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PDK4 / [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 4, mitochondrial · IHC design guide
Design Immunohistochemistry for PDK4
Plan PDK4 paraffin IHC using its general cytoplasmic staining pattern (HPA tissue IHC) and the catalog antibody’s 2–5 μg/ml IHC range (datasheet A02132-1). High-staining glandular or hematopoietic cells offer positive references, while smooth muscle cells are reported as undetected (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
PDK4 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 | General cytoplasmic staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining across multiple cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A02132-1) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Smooth muscle |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Fasting response; intensity effect unknown (UniProt) | |
| Isoform / epitope | No annotated isoforms; epitope impact unknown (UniProt) |
Section 1
Recommended PDK4 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with two published PDK4 chromogenic IHC protocols (datasheet A02132-1; PMC11335245; PMC6461828).
| Sample | Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A02132-1) |
| Fixation | Image fixative and duration unreported (datasheet A02132-1); 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 A02132-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A02132-1) |
| Primary antibody | Rabbit anti-PDK4, 2-5 μg/ml (datasheet A02132-1) |
| Primary incubation | Overnight at 4 °C (datasheet A02132-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A02132-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | PDK4-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet A02132-1); use the published dilutions as tissue-specific starting points (PMC11335245; PMC6461828).
Section 2
What Is the Expected PDK4 Staining Pattern?
PDK4 is a mitochondrial matrix protein with no transmembrane segment (UniProt Q16654: location, topology). In paraffin-section IHC, expect cellular staining that reads as cytoplasmic by light microscopy, consistent with HPA’s general cytoplasmic profile. HPA reports high staining in several cell populations, including adrenal glandular cells and bone-marrow hematopoietic cells. Its tissue IHC antibody is Approved, with medium consistency between staining and RNA data (HPA: tissue IHC).
| Cytoplasmic staining is stronger in adrenal glandular cells or bone-marrow hematopoietic cells than in nearby unstained areas. | This fits the reported high staining in those populations (HPA: High in adrenal glandular and bone-marrow hematopoietic cells). A fine granular appearance may be compatible with mitochondrial localisation, but chromogenic IHC alone cannot resolve the matrix compartment (UniProt Q16654: mitochondrion matrix; standard IHC practice). |
| The strongest signal is confined to nuclei or outlines the plasma membrane, with little cytoplasmic signal. | That distribution conflicts with the general cytoplasmic IHC profile (HPA: tissue IHC) and mitochondrial matrix assignment (UniProt Q16654: location). Treat it as suspect; inspect morphology, counterstain and controls before calling it PDK4 (standard IHC practice). |
| Smooth muscle cells stain strongly while the expected positive cells do not. | HPA reports PDK4 as not detected in smooth muscle cells (HPA: smooth muscle). Strong staining there is a warning for nonspecific binding or endogenous detection activity, especially when a known-positive population fails (standard IHC practice); it does not by itself identify which cause applies. |
| Color appears broadly across tissue, including extracellular spaces, with little cell-to-cell distinction. | A field-wide deposit does not match HPA’s cell-associated cytoplasmic profile (HPA: tissue IHC). It suggests background from detection chemistry, insufficient washing or overly strong staining conditions (standard IHC practice). Judge specific signal only after the background is controlled. |
| No staining appears in a section containing adrenal glandular cells or bone-marrow hematopoietic cells. | Those cells are reported as High (HPA: tissue IHC), so a blank result warrants checking the assay before interpreting the specimen as PDK4-negative (standard IHC practice). Biological and sampling variation remain possible; HPA rates agreement with RNA as medium (HPA: Approved reliability description). |
💡Expected PDK4 appearanceCall a result compatible with PDK4 when staining is predominantly cytoplasmic in an HPA High cell population, such as adrenal glandular cells, with clear cellular contrast; strong isolated nuclear, membrane-edge or extracellular color is suspect (HPA: tissue IHC; UniProt Q16654: mitochondrion matrix; standard IHC practice).
| Cell population and baseline expression | Choose and score the named cell population, not the whole section: HPA reports High adrenal glandular and bone-marrow hematopoietic cells, Low hepatocytes and adipocytes, and Not detected smooth muscle cells (HPA: tissue IHC). A weak result in a Low population is therefore less informative than a failed High control. |
| Compartment and optical resolution | PDK4 is assigned to the mitochondrial matrix and lacks a transmembrane segment (UniProt Q16654: location, topology). HPA describes general cytoplasmic IHC expression (HPA: tissue IHC). Routine chromogenic sections support a cytoplasmic call, but apparent granularity alone does not prove mitochondrial colocalisation (standard IHC practice). |
| Strength of the IHC evidence | The listed antibody HPA056731 has Approved IHC status (HPA: antibodies), and HPA describes medium consistency between staining and RNA expression (HPA: reliability description). Use that evidence as a pattern reference while treating unexpected compartments or cell types as findings to verify, rather than as established PDK4 biology. |
| Target-specific fixation sensitivity | Neither the supplied UniProt record nor HPA tissue IHC profile reports how fixation affects PDK4 staining (UniProt Q16654; HPA: tissue IHC). Optimising retrieval is a general IHC workflow decision; these sources do not support a PDK4-specific fixation or retrieval claim. |
| Situation | Likely cause | Next action |
|---|---|---|
| The known-positive cell population is blank. | The expected population is High in HPA, but the failed signal does not identify a single cause (HPA: tissue IHC; standard IHC practice). | Check section identity and cell preservation, then verify primary-antibody application, detection reagents and retrieval conditions with an assay control (standard IHC practice). Do not infer a PDK4-specific retrieval requirement from HPA. |
| Smooth muscle cells stain strongly. | This conflicts with HPA’s Not detected result for smooth muscle cells (HPA: tissue IHC); nonspecific staining or endogenous detection activity are possibilities (standard IHC practice). | Compare a matched no-primary control, review blocking of endogenous detection activity, and assess whether HPA High cells show the expected cytoplasmic pattern (standard IHC practice; HPA: tissue IHC). |
| Signal is chiefly nuclear or traces cell borders. | The compartment differs from HPA’s general cytoplasmic IHC profile and UniProt’s mitochondrial matrix assignment (HPA: tissue IHC; UniProt Q16654: location). | Review morphology and counterstain, compare controls, and reassess antibody and detection conditions before recording those structures as PDK4-positive (standard IHC practice). |
| The entire section has diffuse color. | Widespread deposit can obscure cell-associated staining; detection background, inadequate washing or excessive signal development are general IHC possibilities (standard IHC practice). | Inspect the no-primary control and adjust washing or detection development as indicated by the controls (standard IHC practice). Recheck for cytoplasmic contrast in an HPA High population (HPA: tissue IHC). |
| Hepatocytes or adipocytes show only weak staining. | Both populations are listed as Low in HPA tissue IHC (HPA: hepatocytes, adipocytes). Weak staining alone is therefore not evidence that the assay failed. | Evaluate a concurrently processed HPA High population and report intensity by cell type (HPA: tissue IHC; standard IHC practice). Avoid scoring an entire mixed section from its faintest cells. |
| Can IF/ICC settle whether the chromogenic signal is mitochondrial? | HPA supplies no ICC-IF images or main subcellular location for this record (HPA: subcellular). UniProt assigns PDK4 to the mitochondrial matrix (UniProt Q16654: location). | Treat mitochondrial colocalisation as an unverified question for the separate IF/ICC guide; this IHC evidence alone does not establish it (HPA: subcellular; standard microscopy practice). |
Experiment evidence
IHC & IF images showing PDK4 staining
A02132-1 · IHCIHC analysis of PDK4 using anti-PDK4 antibody (A02132-1). PDK4 was detected in a paraffin-embedded section of human lung cancer tissue. Heat
A02132-1 · IFIF analysis of PDK4 using anti-PDK4 antibody (A02132-1). PDK4 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/ENSG00000004799-PDK4/tissue/cerebral+cortex#imid_16611747
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000004799-PDK4/tissue/colon#imid_16611689
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000004799-PDK4/tissue/liver#imid_16611674
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000004799-PDK4/tissue/kidney#imid_16611691
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for PDK4 IHC & IF
🧪Run bone marrow first: hematopoietic cells should stain (HPA: High in hematopoietic cells). Run smooth muscle as the negative tissue (HPA: Not detected in smooth muscle cells); on the bone marrow slide, use any cells verified to be unstained as an internal background reference—they should show counterstain without specific DAB deposit, but their PDK4 status cannot be assumed from the supplied HPA row.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Smooth muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for PDK4; 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: Include a no-primary, secondary-only control, a concentration-matched rabbit IgG control, and PDK4-knockout material or validated peptide competition as a biological specificity control (caption: rabbit primary and anti-rabbit secondary). Quench endogenous peroxidase and inspect background carefully in bone marrow before interpreting HRP/DAB staining (HPA: bone marrow hematopoietic cells; caption: HRP/DAB detection).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A02132-1 caption does not state a fixative (caption: fixative unreported). The paraffin-section example uses heat retrieval in EDTA at pH 8.0, but supplies no comparison establishing that retrieval is required (caption: EDTA pH 8.0). Frozen sections or IF cannot be judged easier from the supplied evidence; endogenous peroxidase in bone marrow can complicate chromogenic scoring (HPA: bone marrow hematopoietic cells; caption: HRP/DAB detection).
HPA tissue IHC evidence for PDK4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PDK4 IHC Tips
Use the catalog antibody’s paraffin-section IHC conditions as the starting point, then judge staining against cell type, compartment, and matched controls.
Which retrieval condition should I start with for paraffin-section PDK4 IHC?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin-section PDK4 IHC (datasheet A02132-1). The selected image used this retrieval before incubation with 2 μg/ml catalog antibody overnight at 4°C (datasheet A02132-1). If staining is weak, compare a short and longer heating interval on adjacent sections while keeping antibody concentration and detection constant; excessive heating can damage morphology (standard IHC practice). Assess glandular or respiratory epithelial cells alongside smooth muscle, which has contrasting reported staining (HPA: high in glandular and respiratory epithelial cells; not detected in smooth muscle cells). Record retrieval time and tissue integrity before changing buffer chemistry (standard IHC practice).
How should I troubleshoot fixation-related changes in PDK4 staining?
PDK4-specific sensitivity to fixation is unknown: the selected paraffin-section caption does not state a fixative, so its staining cannot establish an optimal fixation method or duration (datasheet A02132-1). Record the fixative, time before fixation, and fixation duration for each specimen, then compare sections processed together under EDTA pH 8.0 retrieval (standard IHC practice; datasheet A02132-1). If signal differs, first check morphology and staining in matched control tissue before attributing the difference to PDK4 abundance (standard IHC practice). Keep the documented 2 μg/ml primary incubation overnight at 4°C constant during that comparison (datasheet A02132-1).
Where should credible PDK4 staining appear within positive cells?
Expect a predominantly cytoplasmic staining pattern in chromogenic sections, with mitochondrial distribution being biologically plausible because PDK4 resides in the mitochondrial matrix (HPA: general cytoplasmic expression; UniProt Q16654: mitochondrion matrix). At routine light-microscope resolution, DAB signal may not resolve individual mitochondria, so score cellular compartments conservatively (standard IHC practice). PDK4 has no transmembrane segment, making a crisp plasma-membrane-only pattern discordant with its annotated topology (UniProt Q16654 topology). Compare the pattern in reported high-staining respiratory epithelial or glandular cells with adjacent smooth muscle cells, reported as undetected, while accounting for the tissue present on the slide (HPA: tissue IHC). Treat isolated nuclear or luminal deposits as requiring control review (standard IHC practice).
Could isoforms or epitope masking explain variable PDK4 staining?
The supplied record lists 0 isoforms, 0 glycosylation sites, and 0 modified residues, so those annotations do not provide a specific explanation for variable staining (UniProt Q16654 record). It identifies a histidine kinase domain spanning residues 138–368, but supplies no antibody epitope location (UniProt Q16654 record; datasheet A02132-1). Therefore, do not infer whether fixation or retrieval masks this particular antibody’s binding site (datasheet A02132-1: epitope unspecified). Keep EDTA pH 8.0 retrieval and the documented primary incubation constant while comparing independently processed sections (datasheet A02132-1; standard IHC practice). If results remain discordant, check antibody specificity with an appropriate independent control before assigning an isoform explanation (standard IHC practice).
How can I assess PDK4 by IF alongside a cell-type marker?
Treat IF as a separate application requiring its own validation; the supplied A02132-1 evidence shows chromogenic staining of a paraffin section (datasheet A02132-1). For multiplexing, pair PDK4 with a marker identifying the expected cell population, such as respiratory epithelial cells in a relevant section, and use spectrally separated fluorophores (HPA: high in respiratory epithelial cells; standard IF practice). Inspect unstained tissue in each channel and place the weaker signal in a channel with less tissue autofluorescence (standard IF practice). Because PDK4 is in the mitochondrial matrix and lacks a transmembrane segment, optimise permeabilisation for access inside mitochondria without losing cellular structure (UniProt Q16654 topology; standard IF practice). Include single-stain and secondary-only controls to assess bleed-through and nonspecific fluorescence (standard IF practice).
How do I distinguish PDK4 staining from chromogenic background?
The selected section used 10% goat-serum blocking, a rabbit primary at 2 μg/ml, peroxidase-conjugated secondary antibody, and DAB development (datasheet A02132-1). If diffuse brown staining obscures cells, compare a no-primary control and review peroxidase blocking, wash conditions, and DAB development time as routine IHC variables (standard IHC practice). Evaluate background against tissue architecture: PDK4 staining is reported as generally cytoplasmic, whereas smooth muscle cells were reported as undetected in HPA tissue IHC (HPA: tissue IHC). Granular deposits at folds or cut edges, or staining shared by the no-primary control, should not be scored as PDK4-positive cells (standard IHC practice). Change one workflow variable at a time and document exposure to chromogen (standard IHC practice).
What scoring method captures meaningful differences in PDK4 IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring; HPA reports general cytoplasmic expression and different staining levels across cell types (HPA: tissue IHC). For a defined population, record the percentage of positive cells and an H-score from 0–300 using intensity categories 0–3, or measure positive-cell density per mm² when cell counts are the biological endpoint (standard IHC practice). Normalise density to viable, analyzable tissue area and report the number of evaluable cells or area for each section (standard IHC practice). Apply identical retrieval, 2 μg/ml primary concentration, DAB development, and image-analysis thresholds across compared sections; that primary concentration was used in the selected image (datasheet A02132-1; standard IHC practice).
When is a PDK4-positive result convincing rather than artefactual?
A convincing result follows cellular boundaries and a mainly cytoplasmic pattern, consistent with PDK4’s matrix localisation and HPA’s general cytoplasmic staining (UniProt Q16654: mitochondrion matrix; HPA: tissue IHC). Check whether stained cells fit the sampled tissue: HPA reports high staining in respiratory epithelial and several glandular cell populations, but no detection in smooth muscle cells (HPA: tissue IHC). Discount accentuated cut edges, necrotic regions, extracellular DAB deposits, and signal reproduced in a no-primary control; endogenous peroxidase can also contribute chromogenic signal (standard IHC practice). Finally, interpret intensity with restraint because HPA rates its tissue IHC evidence “Approved” with medium consistency between staining and RNA expression (HPA: reliability assessment).
Boster reagents
Best PDK4 / [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 4, mitochondrial IHC Antibodies
A02132-1 has IHC data from a human paraffin lung cancer section and IF data from HELA cells and a human paraffin squamous cell carcinoma section (catalog image captions).
Real IHC data
Anti-PDK4 Antibody ®
Cat # A02132-1
A02132-1 was tested by IHC on a human paraffin lung cancer section (A02132-1 IHC caption). The same SKU has IF images from HELA cells and a human paraffin squamous cell carcinoma section (A02132-1 IF captions).
Which to pick: For tissue IHC, choose A02132-1: its caption documents EDTA pH 8 retrieval, 2 μg/ml primary antibody and HRP/DAB detection in a human paraffin section; the fixative is unreported (A02132-1 IHC caption). For IF/ICC, A02132-1 has images using 5 μg/ml primary antibody in HELA cells and human paraffin tissue (A02132-1 IF captions). For cross-species work, A02132-1 lists Human, Mouse and Rat reactivity, although its supplied IHC image documents only human tissue; clonality is unreported (catalog reactivity; A02132-1 IHC caption; catalog clone field).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q16654 (PDK4_HUMAN, [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 4, mitochondrial).
- Human Protein Atlas. PDK4 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. PDK4 subcellular location (ICC-IF): Highest expression in HeLa: 65.4 nTPM.
- Human Protein Atlas. PDK4 antibody validation summary (1 antibodies).
- Decreased AMPK/SIRT1/PDK4 induced by androgen excess inhibits human endometrial stromal cell decidualization in PCOS. Cellular and molecular life sciences : CMLS 2024 — PMC11335245.
- Multi-dimensional analysis reveals the potential of PDK4 as a tumor biomarker and target for immunotherapy. Discover oncology 2026 — PMC13035999.
- High hepatic expression of PDK4 improves survival upon multimodal treatment of colorectal liver metastases. British journal of cancer 2019 — PMC6461828.
- Deficiency of TOP1MT enhances glycolysis through the stimulation of PDK4 expression in gastric cancer. Cancer & metabolism 2024 — PMC10777619.
- PubMed PMID:8798399 — UniProt-cited evidence.
- PubMed PMID:12853948 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for PDK4 ([Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 4, mitochondrial). Expected localisation General cytoplasmic staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A02132-1). Validated IHC antibody: A02132-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Smooth muscle. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet A02132-1); 2. Fixation: Image fixative and duration unreported (datasheet A02132-1); 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 A02132-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A02132-1); 8. Primary antibody: Rabbit anti-PDK4, 2-5 μg/ml (datasheet A02132-1); 9. Primary incubation: Overnight at 4 °C (datasheet A02132-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: No annotated isoforms; epitope impact unknown (UniProt); Regulation: Fasting response; intensity effect unknown (UniProt). Evidence: UniProt Q16654, HPA tissue IHC · Approved, HPA cell-line RNA · Highest expression in HeLa: 65.4 nTPM, Datasheet A02132-1, PMC11335245, PMC6461828. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PDK4/immunohistochemistry-ihc-guide.