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PHF8 / Histone lysine demethylase PHF8 · IHC design guide
Design Immunohistochemistry for PHF8
Plan paraffin-section PHF8 IHC with the catalog antibody at 1:50 (datasheet M03288). Compare nuclear staining with colon glandular cells or kidney tubular cells, where staining is high, while allowing for occasional cytoplasmic signal (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
PHF8 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 | Predominantly nuclear, with occasional cytoplasmic staining (HPA tissue IHC) | |
| Staining pattern | Variable nuclear staining across cell types; occasional cytoplasm (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M03288) | |
| Positive control | Cerebellum+4 more · see all | |
| Negative control | Liver+1 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M03288) | |
| Caveat | Tissue staining has medium consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Tissue-specific expression regulation is not annotated (UniProt) | |
| Isoform / epitope | 5 isoforms; check epitope coverage across variants (UniProt) |
Section 1
Recommended PHF8 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet M03288) is accompanied by published PHF8 IHC methods for gallbladder carcinoma and melanoma (PMC10047028; PMC8856617).
| Sample | Paraffin-embedded human prostate cancer tissue; fixative not specified (datasheet M03288) |
| Fixation | Image fixative and duration unreported (datasheet M03288); 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 M03288); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M03288) |
| Primary antibody | Rabbit monoclonal (clone ADAI-16) anti-PHF8, 1:50 (datasheet M03288) |
| Primary incubation | Overnight at 4 °C (datasheet M03288) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet M03288) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | PHF8-positive staining in cells in molecular layer of cerebellum (HPA tissue IHC: High). HPA tissue profile: General nuclear with occasional cytoplasmic expression at variable levels. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet M03288); compare the published pH 6.0 and pH 9.0 conditions during optimization (PMC10047028; PMC8856617).
Section 2
What Is the Expected PHF8 Staining Pattern?
PHF8 should stain mainly nuclei in paraffin sections, with high staining reported in colon glandular cells, kidney tubule cells and several other cell types (HPA tissue IHC). HPA describes a general nuclear pattern with occasional cytoplasmic expression, but rates tissue IHC only Approved: antibody staining and RNA show medium consistency, and external verification is pending (HPA tissue IHC). PHF8 is nuclear, can occur in nucleoli, and has no transmembrane segment (UniProt Q9UPP1).
| Clear nuclear chromogen in colon glandular cells or kidney tubule cells. | This matches cell types scored High by HPA (HPA tissue IHC: colon glandular cells; kidney tubule cells). Judge the cell population and nuclear compartment together; occasional cytoplasmic staining can occur, but the reported overall pattern is nuclear (HPA tissue IHC). |
| Predominantly membranous or broadly cytoplasmic staining, with little nuclear signal. | This does not fit PHF8’s nuclear localization or lack of a transmembrane segment (UniProt Q9UPP1). HPA reports occasional cytoplasmic expression, so a small cytoplasmic component alone is inconclusive (HPA tissue IHC). Reassess specificity when the nonnuclear pattern dominates. |
| Strong signal in liver cholangiocytes or oral mucosal squamous cells. | Both populations were scored Not detected by HPA, although esophageal squamous cells were scored High (HPA tissue IHC). Check cell identity and controls; unexpected chromogen may reflect cross-reactivity or endogenous detection activity (general IHC practice). |
| Diffuse chromogen across nuclei, cytoplasm, stroma or empty spaces. | A widespread haze obscures the cell and compartment pattern expected for PHF8 (HPA tissue IHC; UniProt Q9UPP1). Uneven blocking, excess primary antibody or detection background can produce nonspecific staining (general IHC practice); intensity alone cannot confirm a positive result. |
| No nuclear signal in colon glands or kidney tubules. | These are reported High cell populations, so a blank result warrants a technical check (HPA tissue IHC). Examine tissue preservation, retrieval, antibody dilution and detection controls as general IHC checks; HPA does not establish PHF8-specific fixation or retrieval sensitivity. |
💡Expected PHF8 appearanceCall a section positive when identifiable HPA High cells, such as colon glandular cells, show clear nuclear staining (HPA tissue IHC); predominantly membranous staining or diffuse chromogen without a cell-specific nuclear pattern is suspect (UniProt Q9UPP1; general IHC practice).
| Cell population | PHF8 staining varies by cell type: colon glandular cells and kidney tubule cells are High, while liver cholangiocytes are Not detected (HPA tissue IHC). Score named populations rather than treating an entire organ as uniformly positive or negative. |
| Cell-cycle state | PHF8 is recruited to H3K4me3 chromatin in interphase and dissociates from chromatin on entry into mitosis (UniProt Q9UPP1). Nuclear distribution may therefore differ in mitotic cells; this annotation does not predict a specific chromogenic intensity. |
| Subnuclear distribution | UniProt lists nucleus and nucleolus (UniProt Q9UPP1). HPA’s ICC-IF summary instead identifies the nucleoplasm as the supported main location (HPA subcellular ICC-IF). Do not require a prominent nucleolar focus to score tissue IHC. |
| Antibody evidence | The listed IHC antibody HPA038779 is Approved; the HPA tissue profile reports medium staining–RNA consistency and pending external verification (HPA antibodies; HPA tissue IHC). Compare compartment and cell type with controls before treating an isolated signal as specific. |
| Isoforms and processing | UniProt lists five PHF8 isoforms, no signal peptide or propeptide, and a full-length chain (UniProt Q9UPP1). These annotations do not establish which isoforms the IHC antibody detects; interpret unexpected staining through controls, not assumed isoform coverage. |
| IF/ICC Q: Where should PHF8 appear? | A: Mainly in the nucleoplasm (HPA subcellular ICC-IF: supported); UniProt also lists nucleolar localization (UniProt Q9UPP1). This location check is for IF/ICC interpretation, not an alternative tissue IHC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| The expected nuclear signal is absent in a reported High cell population. | Retrieval, primary-antibody dilution or detection may have failed (general IHC practice); the HPA High designation describes an observed population, not a guarantee for every section (HPA tissue IHC). | Check the catalog antibody’s IHC-P instructions and run a reported High population, such as colon glands, beside the specimen (HPA tissue IHC). Verify each detection step with appropriate controls (general IHC practice). |
| Chromogen fills cytoplasm or outlines cell membranes more strongly than nuclei. | The dominant distribution conflicts with nuclear PHF8 and its lack of a transmembrane segment (UniProt Q9UPP1), though occasional cytoplasmic expression is reported (HPA tissue IHC). | Confirm morphology and nuclear counterstain, then compare a no-primary control and a reported High tissue (general IHC practice; HPA tissue IHC). Reassess antibody dilution and detection background before scoring. |
| A reported Not detected population appears strongly positive. | Liver cholangiocytes and oral mucosal squamous cells were Not detected in the HPA profile (HPA tissue IHC). Misidentified cells, nonspecific binding or endogenous detection activity can explain an unexpected IHC signal (general IHC practice). | Recheck the cell population and inspect no-primary and detection controls (general IHC practice). Compare with a reported High population in the same staining run (HPA tissue IHC). |
| The whole section has a brown haze or deposits outside cells. | Diffuse background prevents a compartment call and can arise from excessive antibody or inadequate blocking or washing (general IHC practice). It does not resemble HPA’s general nuclear profile (HPA tissue IHC). | Inspect a no-primary control, review blocking and washes, and adjust antibody dilution within the validated IHC workflow (general IHC practice). Score PHF8 only where individual stained nuclei remain distinguishable. |
| A nucleolar focus is absent despite convincing nuclear staining. | HPA identifies the nucleoplasm as the supported main IF/ICC location, while UniProt also lists nucleoli (HPA subcellular ICC-IF; UniProt Q9UPP1). Neither source requires a visible nucleolar focus in tissue IHC. | Score the nuclear pattern in the correct cell population against tissue controls (HPA tissue IHC; general IHC practice). Avoid rejecting a clear nuclear result solely because nucleoli are not distinct. |
| Replicate sections show different staining intensity. | HPA reports variable tissue levels and only medium agreement between staining and RNA; external verification remains pending (HPA tissue IHC). Section quality or run conditions can also affect chromogenic intensity (general IHC practice). | Compare the same named cell population across runs and include a reported High tissue control (HPA tissue IHC). Review section quality, retrieval, dilution and detection consistency without assuming a PHF8-specific fixation effect (general IHC practice). |
Experiment evidence
IHC & IF images showing PHF8 staining
M03288 · IHCIHC analysis of PHF8 using anti-PHF8 antibody (M03288). PHF8 was detected in a paraffin-embedded section of human prostate cancer tissue. He
M03288 · IHCIHC analysis of PHF8 using anti-PHF8 antibody (M03288). PHF8 was detected in a paraffin-embedded section of human prostate cancer tissue. He
A03288-2 · IFIF analysis of PHF8 using anti-PHF8 antibody (A03288-2) and anti-Beta Tubulin antibody (M01857-3). PHF8 was detected in an immunocytochemica
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000172943-PHF8/tissue/cerebral+cortex#imid_9881164
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000172943-PHF8/tissue/colon#imid_9900026
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000172943-PHF8/tissue/liver#imid_9900011
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000172943-PHF8/tissue/kidney#imid_9900020
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA062015; green = PHF8, blue = nucleus, red = microtubules.
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA062015; green = PHF8, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for PHF8 IHC & IF
🧪Run kidney first and assess nuclear staining in tubular cells (HPA: High in kidney cells in tubules; UniProt Q9UPP1: nucleus). Use liver cholangiocytes as the tissue negative (HPA: Not detected in liver cholangiocytes); within the kidney section, cells without specific PHF8 staining should show counterstain alone, without assuming that every other renal cell type is PHF8-negative (standard IHC interpretation).
Positive control tissue: Cerebellum (Cells in molecular layer, HPA High)
Negative control tissue: Liver (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show PHF8 in HEK293, SiHa, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a concentration-matched rabbit IgG isotype control, and PHF8-knockout tissue or cells as a biological specificity control (M03288 tissue-IHC caption: rabbit primary; standard IHC practice). In kidney, check for endogenous biotin background if using avidin–biotin detection, and block it or use biotin-free detection (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected M03288 tissue-IHC caption does not state the fixative (M03288 tissue-IHC caption: fixative unreported). Heat retrieval with EDTA at pH 8.0 is documented for the caption’s paraffin-section prostate cancer example; whether that retrieval is required for kidney is unreported (M03288 tissue-IHC caption). The supplied evidence does not establish whether frozen sections or IF are easier; endogenous biotin can complicate kidney staining with avidin–biotin detection (standard IHC practice).
HPA tissue IHC evidence for PHF8
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Pending external verification.). 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 |
|---|---|---|---|---|
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Cerebral cortex | Glial cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
Section 3
Advanced PHF8 IHC Tips
Troubleshoot PHF8 staining in paraffin sections using the catalog antibody’s tissue IHC conditions, nuclear localisation evidence, and matched controls.
How should I adjust retrieval when PHF8 nuclear staining is weak?
Start with heat mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet M03288). The selected tissue IHC image used this retrieval before overnight incubation with antibody at 1:50 and 4°C (datasheet M03288). If nuclei remain weak, compare retrieval heating times on adjacent sections while holding antibody concentration and detection conditions constant (standard IHC practice). Inspect morphology alongside staining, because excessive heating can damage sections and make apparent nuclear signal difficult to score (standard IHC practice). Include a known positive cell population in each run so a retrieval failure is distinguishable from a genuinely low signal (standard IHC practice).
Can I change fixation to improve PHF8 staining in paraffin sections?
PHF8 specific sensitivity to fixative type or fixation duration is unknown from the supplied evidence (datasheet M03288: fixative not stated). The catalog image establishes staining in a paraffin section, but its caption does not identify the fixative (datasheet M03288). Record the fixative and fixation interval for each specimen, then compare adjacent sections processed with the same EDTA pH 8.0 retrieval and detection conditions (datasheet M03288; standard IHC practice). If staining differs, assess section morphology and a positive control before attributing the change to PHF8 antigen preservation (standard IHC practice). Keep fixation conditions consistent within any quantitative comparison (standard IHC practice).
Should PHF8 stain nucleoli, nucleoplasm, or cytoplasm in IHC?
Score nuclear staining first: PHF8 is annotated in the nucleus and nucleolus, while cell imaging places it mainly in the nucleoplasm (UniProt Q9UPP1 subcellular; HPA subcellular). HPA tissue IHC reports a general nuclear pattern with occasional cytoplasmic expression at variable levels (HPA tissue IHC). Compare suspected nucleolar enrichment with surrounding nuclear signal and counterstained morphology on the same section (standard IHC practice). Because PHF8 associates with chromatin during interphase and dissociates as cells enter mitosis, inspect cell cycle context before treating a different nuclear pattern as assay failure (UniProt Q9UPP1 subcellular; PubMed:20622854). Predominantly diffuse cytoplasmic staining warrants control review (UniProt Q9UPP1 subcellular; standard IHC practice).
Could PHF8 isoforms or modifications explain uneven nuclear staining?
PHF8 has 5 annotated isoforms, but the supplied antibody caption does not specify its epitope or isoform coverage (UniProt Q9UPP1 isoforms; datasheet M03288). Do not interpret a negative cell as lacking every PHF8 isoform without epitope information or an independent assay (UniProt Q9UPP1 isoforms; standard IHC practice). The protein contains a JmjC domain at residues 231–387 and annotated phosphorylation sites, including CDK1 associated Ser69 and Ser120 (UniProt Q9UPP1 domains; modified residues). Their effect on this antibody’s staining is unreported, so compare sections under identical retrieval and detection conditions before invoking epitope masking or modification (datasheet M03288; standard IHC practice).
How can IF help assess an ambiguous PHF8 IHC pattern?
Use IF as a separate localisation check: PHF8 is mainly nucleoplasmic in the supplied cell imaging evidence (HPA subcellular). Multiplex PHF8 with a marker validated for the cell type under study, and verify that both channels identify the same counterstained cells (standard IF practice). Choose a fluorophore whose emission is separated from the tissue’s measured autofluorescence, with single stain and no primary controls for channel assessment (standard IF practice). PHF8 has no transmembrane segment and is nuclear, so permeabilise fixed cells or sections sufficiently to give antibody access to the nuclear epitope (UniProt Q9UPP1 topology; subcellular; standard IF practice). Optimise IF fixation and permeabilisation independently of the paraffin IHC conditions (standard IF practice).
What should I change if DAB obscures PHF8 nuclear staining?
First compare the tissue with a no primary control to identify signal produced by the detection system (standard IHC practice). The selected image used 10% goat serum blocking, a 1:50 primary antibody overnight at 4°C, and DAB detection (datasheet M03288). For a noisy run, verify the peroxidase block, shorten DAB development, or titrate the primary antibody while retaining a positive control (standard IHC practice). Keep the counterstain light enough to distinguish true nuclear chromogen from densely stained nuclei (standard IHC practice). Treat widespread cytoplasmic color cautiously because HPA reports mainly nuclear staining, although occasional cytoplasmic expression occurs (HPA tissue IHC).
How should I quantify PHF8 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and score nuclear staining separately from cytoplasmic signal, reflecting PHF8’s predominantly nuclear pattern (HPA tissue IHC; HPA subcellular). Report the percentage of positive nuclei and staining intensity, or calculate an H-score from prespecified intensity bins (standard IHC practice). Normalise positive counts to the number of evaluable nuclei in each region; for spatial comparisons, report positive nuclei per mm² of viable tissue (standard IHC practice). Apply identical retrieval, DAB development, counterstaining, and image thresholds across compared sections (datasheet M03288; standard IHC practice). Record excluded damaged or necrotic areas and retain representative images so differences can be reviewed (standard IHC practice).
How can I distinguish true PHF8 staining from section artefact?
A credible result should show interpretable nuclear staining in intact cells, consistent with PHF8 localisation and the general HPA tissue pattern (UniProt Q9UPP1 subcellular; HPA tissue IHC). Check cell identity and morphology: HPA reports high staining in colon glandular cells and kidney tubular cells, while liver cholangiocytes are reported as not detected (HPA tissue IHC). Do not use these patterns as an absolute specificity test because the HPA tissue IHC assessment has medium consistency and awaits external verification (HPA tissue IHC). Discount staining concentrated at section edges, in necrotic regions, or in a no primary control; investigate endogenous peroxidase when DAB persists without primary antibody (standard IHC practice).
Boster reagents
Best PHF8 / Histone lysine demethylase PHF8 IHC Antibodies
PHF8 image data cover human paraffin-embedded prostate cancer tissue and HELA cells; M03288 lists Human, Mouse and Rat reactivity, while A03288-2 lists Human (catalog captions; catalog reactivity).
Real IHC data
Anti-PHF8 Monoclonal Antibody
Cat # M03288
Real IF data
Anti-PHF8 Antibody ®
Cat # A03288-2
M03288 will render with an IHC image from a paraffin-embedded human prostate cancer section (M03288 IHC caption). A03288-2 will render with an IF/ICC image from HELA cells (A03288-2 IF caption).
Which to pick: For tissue IHC, choose rabbit monoclonal M03288: its image documents a 1:50 dilution and EDTA pH 8.0 retrieval on a paraffin section; the fixative is unreported (M03288 catalog; M03288 IHC caption). For human IF/ICC, choose A03288-2, shown in HELA cells at 5 μg/mL (A03288-2 IF caption). For mouse or rat samples, M03288 is the option with listed reactivity, although its depicted IHC sample is human (M03288 catalog reactivity; M03288 IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UPP1 (PHF8_HUMAN, Histone lysine demethylase PHF8).
- Human Protein Atlas. PHF8 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. PHF8 subcellular location (ICC-IF): Mainly localized to the nucleoplasm..
- Human Protein Atlas. PHF8 antibody validation summary (2 antibodies).
- Expression Patterns of PAK4 and PHF8 Are Associated with the Survival of Gallbladder Carcinoma Patients. Diagnostics (Basel, Switzerland) 2023 — PMC10047028.
- The histone demethylase PHF8 regulates TGFβ signaling and promotes melanoma metastasis. Science advances 2022 — PMC8856617.
- Targeting the histone demethylase PHF8-mediated PKCα-Src-PTEN axis in HER2-negative gastric cancer. Proceedings of the National Academy of Sciences of the United States of America 2020 — PMC7547212.
- PHF8 upregulation contributes to autophagic degradation of E-cadherin, epithelial-mesenchymal transition and metastasis in hepatocellular carcinoma. Journal of experimental & clinical cancer research : CR 2018 — PMC6122561.
- PubMed PMID:10470851 — UniProt-cited evidence.
- PubMed PMID:12168954 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for PHF8 (Histone lysine demethylase PHF8). Expected localisation Predominantly nuclear, with occasional cytoplasmic staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M03288). Validated IHC antibody: M03288 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (Cells in molecular layer); negative Liver. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human prostate cancer tissue; fixative not specified (datasheet M03288); 2. Fixation: Image fixative and duration unreported (datasheet M03288); 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 M03288); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M03288); 8. Primary antibody: Rabbit monoclonal (clone ADAI-16) anti-PHF8, 1:50 (datasheet M03288); 9. Primary incubation: Overnight at 4 °C (datasheet M03288). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M03288); Caveat: Tissue staining has medium consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 5 isoforms; check epitope coverage across variants (UniProt); Regulation: Tissue-specific expression regulation is not annotated (UniProt). Evidence: UniProt Q9UPP1, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm., Datasheet M03288, PMC10047028, PMC8856617. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PHF8/immunohistochemistry-ihc-guide.