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PLD6 / Mitochondrial cardiolipin hydrolase · IHC design guide
Design Immunohistochemistry for PLD6
Plan chromogenic PLD6 IHC on paraffin sections using the catalog antibody’s 2–5 μg/ml range (datasheet A10904-1). Assess granular cytoplasmic staining against the reported high signal in testis pachytene spermatocytes and glandular cells (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
PLD6 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 outer membrane or Golgi (UniProt) | |
| Staining pattern | Granular cytoplasm in glandular cells and pachytene spermatocytes (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A10904-1) | |
| Positive control | Bronchus+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 | Cell type and developmental stage can change PLD6 location (UniProt) | |
| Regulation | High in pachytene spermatocytes (HPA tissue IHC) | |
| Isoform / epitope | 0 isoforms or processing events; residues 28–252 face cytoplasm (UniProt) |
Section 1
Recommended PLD6 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet A10904-1) is accompanied by one published chromogenic PLD6 IHC protocol in colorectal specimens (PMC12046002).
| Sample | Paraffin-embedded human endometrial adenocarcinoma tissue; fixative not specified (datasheet A10904-1) |
| Fixation | Image fixative and duration unreported (datasheet A10904-1); 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 A10904-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A10904-1) |
| Primary antibody | Rabbit anti-PLD6, 2-5 μg/ml (datasheet A10904-1) |
| Primary incubation | Overnight at 4 °C (datasheet A10904-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A10904-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | PLD6-positive staining in respiratory epithelial cells of bronchus (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 (datasheet A10904-1); the colorectal IHC report does not specify retrieval conditions (PMC12046002).
Section 2
What Is the Expected PLD6 Staining Pattern?
PLD6 is an outer mitochondrial membrane protein, with its main domain facing the cytoplasm; UniProt also reports Golgi localisation in mouse germ cells at specific stages (UniProt Q8N2A8 topology and localisation). In paraffin sections, expect granular cytoplasmic staining, especially in HPA high-staining glandular and respiratory epithelial cells and pachytene spermatocytes (HPA tissue IHC). HPA rates the tissue antibody Approved, with medium consistency against RNA data (HPA tissue IHC).
| Granular cytoplasmic staining is strong in pancreatic exocrine cells, duodenal glands, or bronchial respiratory epithelium. | This matches the general cytoplasmic granular pattern and High staining reported for those cell populations (HPA tissue IHC). Assess the named cells within each tissue rather than treating every neighbouring cell as a positive control (HPA tissue IHC). |
| Signal is predominantly nuclear, extracellular, or uniformly spread across the section. | This departs from HPA's granular cytoplasmic tissue pattern and UniProt's outer mitochondrial membrane and Golgi localisation (HPA tissue IHC; UniProt Q8N2A8 localisation). Check whether the same pattern appears in a no-primary control before calling it PLD6 (general IHC practice). |
| Strong staining appears in adipocytes, adrenal glandular cells, or ovarian stromal cells. | Those specific cell populations are Not detected in HPA tissue IHC; strong signal there warrants a cross-reactivity or endogenous detection check (HPA tissue IHC; general IHC practice). The ovarian-stroma result does not negate UniProt's broader report of ovarian PLD6 protein (UniProt Q8N2A8 tissue specificity). |
| A diffuse chromogenic haze obscures cell boundaries and granular detail. | The slide cannot be judged against HPA's granular cytoplasmic pattern while background dominates (HPA tissue IHC). Background may reflect detection chemistry or inadequate blocking or washing; use control sections to distinguish these possibilities (general IHC practice). |
| A validated run shows no staining in pancreatic exocrine cells or pachytene spermatocytes. | Both are High-staining populations in HPA tissue IHC, so an absent signal calls for an assay check (HPA tissue IHC). Review the antibody and detection steps alongside a positive control; a negative slide alone does not establish absent PLD6 expression (general IHC practice). |
💡Expected PLD6 appearanceA convincing positive is granular cytoplasmic staining in the named High-staining epithelial, glandular, or pachytene-spermatocyte populations (HPA tissue IHC); dominant nuclear, extracellular, or diffuse section-wide signal is discordant with the reported pattern (HPA tissue IHC; UniProt Q8N2A8 localisation).
| Membrane topology | PLD6 spans residues 5–27, while residues 28–252 face the cytoplasm (UniProt Q8N2A8 topology). Antibody epitope position is not supplied, so topology alone cannot predict retrieval needs or antibody access. |
| Cell and stage context | UniProt places PLD6 at the outer mitochondrial membrane and reports Golgi restriction in mouse pachytene spermatocytes and spermatids under nuclease-predominant conditions (UniProt Q8N2A8 localisation). Do not assign that mouse stage-specific distribution to every human section. |
| Tissue and cell selection | HPA reports High staining in several glandular or respiratory epithelial populations and pachytene spermatocytes, but Not detected in ovarian stroma and adipocytes (HPA tissue IHC). These are cell-specific comparisons, not whole-organ positive or negative labels. |
| IHC evidence strength | The HPA tissue antibody HPA049345 is Approved, and staining has medium consistency with RNA data (HPA antibody validation; HPA tissue IHC). Treat agreement with its recorded pattern as supporting evidence, while resolving unexpected staining with controls. |
| Processing and modifications | UniProt lists one 1–252 chain, no signal peptide or propeptide, and no glycosylation sites (UniProt Q8N2A8 processing and glycosylation). These annotations do not establish a target-specific fixation effect or a required antigen retrieval condition. |
| IF/ICC evidence limit | HPA's subcellular summary says Membrane but supplies no main location or ICC-IF image-bearing cell lines; its listed antibody has no ICC validation status (HPA subcellular; HPA antibody validation). A precise IF puncta pattern is therefore unconfirmed by these payloads. |
| Situation | Likely cause | Next action |
|---|---|---|
| No chromogenic signal in a High-staining reference population. | The result conflicts with HPA's pancreatic exocrine or pachytene-spermatocyte observations, but the failed step is unknown (HPA tissue IHC). | Check the positive-control section, antibody identity and dilution, and detection reagents; review retrieval against the assay instructions (general IHC practice). |
| Granular signal is lost beneath broad brown background. | Background obscures the HPA granular cytoplasmic pattern; endogenous peroxidase or nonspecific detection is possible in chromogenic IHC (HPA tissue IHC; general IHC practice). | Run a no-primary control, verify endogenous-peroxidase blocking where relevant, and review blocking and washes (general IHC practice). |
| Nuclei dominate the stain in otherwise positive tissue. | Nuclear dominance is discordant with HPA's cytoplasmic pattern and UniProt's membrane-associated locations (HPA tissue IHC; UniProt Q8N2A8 localisation). | Compare a no-primary control and repeat with the IHC-validated antibody under its documented conditions (general IHC practice). |
| Adipocytes or ovarian stromal cells stain as strongly as the positive control. | HPA lists those cells as Not detected; cross-reactivity or detection background should be considered (HPA tissue IHC; general IHC practice). | Inspect matched negative and no-primary controls before scoring these cells as PLD6-positive (general IHC practice). |
| Testis staining varies between germ-cell stages. | HPA identifies pachytene spermatocytes as High; UniProt describes stage-dependent localisation in mouse testis (HPA tissue IHC; UniProt Q8N2A8 localisation). | Score identified cell populations separately and record the cytoplasmic pattern; avoid assigning a mouse Golgi pattern to the human section (HPA tissue IHC; UniProt Q8N2A8 localisation). |
| IF/ICC: should every PLD6-positive cell show mitochondrial puncta? | UniProt also reports context-dependent Golgi localisation, while HPA supplies no ICC-IF image-bearing cell lines (UniProt Q8N2A8 localisation; HPA subcellular). | Use the dedicated IF/ICC guide and validate localisation in the chosen cells; do not infer an IF protocol or universal puncta pattern from the IHC record (HPA antibody validation; general IF practice). |
Experiment evidence
IHC & IF images showing PLD6 staining
A10904-1 · IHCIHC analysis of PLD6 using anti-PLD6 antibody (A10904-1). PLD6 was detected in a paraffin-embedded section of human endometrial adenocarcino
A10904-1 · IHCIHC analysis of PLD6 using anti-PLD6 antibody (A10904-1). PLD6 was detected in a paraffin-embedded section of human colon adenocarcinoma tis
A10904-1 · IFIF analysis of PLD6 using anti-PLD6 antibody (A10904-1). PLD6 was detected in a paraffin-embedded section of human oesophagus squama cancer
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000179598-PLD6/tissue/cerebral+cortex#imid_13502123
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000179598-PLD6/tissue/colon#imid_13499429
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000179598-PLD6/tissue/liver#imid_13499446
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000179598-PLD6/tissue/kidney#imid_13499439
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for PLD6 IHC & IF
🧪Run pancreas first and score its exocrine glandular cells for PLD6 staining (HPA: High in pancreatic exocrine glandular cells). Use adipose tissue as the biological negative (HPA: Not detected in adipocytes); on the pancreatic slide, candidate internal negative stromal cells should show only background chromogen and intact counterstained nuclei (standard IHC practice; HPA does not assign those cells a PLD6 level).
Positive control tissue: Bronchus (Respiratory epithelial cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for PLD6; derive a cell-line control from the positive tissue's cell type (Respiratory epithelial cells) and confirm it by RNA or western blot first.
Technical controls: Include no-primary (secondary-only) and concentration-matched rabbit IgG isotype controls, plus PLD6-knockout tissue if available as a biological specificity control (selected-SKU caption: rabbit primary antibody; standard IHC practice). Quench endogenous peroxidase and check the no-primary pancreatic section for residual HRP/DAB background (selected-SKU caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported in the supplied evidence, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU tissue-IHC caption: fixative unreported). The caption uses heat retrieval in EDTA at pH 8.0, a starting condition to verify for pancreatic IHC rather than a demonstrated requirement for that tissue (selected-SKU tissue-IHC caption: EDTA retrieval in a human endometrial adenocarcinoma section). Frozen sections and IF are not established as easier by these data; assess pancreatic granular staining against the no-primary control before scoring it as specific (HPA: High in pancreatic exocrine glandular cells; HPA subcellular: no ICC-IF image cell lines; standard IHC practice).
HPA tissue IHC evidence for PLD6
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 |
|---|---|---|---|---|
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Nasopharynx | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Pancreas | Exocrine glandular cells | High | Protein (IHC) | HPA → |
| Salivary gland | 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 → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PLD6 IHC Tips
Troubleshoot PLD6 chromogenic IHC by checking retrieval, staining pattern, controls and scoring against the available tissue and protein evidence.
What retrieval should I try first for weak PLD6 staining in paraffin sections?
Start with heat mediated retrieval in EDTA at pH 8.0 (datasheet A10904-1). The selected paraffin section example used this retrieval before 2 μg/ml primary antibody overnight at 4°C, so reproduce that sequence when assessing weak staining (datasheet A10904-1). Keep section thickness, heating and cooling consistent across the test and control slides; changes in retrieval can alter staining intensity and tissue integrity (standard IHC practice). If staining remains weak, compare a limited alternative retrieval condition on adjacent sections, recording its buffer, pH and heating time rather than treating it as validated for this antibody (standard IHC practice).
How should I handle variable fixation when PLD6 staining is inconsistent?
The selected paraffin section caption does not state a fixative, so PLD6 sensitivity to fixation is unknown for this antibody (datasheet A10904-1). Record the fixative, time before fixation, fixation duration and tissue processing for each block; compare sections processed as similarly as possible (standard IHC practice). Run a known staining control alongside affected sections and apply the same EDTA pH 8.0 retrieval before changing antibody concentration (datasheet A10904-1; standard IHC practice). If staining differs between blocks, report the processing difference as a possible explanation, since neither tissue staining patterns nor protein topology establishes a target specific fixation effect (standard IHC practice).
Where should PLD6 signal appear, and how much does IHC resolve?
Expect a granular cytoplasmic pattern in tissue sections, consistent with the reported PLD6 IHC profile (HPA: general cytoplasmic expression with a granular pattern). PLD6 is assigned to the mitochondrial outer membrane and Golgi, with a predicted transmembrane segment at residues 5–27 and a cytoplasmic region at 28–252 (UniProt Q8N2A8 subcellular location and topology). Mouse pachytene spermatocytes and spermatids are reported to show Golgi restricted localisation, so interpret germ cell patterns in their developmental context (UniProt Q8N2A8 subcellular location). Chromogenic granules alone cannot establish mitochondrial or Golgi identity; assess cellular context and use compartment markers in a separate localisation experiment (standard IHC practice).
Can a hidden isoform or epitope explain discordant PLD6 staining?
The supplied record lists 0 isoforms, a 252 residue chain and no annotated glycosylation or modified residues (UniProt Q8N2A8 isoforms, processing and modifications). It places the membrane segment at residues 5–27 and the larger cytoplasmic region at 28–252, but the catalog antibody epitope is not specified (UniProt Q8N2A8 topology; datasheet A10904-1). Therefore, do not assign weak staining to a particular isoform, modification or inaccessible epitope without independent epitope information (standard IHC interpretation). Compare retrieval matched control sections and, if available, an antibody recognizing a documented different epitope; concordant cellular staining strengthens the interpretation (standard IHC practice).
How can I investigate PLD6 localisation with a separate IF experiment?
Treat IF as a separate validation experiment; the supplied antibody example documents paraffin section chromogenic IHC, while the subcellular record supplies no ICC/IF images (datasheet A10904-1; HPA subcellular record). Multiplex PLD6 with a marker for the expected cell population, such as a pachytene spermatocyte marker in testis, and include single stain controls (HPA: high in pachytene spermatocytes; standard IF practice). Choose fluorophores after checking tissue autofluorescence in an unstained section, keeping the weaker signal in a cleaner spectral channel (standard IF practice). Because the predicted region at residues 28–252 faces the cytoplasm, test permeabilisation if the antibody epitope lies there; its location is currently unspecified (UniProt Q8N2A8 topology; datasheet A10904-1).
How can I reduce diffuse brown staining without suppressing PLD6 signal?
First compare a no primary control with the stained section to distinguish detection background from antibody associated signal (standard IHC practice). The selected example used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and a peroxidase linked secondary for 30 minutes at 37°C (datasheet A10904-1). Peroxidase blocking before HRP detection and careful DAB development are general controls for endogenous enzyme activity and excessive chromogen deposition (standard IHC practice). Recheck washes and titrate the primary around the documented concentration while retaining a positive tissue control, since PLD6 can produce granular cytoplasmic staining (standard IHC practice; HPA: granular cytoplasmic expression).
How should I score PLD6 staining across heterogeneous tissue regions? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, since PLD6 IHC is described as granular cytoplasmic staining (HPA: general cytoplasmic expression with a granular pattern). For each region, record the percentage of positive target cells and an intensity grade of 0–3; an H-score then spans 0–300 (standard IHC scoring practice). If using positive cell density per mm², divide the count by viable tissue area and report the cell selection rule (standard IHC scoring practice). Apply one threshold and imaging setting across sections, normalise to the number of eligible cells or viable area, and report controls and regional variability (standard IHC scoring practice).
What distinguishes credible PLD6 staining from artefact in a tissue section?
Credible staining should be granular and cytoplasmic in the scored cells, rather than predominantly nuclear or confined to tissue edges (HPA: granular cytoplasmic expression; standard IHC interpretation). High staining in pachytene spermatocytes or pancreatic exocrine glandular cells has tissue IHC support, while the listed adipocytes are not detected (HPA: testis, pancreas and adipose tissue profiles). Examine viable interiors separately from folds, cut edges and necrotic regions, where chromogen can mislead interpretation (standard IHC practice). A no primary control helps identify endogenous peroxidase or detection background; confirm unexpected cell populations with an independent control before attributing their staining to PLD6 (standard IHC practice).
Boster reagents
Best PLD6 / Mitochondrial cardiolipin hydrolase IHC Antibodies
A10904-1 has IHC images from human paraffin sections of endometrial and colon adenocarcinoma, plus an IF image from a human paraffin section of oesophagus squama cancer (catalog image captions).
Real IHC data
Anti-PLD6 Antibody ®
Cat # A10904-1
A10904-1 is listed for human IHC and IF (catalog applications; reactivity). Its captions show IHC in human paraffin sections of endometrial and colon adenocarcinoma and IF in a human paraffin section of oesophagus squama cancer (catalog image captions).
Which to pick: For tissue IHC, choose A10904-1 for human paraffin sections; its own IHC captions show both tissues, but report no fixative (catalog IHC captions). For IF, A10904-1 has a human paraffin-section example; ICC validation is unreported (catalog IF caption; applications). No cross-species choice is supported because A10904-1 lists Human reactivity only (catalog reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8N2A8 (PLD6_HUMAN, Mitochondrial cardiolipin hydrolase).
- Human Protein Atlas. PLD6 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. PLD6 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. PLD6 antibody validation summary (1 antibodies).
- Expression of Phospholipase D Family Member 6 in Bovine Testes and Its Molecular Characteristics. International journal of molecular sciences 2023 — PMC10418416.
- Phospholipase D6 activates Wnt/β-catenin signaling through mitochondrial metabolic reprogramming to promote tumorigenesis in colorectal cancer. Experimental & molecular medicine 2025 — PMC12046002.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16625196 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for PLD6 (Mitochondrial cardiolipin hydrolase). Expected localisation Granular cytoplasm (HPA tissue IHC); mitochondrial outer membrane or Golgi (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A10904-1). Validated IHC antibody: A10904-1 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bronchus (Respiratory epithelial cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human endometrial adenocarcinoma tissue; fixative not specified (datasheet A10904-1); 2. Fixation: Image fixative and duration unreported (datasheet A10904-1); 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 A10904-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A10904-1); 8. Primary antibody: Rabbit anti-PLD6, 2-5 μg/ml (datasheet A10904-1); 9. Primary incubation: Overnight at 4 °C (datasheet A10904-1). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Cell type and developmental stage can change PLD6 location (UniProt); Isoform / epitope: 0 isoforms or processing events; residues 28–252 face cytoplasm (UniProt); Regulation: High in pachytene spermatocytes (HPA tissue IHC). Evidence: UniProt Q8N2A8, HPA tissue IHC · Approved, HPA ICC-IF · Membrane, Datasheet A10904-1, PMC12046002. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PLD6/immunohistochemistry-ihc-guide.