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PNPLA8 / Calcium-independent phospholipase A2-gamma · IHC design guide
Design Immunohistochemistry for PNPLA8
Plan PNPLA8 chromogenic IHC on paraffin sections with the IHC-validated antibody, starting at 1:50 (datasheet A30583). Expect cytoplasmic tissue staining (HPA tissue IHC); bronchial respiratory epithelium stains strongly, while vaginal squamous epithelium is not detected (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
PNPLA8 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 | Cytoplasm (HPA tissue IHC); ER, mitochondrial and peroxisomal membranes (UniProt) | |
| Staining pattern | Cytoplasmic staining in glandular, respiratory epithelial and neuronal cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A30583) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Vagina |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent across samples (standard IHC practice; not target-specific) | |
| Caveat | Tissue staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Expression regulation is not annotated (UniProt) | |
| Isoform / epitope | 3 isoforms; check epitope coverage and membrane orientation (UniProt) |
Section 1
Recommended PNPLA8 IHC & IF Protocols
The catalog antibody’s IHC-P protocol uses Tris-EDTA retrieval (datasheet A30583). Two published PNPLA8 tissue microarray protocols provide citrate-retrieval alternatives (PMC10989391; PMC10683240).
| Sample | Paraffin-embedded human Gastric adenocarcinoma tissue; fixative not specified (datasheet A30583) |
| Fixation | Image fixative and duration unreported (datasheet A30583); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 9.0 (datasheet A30583); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-PNPLA8, 1:50 recommended; image 1:200 (datasheet A30583) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | PNPLA8-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 Tris-EDTA, pH 9.0 (datasheet A30583); if optimizing retrieval, both published protocols used citrate at pH 6.0 (PMC10989391; PMC10683240).
Section 2
What Is the Expected PNPLA8 Staining Pattern?
PNPLA8 IHC should show general cytoplasmic staining in glandular cells of several organs, bronchial respiratory epithelium, and selected brain cells (HPA tissue IHC). PNPLA8 is annotated on endoplasmic reticulum, mitochondrial, and peroxisomal membranes, with one transmembrane segment at residues 475–495 (UniProt Q9NP80 topology). HPA rates the tissue staining Approved, with medium consistency between staining and RNA expression (HPA tissue IHC).
| Clear cytoplasmic staining in bronchial respiratory epithelial cells or colon glandular cells. | This fits the reported High staining in those cell types (HPA tissue IHC). Score the relevant cells and their cytoplasmic signal; ordinary chromogenic IHC does not resolve which PNPLA8-associated organelle produced it (UniProt Q9NP80 subcellular location; general IHC practice). |
| Staining appears mainly extracellular or in glandular lumens, without convincing cellular cytoplasmic signal. | That distribution does not match HPA’s general cytoplasmic IHC profile (HPA tissue IHC). Inspect a no-primary control and section quality before assigning it to PNPLA8; deposited chromogen or nonspecific staining can mislead interpretation (general IHC practice). |
| Strong staining appears in vaginal squamous epithelium or broadly across unrelated cell types. | Vaginal squamous epithelial cells are reported as Not detected (HPA tissue IHC). Treat unexpected staining as unconfirmed: cross-reactivity or endogenous detection activity are possibilities, and morphology alone cannot distinguish them (general IHC practice). |
| Diffuse colour covers the section, obscuring boundaries between positive cells and surrounding tissue. | A section-wide haze prevents comparison with HPA’s cell-specific observations (HPA tissue IHC). Check the no-primary control, detection background, blocking, washing, and exposure to chromogen before scoring intensity (general IHC practice). |
| A bronchus or colon section shows no convincing signal in the expected epithelial or glandular cells. | That conflicts with the reported High staining in those cells (HPA tissue IHC), but a single negative slide cannot establish absent PNPLA8. Check tissue identity and run a documented positive section alongside it; then assess the IHC workflow (general IHC practice). |
💡Expected PNPLA8 appearanceCall a section positive when identifiable glandular or bronchial epithelial cells show clear cytoplasmic staining, potentially High in the cited HPA examples; extracellular colour or uniform haze without cell-specific signal is suspect (HPA tissue IHC; general IHC practice).
| Membrane location and light-microscopy resolution | PNPLA8 has one transmembrane segment at 475–495 and is annotated on three intracellular membrane systems (UniProt Q9NP80 topology and subcellular location). HPA describes the tissue IHC appearance as general cytoplasmic expression; chromogenic IHC alone cannot assign an individual organelle (HPA tissue IHC; general IHC practice). |
| Tissue selection and validation strength | Adrenal, appendix, colon, and duodenal glandular cells, bronchial epithelium, and selected brain cells are reported High; vaginal squamous cells are Not detected (HPA tissue IHC). The listed antibody, HPA020083, is IHC Approved; no IHC Enhanced designation is supplied (HPA antibodies). |
| Isoforms and unknown epitope | UniProt lists 3 PNPLA8 isoforms (UniProt Q9NP80). The supplied evidence does not identify the IHC antibody’s epitope or establish which isoforms it detects, so isoform-specific staining or loss of signal cannot be inferred from this record. |
| Does IF/ICC predict the IHC compartment? | HPA ICC-IF reports approved nucleoplasm and cytosol locations, plus Golgi localization (HPA subcellular ICC-IF). HPA tissue IHC instead summarizes a general cytoplasmic pattern (HPA tissue IHC); evaluate a nuclear-only IHC result with controls rather than treating either assay’s compartment call as interchangeable. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive epithelial cells are blank. | The result conflicts with HPA’s High bronchial or colon cell staining, but the failed step is undetermined (HPA tissue IHC). | Confirm the cell type on the section; compare a documented positive section processed in the same IHC run, then review retrieval, antibody incubation, and detection as general IHC practice. |
| The no-primary control develops brown signal. | Primary-antibody-independent staining suggests detection-system background, including possible endogenous enzyme activity (general chromogenic IHC practice). | Resolve the control signal before interpreting PNPLA8; review the detection system, endogenous-activity control, washing, and chromogen development (general IHC practice). |
| Vaginal squamous cells stain strongly. | Those cells are listed as Not detected (HPA tissue IHC); the unexpected signal could reflect nonspecific binding or detection background (general IHC practice). | Check cell identity and no-primary staining, then compare with a documented HPA-positive tissue before assigning PNPLA8 positivity (HPA tissue IHC; general IHC practice). |
| All compartments look uniformly stained. | Uniform haze does not permit the cell-specific, general cytoplasmic assessment reported for PNPLA8 (HPA tissue IHC). | Use the no-primary control to assess background; review blocking, washes, antibody concentration, and chromogen development as general IHC practice, without assuming a PNPLA8-specific fixation effect. |
| Only nuclei stain in tissue IHC. | HPA ICC-IF reports nucleoplasm localization, while its tissue IHC summary is general cytoplasmic expression (HPA subcellular ICC-IF; HPA tissue IHC). The assays do not establish the cause of this slide’s discrepancy. | Check the cytoplasmic signal in a documented positive tissue and examine no-primary staining; record nuclear-only IHC as unresolved until controlled (general IHC practice). |
| A negative result is attributed to fixation. | The supplied HPA tissue IHC and UniProt records provide no target-specific evidence that fixation changes PNPLA8 staining (HPA tissue IHC; UniProt Q9NP80). | Keep fixation sensitivity unassigned; first verify a positive control and routine retrieval and detection performance under the same IHC conditions (general IHC practice). |
Experiment evidence
IHC & IF images showing PNPLA8 staining
A30583 · IHCImmunohistochemical analysis of paraffin-embedded human Gastric adenocarcinoma. 1, Antibody was diluted at 1:200(4° overnight). 2, Tris-EDTA
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000135241-PNPLA8/tissue/cerebral+cortex#imid_5490576
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000135241-PNPLA8/tissue/colon#imid_5490510
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000135241-PNPLA8/tissue/liver#imid_5490519
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000135241-PNPLA8/tissue/kidney#imid_5490504
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA020083; green = PNPLA8, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA020083; green = PNPLA8, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for PNPLA8 IHC & IF
🧪Run bronchus first and require staining in respiratory epithelial cells (HPA: High in bronchus respiratory epithelial cells). Run vagina as the negative tissue (HPA: Not detected in vaginal squamous epithelial cells); on the bronchus slide, use cells that remain unstained as a background reference, without assuming a particular cell type is PNPLA8-negative.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Vagina (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show PNPLA8 in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host-species-matched, clonality-appropriate isotype controls, plus PNPLA8 knockout tissue or a peptide-block control when suitable material is available (standard IHC practice). For chromogenic bronchus IHC, quench endogenous peroxidase and block endogenous biotin if using avidin–biotin detection (standard IHC practice).
⚠️Feasibility: A PNPLA8-specific fixation window is unreported, and the selected paraffin-section caption does not state its fixative (catalog antibody caption: A30583). That caption uses Tris-EDTA at pH 9.0 for retrieval, but does not establish that PNPLA8 staining depends on retrieval (catalog antibody caption: A30583). The supplied evidence does not establish whether frozen sections or IF are easier; check bronchial mucus for trapped chromogen when scoring epithelial staining (standard IHC practice).
HPA tissue IHC evidence for PNPLA8
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 → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Vagina | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PNPLA8 IHC Tips
Use the catalog antibody’s paraffin-section IHC conditions as a starting point, then judge PNPLA8 staining against tissue morphology and controls.
What retrieval conditions should I try first for weak PNPLA8 staining?
Start with heat-mediated Tris-EDTA at pH 9.0 for PNPLA8 paraffin-section IHC (datasheet A30583). The selected image used this retrieval with primary antibody at 1:200, incubated at 4°C overnight (caption A30583). Keep heating, cooling, detection, and counterstaining consistent across adjacent sections, with a no-primary control (standard IHC practice). If staining remains weak, test citrate at pH 6.0 as a fallback and compare signal, background, and morphology (standard IHC practice). PNPLA8 has a membrane-spanning segment at residues 475–495, but the antibody’s epitope is unspecified, so retrieval behavior cannot be predicted from topology alone (UniProt Q9NP80 topology; supplied antibody evidence).
How should I troubleshoot fixation-related loss of PNPLA8 staining?
Target-specific fixation sensitivity is unknown; the selected paraffin-section caption does not state a fixative (caption A30583). Record the fixative and fixation duration for each specimen before comparing staining across cases (standard IHC practice). If fixation is suspected, compare matched tissue sections processed under documented conditions while holding Tris-EDTA retrieval at pH 9.0 and primary incubation constant (datasheet A30583; standard IHC practice). Examine morphology and no-primary controls alongside PNPLA8 signal to distinguish poor antigen detection from tissue damage or detection background (standard IHC practice). Do not infer a preferred fixation duration from tissue staining patterns or PNPLA8’s membrane topology (HPA tissue IHC; UniProt Q9NP80 topology).
Which compartments should count as PNPLA8-positive in chromogenic sections?
Assess cytoplasmic staining first: tissue IHC is described as showing general cytoplasmic expression (HPA tissue IHC). PNPLA8 is annotated at endoplasmic reticulum, mitochondrial, and peroxisomal membranes, with a transmembrane segment at residues 475–495 (UniProt Q9NP80). Routine chromogenic sections may not resolve those organelles, so score a reproducible cytoplasmic pattern in the appropriate cell population (standard IHC practice). Cell-line IF also reports nucleoplasm and cytosol as main locations, plus Golgi localisation, but that observation needs separate evaluation in tissue IHC (HPA subcellular). Compare any prominent nuclear staining with no-primary controls and tissue morphology before assigning it to PNPLA8 (standard IHC practice).
Could isoforms or epitope accessibility explain inconsistent PNPLA8 IHC?
PNPLA8 has 3 annotated isoforms, so confirm which sequences the antibody’s stated immunogen could recognize before comparing specimens (UniProt Q9NP80; standard IHC practice). Its PNPLA domain spans residues 445–640, and its membrane-spanning segment lies at 475–495 (UniProt Q9NP80). Glycosylation sites are annotated at residues 4 and 361, but no antibody epitope is supplied, so a modification-dependent staining effect remains unproven (UniProt Q9NP80; supplied antibody evidence). Compare serial sections under identical retrieval and detection conditions, using a second independently mapped antibody if available (standard IHC practice). Report the tested antibody and retrieval conditions when discussing isoform coverage or epitope accessibility (standard IHC practice).
How can IF help investigate a questionable PNPLA8 IHC pattern?
Use IF as a separate localisation check rather than transferring the paraffin-section antibody dilution into an untested IF protocol (caption A30583; standard IF practice). Multiplex PNPLA8 with an epithelial marker when examining bronchial respiratory epithelium, a high-staining population in tissue IHC (HPA tissue IHC; standard IF practice). Choose a far-red fluorophore if shorter-wavelength channels show tissue autofluorescence, and include single-label and no-primary controls (standard IF practice). Because PNPLA8 associates with intracellular membranes, choose permeabilisation according to the mapped epitope’s membrane-facing side; that side is not specified for this antibody (UniProt Q9NP80 topology; supplied antibody evidence). Compare compartment patterns with reported nucleoplasm, cytosol, and Golgi IF localisation cautiously (HPA subcellular).
How do I reduce diffuse brown staining without losing PNPLA8 signal?
Run a no-primary section to identify detection-system staining, and block endogenous peroxidase before chromogenic detection (standard IHC practice). Evaluate DAB development and counterstaining on adjacent sections processed with the same Tris-EDTA retrieval at pH 9.0 (datasheet A30583; standard IHC practice). The selected image used primary antibody at 1:200 and a 4°C overnight incubation; treat these as documented starting conditions, not a background threshold (caption A30583). If diffuse signal persists, adjust blocking, washing, or antibody concentration systematically while preserving a positive tissue control (standard IHC practice). Compare the cellular pattern with HPA’s general cytoplasmic staining description before interpreting residual brown pigment as PNPLA8 (HPA tissue IHC).
How should I quantify PNPLA8 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because tissue IHC describes general cytoplasmic PNPLA8 expression (HPA tissue IHC). An H-score combines the percentage of cells at each intensity grade, 0–3, into a 0–300 score (standard IHC practice). Alternatively, report percent positive cells or positive-cell density per mm², with a fixed threshold and documented sampling fields (standard IHC practice). Normalize cell-based scores to viable cells of the specified type, or area-based scores to viable tissue area, excluding necrosis and section edges (standard IHC practice). Use matched retrieval, detection, and counterstaining conditions across compared sections; the documented retrieval is Tris-EDTA at pH 9.0 (datasheet A30583; standard IHC practice).
How can I distinguish genuine PNPLA8 staining from artefact?
Look for a reproducible cellular pattern, especially cytoplasmic signal, rather than interpreting isolated brown deposits as PNPLA8 (HPA tissue IHC; standard IHC practice). High staining is reported in bronchial respiratory epithelial cells and several glandular-cell populations; vaginal squamous epithelial cells are reported as not detected (HPA tissue IHC). Treat those patterns as reference observations, not definitive positive or negative controls for every specimen (HPA tissue IHC; standard IHC practice). Question signal confined to section edges, necrotic regions, or no-primary controls, and assess endogenous peroxidase before attributing DAB deposits to the target (standard IHC practice). Prominent nuclear staining warrants separate validation because tissue IHC describes general cytoplasmic expression, although cell-line IF reports nucleoplasmic localisation (HPA tissue IHC; HPA subcellular).
Boster reagents
Best PNPLA8 / Calcium-independent phospholipase A2-gamma IHC Antibodies
A30583 has IHC data from paraffin-embedded human gastric adenocarcinoma (A30583 image caption). Its listed reactivity covers human and mouse (A30583 catalog).
Real IHC data
Anti-PNPLA8/Ipla2 Gamma Antibody
Cat # A30583
A30583 is listed for IHC and has an IHC image from paraffin-embedded human gastric adenocarcinoma (A30583 catalog; A30583 image caption). It is listed as human- and mouse-reactive; the supplied image shows human tissue only (A30583 catalog; A30583 image caption).
Which to pick: For tissue IHC, choose A30583: it is a rabbit polyclonal listed for IHC, with a paraffin-section example; the fixative is unreported (A30583 catalog; A30583 image caption). For IF/ICC, A30583 has neither a listed IF application nor an IF image (A30583 catalog). For cross-species work, A30583 lists human and mouse reactivity, while its supplied IHC image shows human tissue only (A30583 catalog; A30583 image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9NP80 (PLPL8_HUMAN, Calcium-independent phospholipase A2-gamma).
- Human Protein Atlas. PNPLA8 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. PNPLA8 subcellular location (ICC-IF): Mainly localized to the nucleoplasm and cytosol. In addition localized to the Golgi apparatus..
- Human Protein Atlas. PNPLA8 antibody validation summary (1 antibodies).
- High patatin like phospholipase domain containing 8 expression as a biomarker for poor prognosis of colorectal cancer. World journal of gastrointestinal oncology 2024 — PMC10989391.
- GPD1L as a potential biomarker associated with Treg cell infiltration and lipid metabolism in clear cell renal cell carcinoma. Journal of translational medicine 2025 — PMC12329951.
- Key regulator PNPLA8 drives phospholipid reprogramming induced proliferation and migration in triple-negative breast cancer. Breast cancer research : BCR 2023 — PMC10683240.
- PubMed PMID:10833412 — UniProt-cited evidence.
- PubMed PMID:10744668 — UniProt-cited evidence.
- PubMed PMID:17974005 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for PNPLA8 (Calcium-independent phospholipase A2-gamma). Expected localisation Cytoplasm (HPA tissue IHC); ER, mitochondrial and peroxisomal membranes (UniProt). Antigen retrieval: Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A30583). Validated IHC antibody: A30583 at 1:50 recommended; image 1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Vagina. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human Gastric adenocarcinoma tissue; fixative not specified (datasheet A30583); 2. Fixation: Image fixative and duration unreported (datasheet A30583); 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: Tris-EDTA pH 9.0 (datasheet A30583); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-PNPLA8, 1:50 recommended; image 1:200 (datasheet A30583); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep paraffin-section fixation consistent across samples (standard IHC practice; not target-specific); Caveat: Tissue staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 3 isoforms; check epitope coverage and membrane orientation (UniProt); Regulation: Expression regulation is not annotated (UniProt). Evidence: UniProt Q9NP80, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm and cytosol. In addition…, Datasheet A30583, PMC10989391, PMC10683240. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PNPLA8/immunohistochemistry-ihc-guide.