain and no-primary controls before interpreting overlap (standard IF practice). Because BCKDHA is in the mitochondrial matrix and has no transmembrane segment, intracellular epitope access requires suitable permeabilisation; optimize it against tissue preservation (UniProt P12694 localisation and topology; standard IF practice).
How can I distinguish BCKDHA staining from chromogenic background?
Inspect a no-primary section and the tissue morphology before treating brown deposits as BCKDHA signal (standard IHC practice). The selected method used biotinylated secondary antibody, a streptavidin–biotin complex and DAB, so assess detection-system background with appropriate controls (datasheet A04561-1; standard IHC practice). Peroxidase blocking is a general precaution for DAB workflows, especially when endogenous enzyme activity could contribute color (standard IHC practice). If background is widespread, reassess blocking, washing and antibody concentration against the documented 10% goat serum block and 2 μg/ml primary condition (datasheet A04561-1; standard IHC practice). Give greatest weight to reproducible granular cytoplasmic signal, while retaining the HPA off-target caveat (HPA: tissue IHC profile and reliability).
How should I quantify BCKDHA IHC across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and analysis regions before scoring, using matched section area and excluding damaged or necrotic tissue (standard IHC practice). For a chromogenic readout, record the percentage of positive cells and staining intensity, or calculate an H-score from intensity categories; use one method consistently (standard IHC practice). Normalize positive-cell counts to all evaluable cells of the same type, or report positive-cell density per mm² of evaluable tissue (standard IHC practice). Restrict the positive call to the granular cytoplasmic pattern reported for BCKDHA rather than diffuse color alone (HPA: tissue IHC profile). Keep retrieval, imaging and scoring thresholds constant across comparison groups, and review blinded fields when feasible (standard IHC practice).
When is an apparent BCKDHA-positive cell likely to be an artefact?
A credible positive cell shows granular cytoplasmic staining consistent with mitochondrial matrix localisation, with intact morphology and a clean detection control (UniProt P12694 localisation; HPA: tissue IHC profile; standard IHC practice). Recheck a signal confined to nuclei or cell borders, because those compartments conflict with the expected distribution (UniProt P12694 localisation and topology). Edge staining, necrotic deposits and color reproduced in a no-primary control favor processing or detection artefact (standard IHC practice). Cell identity also matters: HPA reports high staining in breast adipocytes and several glandular cell populations, but its Supported tissue assay cautions that presumed off-target binding occurs (HPA: tissue IHC positive entries and reliability). Confirm a consequential interpretation with independent evidence (standard IHC practice).
Boster reagents
Best BCKDHA / 2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial IHC Antibodies
A04561-1 has IHC images from paraffin-embedded human breast, colon and liver cancer sections (IHC image captions) and an IF image from A549 cells (IF image caption).
Real IHC data
Anti-BCKDHA Antibody ®
Cat # A04561-1
A04561-1 is listed for IHC, ICC and IF, with human, mouse and rat reactivity (catalog applications and reactivity). Its IHC images show paraffin-embedded human breast, colon and liver cancer sections, while its IF image shows A549 cells (IHC and IF image captions).
Which to pick: For tissue IHC, choose A04561-1: its images document paraffin-embedded human sections with EDTA pH 8 retrieval; the fixative is unreported (IHC image captions). For IF/ICC, the same SKU has an A549 cell IF image and a listed concentration of 5 μg/ml (IF image caption; catalog dilution). For mouse or rat work, A04561-1 lists both species for IHC, although the supplied IHC images show human tissue only; its host is rabbit and clonality is unreported (catalog reactivity, host and clone fields; IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P12694 (ODBA_HUMAN, 2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial).
- Human Protein Atlas. BCKDHA tissue IHC expression (reliability: Supported).
- Human Protein Atlas. BCKDHA subcellular location (ICC-IF): Highest expression in HEL: 69.8 nTPM.
- Human Protein Atlas. BCKDHA antibody validation summary (1 antibodies).
- Identification of neoadjuvant chemoradiotherapy resistance-associated proteins in locally advanced rectal cancer: A pilot study. Oncology letters 2026 — PMC13324575.
- PPM1K mediates metabolic disorder of branched-chain amino acid and regulates cerebral ischemia-reperfusion injury by activating ferroptosis in neurons. Cell death & disease 2023 — PMC10522625.
- Loss of BCAA catabolism enhances Rab1A-mTORC1 signaling activity and promotes tumor proliferation in NSCLC. Translational oncology 2023 — PMC10209880.
- PubMed PMID:1420356 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15057824 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for BCKDHA (2-oxoisovalerate dehydrogenase subunit alpha, mitochondrial). Expected localisation Granular cytoplasm in tissue (HPA tissue IHC); mitochondrial matrix expected (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A04561-1). Validated IHC antibody: A04561-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A04561-1); 2. Fixation: Image fixative and duration unreported (datasheet A04561-1); 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 A04561-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A04561-1); 8. Primary antibody: Rabbit anti-BCKDHA, 2-5 μg/ml (datasheet A04561-1); 9. Primary incubation: Overnight at 4 °C (datasheet A04561-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Presumed off-target staining may complicate interpretation (HPA tissue IHC); Isoform / epitope: 2 isoforms; mature chain spans aa 46–445 ; epitope map unknown (UniProt); Regulation: No expression regulator annotated (UniProt). Evidence: UniProt P12694, HPA tissue IHC · Supported, HPA cell-line RNA · Highest expression in HEL: 69.8 nTPM, Datasheet A04561-1, PMC13324575. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/BCKDHA/immunohistochemistry-ihc-guide.