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BCKDK / Branched-chain alpha-ketoacid dehydrogenase kinase · IHC design guide
Design Immunohistochemistry for BCKDK
Plan BCKDK chromogenic IHC in paraffin sections using the catalog antibody’s IHC-P conditions. Compare granular cytoplasmic staining with HPA tissue IHC reference patterns, including high staining in glandular cells and lung macrophages (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
BCKDK 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 expected (UniProt) | |
| Staining pattern | Granular cytoplasm in glandular cells and macrophages (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A07798-2) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+4 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 | High lung macrophage staining can confound scoring (HPA tissue IHC) | |
| Regulation | Refeeding raises the BCKDK:PPM1K ratio (UniProt) | |
| Isoform / epitope | 3 isoforms; mature chain spans residues 31–412 (UniProt) |
Section 1
Recommended BCKDK IHC & IF Protocols
The catalog antibody uses EDTA pH 8.0 retrieval (datasheet A07798-2). The published IHC protocols below cover renal carcinoma tissue, liver specimens, and glioma tissue microarrays (PMC10637060; PMC6506390; PMC11336342).
| Sample | Paraffin-embedded human gall bladder adenosquamous carcinoma tissue; fixative not specified (datasheet A07798-2) |
| Fixation | Image fixative and duration unreported (datasheet A07798-2); 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 A07798-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A07798-2) |
| Primary antibody | Rabbit anti-BCKDK, 2-5 μg/ml (datasheet A07798-2) |
| Primary incubation | Overnight at 4 °C (datasheet A07798-2) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A07798-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | BCKDK-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression with a granular pattern. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet A07798-2). Citrate retrieval is a published alternative for liver sections (PMC6506390).
Section 2
What Is the Expected BCKDK Staining Pattern?
BCKDK is a mitochondrial matrix protein with no transmembrane segment (UniProt O14874 topology; UniProt O14874 subcellular location). In paraffin-section IHC, expect granular cytoplasmic staining, particularly in glandular and respiratory epithelial cells and lung macrophages (HPA tissue IHC: Supported; medium consistency with RNA). Expression is widespread, but staining varies by cell type; an unstained cell in a positive tissue is therefore possible (UniProt O14874 tissue specificity; HPA tissue IHC).
| Granular cytoplasmic staining in pancreatic exocrine cells, colonic glands, or lung macrophages. | This fits the reported distribution: each named cell population has High staining, and the overall IHC profile is granular and cytoplasmic (HPA tissue IHC). Score the intended cell population separately from its neighbors; tissue-wide intensity alone can obscure that distinction (general IHC practice). |
| Predominantly nuclear or membrane-rim staining, with little granular cytoplasmic signal. | The compartment disagrees with the expected matrix location and observed tissue pattern (UniProt O14874 subcellular location and topology; HPA tissue IHC). Treat it as suspect antibody or detection staining until a suitable positive section and omission control clarify the pattern (general IHC practice). |
| Strong signal in adipocytes, cardiomyocytes, skeletal myocytes, or smooth muscle cells. | Those particular cell types are listed as Not detected, even though BCKDK has low tissue specificity overall (HPA tissue IHC: Not detected; HPA tissue RNA: low tissue specificity). Check whether the signal belongs to adjacent cells, then assess cross-reactivity or endogenous detection activity with controls (general IHC practice). |
| Uniform brown haze across cells and extracellular spaces. | A uniform deposit does not resemble the reported granular cytoplasmic pattern (HPA tissue IHC). Background from detection reagents or incomplete washing is possible; an omission control and review of blocking, washing, and chromogen development can distinguish it from cell-associated staining (general IHC practice). |
| No stain in the expected cells of a positive control section. | Absence in a High-staining population, such as pancreatic exocrine cells, makes this run difficult to interpret (HPA tissue IHC: High in pancreatic exocrine cells). First check section quality and the antibody and detection steps; an absent signal alone does not establish biological loss (general IHC practice). |
💡Expected BCKDK appearanceCall a result positive when the relevant glandular or respiratory epithelial cells, or lung macrophages, show cell-associated granular cytoplasmic staining; HPA lists these populations as High, while dominant nuclear signal, uniform haze, or strong myocyte staining is suspect (HPA tissue IHC; UniProt O14874 subcellular location).
| Cell population chosen for scoring | HPA reports High staining in several epithelial populations and lung macrophages, but Not detected in adipocytes and several muscle cell types (HPA tissue IHC). Identify the cells before comparing intensity across sections. |
| Antibody and application | HPA017995 has Supported IHC validation; HPA056067 has Supported ICC validation, with no IHC status supplied for that antibody (HPA antibody validation). Use application-specific evidence when interpreting a paraffin-section result. |
| Isoforms and mature protein | UniProt lists 3 isoforms and a chain spanning residues 31–412 (UniProt O14874 isoforms and processing). The supplied evidence gives no antibody epitope, so isoform coverage and recognition of the mature chain cannot be predicted. |
| Detection chemistry | Endogenous enzyme activity or nonspecific reagent deposition can mimic chromogenic staining; an omission control helps assess that contribution (general IHC practice). This is a general workflow consideration, not a reported BCKDK-specific effect. |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive control is blank in the expected cells. | The run may have failed at antibody incubation or detection; the absent stain does not by itself prove low BCKDK expression (general IHC practice). | Confirm the expected cell population using a reported High example, then review reagent preparation, retrieval and detection against the validated IHC procedure (HPA tissue IHC; general IHC practice). No target-specific retrieval setting is supplied. |
| Granular signal is weak, but tissue and cells are identifiable. | HPA tissue staining is variable, and its IHC reliability is Supported with medium consistency against RNA (HPA tissue IHC). Weak signal alone cannot establish a technical failure. | Compare the same cell type with a High-staining control processed in the same run; inspect counterstain and detection before changing scoring thresholds (HPA tissue IHC; general IHC practice). |
| Every compartment has diffuse brown staining. | General background can arise from detection reagents, insufficient blocking or washing, or excessive chromogen development (general IHC practice). The pattern differs from HPA's granular cytoplasmic profile (HPA tissue IHC). | Run a primary-antibody omission control and review blocking, washes, and development time; score only cell-associated signal above that background (general IHC practice). |
| Muscle or adipose cells appear strongly positive. | These cell types are reported as Not detected; staining could reflect cell misidentification, cross-reactivity, or endogenous detection activity (HPA tissue IHC; general IHC practice). | Check cell boundaries and neighboring populations, compare a reported High-staining control, and inspect the omission control before accepting the signal as BCKDK (HPA tissue IHC; general IHC practice). |
| Nuclear staining dominates a positive section. | Predominant nuclear localization conflicts with mitochondrial matrix annotation and the tissue IHC pattern (UniProt O14874 subcellular location; HPA tissue IHC). | Treat nuclear signal as unconfirmed; inspect a positive control for granular cytoplasmic staining and compare the omission control before revising the interpretation (HPA tissue IHC; general IHC practice). |
| What should an IF/ICC image show? | The application has separate validation: HPA reports supported mitochondrial localization in ICC-IF, while the tissue IHC profile is granular cytoplasmic (HPA subcellular ICC-IF; HPA tissue IHC). | Expect mitochondrial staining and assess localization against an appropriate mitochondrial reference (HPA subcellular ICC-IF; general IF practice). Use the separate IF/ICC guide for experimental setup; this IHC section supplies no IF protocol. |
Experiment evidence
IHC & IF images showing BCKDK staining
A07798-2 · IHCIHC analysis of BCKDK using anti-BCKDK antibody (A07798-2). BCKDK was detected in a paraffin-embedded section of human gall bladder adenosqu
A07798-2 · IHCIHC analysis of BCKDK using anti-BCKDK antibody (A07798-2). BCKDK was detected in a paraffin-embedded section of human breast cancer tissue.
A07798-2 · IFIF analysis of BCKDK using anti-BCKDK antibody (A07798-2). BCKDK was detected in an immunocytochemical section of MCF-7 cells. Enzyme antige
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000103507-BCKDK/tissue/cerebral+cortex#imid_4617768
HPA · Salivary glandHuman Protein Atlas IHC, Salivary gland. Source: https://www.proteinatlas.org/ENSG00000103507-BCKDK/tissue/salivary+gland#imid_4617732
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000103507-BCKDK/tissue/colon#imid_4617710
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000103507-BCKDK/tissue/liver#imid_4617711
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA056067; green = BCKDK, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA056067; green = BCKDK, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for BCKDK IHC & IF
🧪Start with colon: glandular cells should stain (HPA: High in colon glandular cells). Run skeletal muscle as a negative tissue, where myocytes are listed as not detected (HPA: Not detected in skeletal muscle myocytes); adjacent colon stromal cells can help assess background, but their BCKDK status is unreported in the supplied HPA rows.
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 BCKDK in A-431, U-251MG, U2OS, with annotated localisation: Mitochondria (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a rabbit IgG isotype control matched to the primary antibody’s clonality where applicable (selected-SKU caption: rabbit anti-BCKDK primary). Use a BCKDK knockout specimen as a biological negative if available; with the caption’s biotin-based DAB detection, check endogenous peroxidase and biotin background (selected-SKU caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: The selected-SKU paraffin-section caption does not report a fixative, and no target-specific fixation window or fixation effect is reported in the supplied evidence (selected-SKU caption: fixative not stated). Heat retrieval in EDTA at pH 8.0 was used for the illustrated IHC section, but its necessity for BCKDK is unreported (selected-SKU caption: EDTA retrieval). Frozen-section ease is unreported; ICC-IF images exist for A-431, U-251MG and U2OS, but they do not establish an IF protocol or a relative ease advantage (HPA subcellular: ICC-IF image cell lines).
HPA tissue IHC evidence for BCKDK
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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 → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Endometrium | 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 → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced BCKDK IHC Tips
Troubleshoot BCKDK staining in paraffin sections by checking retrieval, cell identity and the expected granular cytoplasmic pattern before scoring signal.
How should I troubleshoot weak BCKDK staining after antigen retrieval?
Start with heat-mediated EDTA at pH 8.0 for paraffin sections stained with A07798-2 (datasheet A07798-2). The documented tissue example used 2 μg/ml primary antibody overnight at 4°C after this retrieval, so hold those conditions constant while assessing retrieval (datasheet A07798-2). If staining is weak, compare shorter and longer heating intervals in the same buffer on adjacent sections, checking morphology and granular cytoplasmic signal after each trial (standard IHC practice; HPA: general cytoplasmic granular pattern). If that fails, try another retrieval buffer as a fallback and compare it with the EDTA condition using a matched positive section and a no-primary control (standard IHC practice).
Could fixation explain weak or uneven BCKDK IHC staining?
The A07798-2 tissue caption identifies a paraffin section but does not report its fixative, so BCKDK-specific fixation sensitivity is unknown (datasheet A07798-2). Record the fixative, fixation duration and processing history for each block before attributing different staining intensities to biology (standard IHC practice). Compare similarly processed sections with the same EDTA pH 8.0 retrieval and 2 μg/ml primary condition used in the documented example (datasheet A07798-2). If one block stains poorly, assess tissue preservation and staining uniformity alongside a matched positive control; these checks cannot establish a target-specific fixation effect (standard IHC practice).
What localisation should a convincing BCKDK signal show?
Look for granular cytoplasmic staining in tissue sections, the reported IHC pattern for BCKDK (HPA: general cytoplasmic expression with a granular pattern). A mitochondrial matrix location supports that expectation, while liver cells may also show cytosolic BCKDK (UniProt O14874: subcellular location). Because BCKDK has no transmembrane segment, a sharp cell-surface rim alone warrants scrutiny rather than automatic scoring as target signal (UniProt O14874: topology; standard IHC interpretation). Compare the same cell type in adjacent sections, document whether granules remain inside viable cell boundaries, and check a no-primary control when staining appears nuclear or restricted to section edges (standard IHC practice).
How do isoforms and processing affect interpretation of this antibody?
BCKDK has 3 reported isoforms and a processed chain spanning residues 31–412, but the supplied caption does not identify the A07798-2 epitope (UniProt O14874: isoforms and processing; datasheet A07798-2). Therefore, staining cannot establish which isoform is present or whether processing has exposed or removed the recognized sequence (standard antibody interpretation). Report the antibody identifier, EDTA pH 8.0 retrieval and scored compartment so comparisons can be reproduced (datasheet A07798-2; standard IHC practice). If an epitope map becomes available, check its position against the processed chain and reported phosphorylation or acetylation sites before proposing an epitope-specific explanation for discordant staining (UniProt O14874: processing and modified residues).
How can IF help assess cell identity and BCKDK localisation?
For IF follow-up, multiplex BCKDK with CD68 when examining lung macrophages, which show high tissue IHC staining, and score the markers within the same cells (HPA: High in lung macrophages; standard IF practice). Choose a BCKDK fluorophore in a channel with low tissue autofluorescence, then inspect single-stain and no-primary controls before interpreting overlap (standard IF practice). BCKDK is assigned to the mitochondrial matrix and has no transmembrane segment, so permeabilisation must allow antibody access beyond the plasma membrane and mitochondrial boundaries; titrate detergent conditions because the antibody epitope is unspecified (UniProt O14874: location and topology; datasheet A07798-2; standard IF practice). Use the granular cytoplasmic tissue pattern as context, not as proof of IF specificity (HPA: tissue IHC profile).
How do I separate BCKDK staining from chromogenic background?
The documented workflow uses 10% goat serum block, biotinylated secondary antibody, a streptavidin–biotin complex and DAB, so assess background at both antibody and detection steps (datasheet A07798-2). Run a no-primary section to reveal signal from secondary reagents, endogenous biotin or chromogenic detection, and inspect a matched section for tissue pigment (standard IHC practice). For a peroxidase-based DAB workflow, include an appropriate endogenous peroxidase block and check its effectiveness in the no-primary control (standard IHC practice). Score cellular granular cytoplasmic staining separately from diffuse deposits, edge staining and pigment that persists without primary antibody (HPA: granular cytoplasmic profile; standard IHC practice).
How should BCKDK IHC staining be quantified across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and viable tissue area before scoring, because the reported tissue pattern is granular cytoplasmic and expression differs among cell types (HPA: tissue IHC profile). For a selected cell population, record the percentage of positive cells and intensity, or calculate an H-score from intensity categories 0–3 and their cell percentages, yielding 0–300 (standard IHC scoring practice). If counting discrete positive cells, report density per mm² of viable tissue and retain cell-type labels rather than pooling unlike compartments (standard IHC scoring practice). Normalize comparisons to the same eligible cell population or viable area, with matched staining conditions and a prespecified positivity threshold (standard IHC practice).
What distinguishes a true BCKDK-positive cell from an artefact?
A plausible positive cell shows intracellular granular cytoplasmic staining, consistent with the tissue IHC profile and mitochondrial localisation (HPA: general cytoplasmic granular pattern; UniProt O14874: mitochondrial matrix). Cell identity matters: lung macrophages are reported high, whereas cardiomyocytes are reported not detected, so annotate the stained population before interpreting a tissue-wide score (HPA: High in lung macrophages; HPA: Not detected in cardiomyocytes). Do not reject every diffuse cytosolic signal solely on compartment grounds, because BCKDK has also been detected in the cytosolic compartment of liver cells (UniProt O14874: subcellular location). Treat staining concentrated at section edges, in necrotic areas or in a no-primary control as suspect, and check endogenous peroxidase contribution to DAB signal (standard IHC practice).
Boster reagents
Best BCKDK / Branched-chain alpha-ketoacid dehydrogenase kinase IHC Antibodies
A07798-2 has documented BCKDK staining in human paraffin tissue and MCF-7 cells (IHC and IF image captions); the catalog lists human, mouse, and rat reactivity (datasheet).
Real IHC data
Anti-BCKDK Antibody ®
Cat # A07798-2
A07798-2 has IHC images from human paraffin sections of gall bladder adenosquamous carcinoma, breast cancer, colonic adenocarcinoma, and gastric adenocarcinoma (IHC image captions). Its IF image uses MCF-7 cells (IF image caption), while its application list includes IHC, ICC, and IF (datasheet).
Which to pick: For tissue IHC, choose A07798-2: its own caption documents a human paraffin section, EDTA retrieval at pH 8.0, and 2 μg/ml primary antibody; the fixative is unreported (IHC image caption). For IF/ICC, the same SKU has an MCF-7 IF image at 5 μg/ml (IF image caption). For cross-species work, A07798-2 lists human, mouse, and rat reactivity, but the supplied IHC and IF images document human samples only; its host is rabbit and its clone is unspecified (datasheet; IHC and IF image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O14874 (BCKD_HUMAN, Branched-chain alpha-ketoacid dehydrogenase kinase).
- Human Protein Atlas. BCKDK tissue IHC expression (reliability: Supported).
- Human Protein Atlas. BCKDK subcellular location (ICC-IF): Localized to the mitochondria..
- Human Protein Atlas. BCKDK antibody validation summary (2 antibodies).
- Branched-chain keto-acid dehydrogenase kinase regulates vascular permeability and angiogenesis to facilitate tumor metastasis in renal cell carcinoma. Cancer science 2023 — PMC10637060.
- Loss of BCAA Catabolism during Carcinogenesis Enhances mTORC1 Activity and Promotes Tumor Development and Progression. Cell metabolism 2019 — PMC6506390.
- MAZ-mediated up-regulation of BCKDK reprograms glucose metabolism and promotes growth by regulating glucose-6-phosphate dehydrogenase stability in triple-negative breast cancer. Cell death & disease 2024 — PMC11258276.
- FYN-mediated phosphorylation of BCKDK at Y151 promotes GBM proliferation by increasing the oncogenic metabolite N-acetyl-L-alanine. Heliyon 2024 — PMC11336342.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15616553 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for BCKDK (Branched-chain alpha-ketoacid dehydrogenase kinase). Expected localisation Granular cytoplasm (HPA tissue IHC); mitochondrial matrix expected (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A07798-2). Validated IHC antibody: A07798-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human gall bladder adenosquamous carcinoma tissue; fixative not specified…; 2. Fixation: Image fixative and duration unreported (datasheet A07798-2); 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 A07798-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A07798-2); 8. Primary antibody: Rabbit anti-BCKDK, 2-5 μg/ml (datasheet A07798-2); 9. Primary incubation: Overnight at 4 °C (datasheet A07798-2). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: High lung macrophage staining can confound scoring (HPA tissue IHC); Isoform / epitope: 3 isoforms; mature chain spans residues 31–412 (UniProt); Regulation: Refeeding raises the BCKDK:PPM1K ratio (UniProt). Evidence: UniProt O14874, HPA tissue IHC · Supported, HPA ICC-IF · mitochondria., Datasheet A07798-2, PMC10637060, PMC6506390. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/BCKDK/immunohistochemistry-ihc-guide.