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PYGO2 / Pygopus homolog 2 · IHC design guide
Design Immunohistochemistry for PYGO2
Plan chromogenic PYGO2 IHC on paraffin sections using the general nuclear tissue pattern (HPA tissue IHC). High staining in adrenal glandular cells and undetected staining in heart cardiomyocytes offer candidate comparison tissues (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
PYGO2 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 nuclear staining (HPA tissue IHC) | |
| Staining pattern | Nuclear staining in glandular and squamous epithelial cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Heart muscle+3 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep formalin fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA expression have medium consistency (HPA tissue IHC) | |
| Regulation | Expression regulation is not annotated (UniProt) | |
| Isoform / epitope | No isoforms annotated; mature chain spans residues 2–406 (UniProt) |
Section 1
Recommended PYGO2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by three published PYGO2 IHC methods, including one performed on OCT sections (PMC3913260).
| Sample | Paraffin-embedded human uterus tissue; fixative not specified (datasheet M05260) |
| Fixation | Image fixative and duration unreported (datasheet M05260); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen) |
| 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 monoclonal (clone ABI-16) anti-PYGO2, 1:50 (datasheet M05260) |
| 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 | PYGO2-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General nuclear expression. No signal in the no-primary control. |
💡Decision noteFor paraffin sections, start with Tris-EDTA pH 9.0 HIER at 95–98 °C for 20 min (page retrieval); the ESCC study used pH 6–7 (PMC11872025).
Section 2
What Is the Expected PYGO2 Staining Pattern?
PYGO2 is expected mainly in nuclei, with HPA reporting general nuclear tissue staining and supported nucleoplasmic IF localisation (HPA: tissue IHC; HPA: subcellular). UniProt places PYGO2 in the nucleus and annotates no transmembrane segment (UniProt Q9BRQ0). High IHC staining is reported in selected glandular, squamous epithelial, basal and cytotrophoblast cells (HPA: tissue IHC). The tissue profile is Supported, with medium consistency between antibody staining and RNA data (HPA: tissue IHC reliability).
| Distinct chromogenic staining within nuclei of adrenal or stomach glandular cells. | This fits the expected compartment and a reported High tissue pattern (UniProt Q9BRQ0: nucleus; HPA: High in adrenal and stomach glandular cells). Compare nuclei with nearby background on the same section; HPA intensity labels describe observed staining, not a required numerical cutoff (HPA: tissue IHC). |
| Predominantly cytoplasmic or membranous staining with little nuclear signal. | Question target attribution: the supported pattern is nuclear or nucleoplasmic (UniProt Q9BRQ0; HPA: subcellular). HPA also reports plasma membrane IF localisation as uncertain, so membrane signal alone cannot establish a PYGO2-positive IHC result (HPA: subcellular). |
| Strong staining of cardiomyocytes, skeletal myocytes or spleen red-pulp cells. | These cells are reported as Not detected by HPA IHC (HPA: tissue IHC). Unexpected staining raises concern for cross-reactivity or detection-system activity (general IHC practice). It needs control-based investigation; an HPA negative observation is not proof that every sample must be negative. |
| Diffuse chromogen across nuclei, cytoplasm and tissue background. | The spread obscures the expected nuclear pattern (HPA: general nuclear expression; UniProt Q9BRQ0: nucleus). Excess antibody or incomplete blocking or washing can produce nonspecific background (general IHC practice). Do not score indistinct colour as PYGO2-positive cells. |
| No nuclear staining in an adequately sampled HPA high-expression cell population. | Absence in adrenal glandular cells, cervical squamous epithelial cells or placental cytotrophoblasts conflicts with HPA's reported High patterns (HPA: tissue IHC). Check controls and detection before concluding biological absence; HPA assigns the overall tissue IHC profile Supported with medium RNA–staining consistency (HPA: reliability). |
💡Expected PYGO2 appearanceCall a section positive when discrete nuclear chromogen appears in a reported positive cell population, especially one labelled High by HPA; isolated diffuse cytoplasmic, background or membrane-only colour is insufficient (HPA: tissue IHC and subcellular; UniProt Q9BRQ0: nucleus).
| Compartment and topology | UniProt assigns PYGO2 to the nucleus and annotates no transmembrane segment (UniProt Q9BRQ0). HPA supports nucleoplasmic IF localisation but marks additional plasma membrane localisation uncertain (HPA: subcellular); prioritize nuclear IHC staining when interpreting a slide. |
| Cell population and tissue choice | HPA reports High staining in gallbladder and stomach glandular cells, oral mucosal squamous cells, nasopharyngeal basal cells and placental cytotrophoblasts (HPA: tissue IHC). Skin keratinocytes are Medium; liver hepatocytes and ovarian follicle cells are Low (HPA: tissue IHC). Compare the named cell population, not the entire tissue. |
| Strength of tissue and antibody evidence | The HPA tissue IHC profile is Supported, with medium consistency against RNA expression (HPA: reliability). HPA023689 is IHC Supported; HPA074813 has no listed IHC status in the supplied record (HPA: antibodies). These labels guide confidence but do not guarantee identical staining across specimens. |
| IF/ICC: what localisation should be expected? | Predominantly nucleoplasmic signal is supported; an additional plasma membrane location is uncertain (HPA: subcellular). HPA lists ICC Approved for HPA023689 and HPA074813 (HPA: antibodies). This IF observation does not establish an IHC membrane pattern or an IF protocol. |
| Detection background | Chromogenic IHC can show signal from the detection system or endogenous activity, depending on the reagents and tissue (general IHC practice). A no-primary control tests this contribution. No PYGO2-specific endogenous detection activity or fixation sensitivity is established by the supplied HPA or UniProt records. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known HPA high-expression cells show no nuclear signal. | The staining run may have failed, or the sampled cells may differ from those in HPA images (general IHC practice; HPA: tissue IHC). | Confirm the intended cell population and inspect a positive-control section and run controls; then review retrieval, antibody application and detection as general IHC workflow steps. Do not infer PYGO2-specific retrieval or fixation requirements from HPA staining levels. |
| Chromogen is widespread outside cell nuclei. | Nonspecific primary binding or detection background may mask the expected pattern (general IHC practice; HPA: general nuclear expression). | Compare a no-primary control, review blocking and washes, and optimize primary antibody concentration within a validated IHC procedure (general IHC practice). Score only interpretable nuclear staining. |
| Membrane staining dominates while nuclei remain blank. | The proposed membrane IF location is uncertain, whereas nucleoplasmic localisation is supported (HPA: subcellular). | Treat membrane-only colour as inconclusive. Check the no-primary control and a reported nuclear-positive cell population before attributing the signal to PYGO2 (HPA: tissue IHC; general IHC practice). |
| Cardiomyocytes or skeletal myocytes appear strongly positive. | That conflicts with HPA's Not detected IHC observations and may reflect nonspecific staining (HPA: tissue IHC; general IHC practice). | Recheck cell identity and controls, then compare nuclear staining in an HPA high-expression population. Report the discrepancy without treating HPA's negative observations as universal exclusion rules. |
| Only a few cells stain in a tissue reported as positive. | HPA levels refer to specified cell types, and the tissue profile has medium RNA–staining consistency (HPA: tissue IHC reliability). | Identify the relevant cells before scoring: for example, HPA specifies cytotrophoblasts in placenta and basal cells in nasopharynx as High (HPA: tissue IHC). Document the fraction and compartment of stained cells. |
| An IF image suggests a pattern different from the IHC section. | HPA supports nucleoplasmic ICC-IF localisation but calls additional plasma membrane localisation uncertain (HPA: subcellular); the assays and specimens differ (general IHC/IF practice). | Interpret each assay with its own controls and compartment evidence. For this paraffin-section IHC result, use the HPA nuclear tissue pattern and the IHC antibody status; do not transfer an IF observation into an IHC protocol (HPA: tissue IHC; HPA: antibodies). |
Experiment evidence
IHC & IF images showing PYGO2 staining
M05260 · IHCImmunohistochemical analysis of paraffin-embedded human uterus, using Pygopus 2 Antibody.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000163348-PYGO2/tissue/cerebral+cortex#imid_6152264
HPA · Oral mucosaHuman Protein Atlas IHC, Oral mucosa. Source: https://www.proteinatlas.org/ENSG00000163348-PYGO2/tissue/oral+mucosa#imid_6152180
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000163348-PYGO2/tissue/colon#imid_6152198
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000163348-PYGO2/tissue/liver#imid_6152183
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA023689; green = PYGO2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA023689; green = PYGO2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for PYGO2 IHC & IF
🧪Run cervix first: squamous epithelial cells should stain for PYGO2 (HPA: High in cervical squamous epithelial cells). Use skeletal muscle as the negative tissue (HPA: Not detected in myocytes); cells without PYGO2 signal within the cervical section should lack nuclear chromogen above background (UniProt: nucleus).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Heart muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show PYGO2 in A-431, U-251MG, U2OS, MCF-7, SH-SY5Y, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide, a host-species- and clonality-matched isotype control, and a PYGO2 knockout sample or peptide-blocked antibody where available (standard IHC controls). Block endogenous peroxidase for chromogenic detection and compare cervical epithelial background with the no-primary slide (standard IHC practice).
⚠️Feasibility: A PYGO2-specific fixation window and retrieval requirement are unreported in the supplied evidence; optimize antigen retrieval empirically for paraffin sections. The selected M05260 paraffin-section uterus caption does not report a fixative (selected SKU caption: fixative not stated). HPA provides ICC-IF images in several cell lines (HPA: ICC-IF image cell lines), but the evidence does not establish that frozen sections or IF are easier; assess cervical epithelial background that could obscure nuclear chromogen (standard IHC practice).
HPA tissue IHC evidence for PYGO2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Medium consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Nasopharynx | Basal cells | High | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PYGO2 IHC Tips
Troubleshoot PYGO2 staining in paraffin sections by checking retrieval, nuclear localisation, background controls, and cell-specific scoring (UniProt Q9BRQ0; HPA tissue IHC).
How should I retrieve PYGO2 in paraffin sections when nuclear staining is weak?
Use Tris-EDTA pH 9.0 heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval specification: nuclear antigen). Let slides cool in retrieval buffer, then compare a positive tissue and a matched control processed in the same run (general IHC practice; HPA tissue IHC: high staining in placental cytotrophoblasts). If staining remains weak, check temperature at the slides, buffer pH, section adhesion, and primary-antibody incubation before changing retrieval conditions (general IHC practice). Score nuclei separately from cytoplasm because PYGO2 is nuclear and HPA describes general nuclear tissue expression (UniProt Q9BRQ0: nucleus; HPA tissue IHC: general nuclear expression).
Can fixation explain variable PYGO2 staining between paraffin blocks?
The M05260 uterus image identifies paraffin sections but gives no fixative, so PYGO2-specific fixation sensitivity is unknown (catalog image caption: M05260). Record the fixative and exposure time for each block; if using 10% neutral buffered formalin, compare blocks only after applying the same retrieval and staining run (general IHC practice). Differences between blocks can reflect preanalytic handling, so include a control section from a consistently processed block (general IHC practice). Do not infer fixation tolerance from tissue staining patterns or PYGO2 modifications: those sources provide localisation and residue annotations, not a fixation comparison (HPA tissue IHC; UniProt Q9BRQ0: modified residues).
Should PYGO2 staining outside nuclei count as positive IHC?
Treat nuclear staining as the primary readout: PYGO2 is assigned to the nucleus, and tissue IHC describes general nuclear expression (UniProt Q9BRQ0: nucleus; HPA tissue IHC: general nuclear expression). HPA places it mainly in the nucleoplasm and reports an additional plasma-membrane location as uncertain (HPA subcellular: nucleoplasm supported; plasma membrane uncertain). Because PYGO2 has no annotated transmembrane segment, score isolated membrane staining cautiously and check whether it persists in an independent control (UniProt Q9BRQ0: topology; general IHC practice). Compare the nuclear pattern within the expected cell population, such as placental cytotrophoblasts, using matched processing and exposure to chromogen (HPA tissue IHC: high in cytotrophoblasts; general IHC practice).
How do epitope uncertainty and PYGO2 modifications affect interpretation?
No isoforms are annotated for the 406-amino-acid PYGO2 record; that annotation does not establish which sequence the catalog antibody recognizes (UniProt Q9BRQ0: length and isoforms; catalog image caption: M05260). The record lists acetylation at residue 2 and phosphorylation at residues 40 and 302, but supplies no evidence that these alter this antibody’s IHC staining (UniProt Q9BRQ0: modified residues). Check the antibody’s stated immunogen or epitope before interpreting loss of staining in an altered sample (general IHC practice). If specificity is uncertain, compare nuclear staining with an independent antibody recognizing a documented different epitope and include appropriate controls (UniProt Q9BRQ0: nucleus; general IHC practice).
How can IF help check a chromogenic PYGO2 result?
Use IF as a separate cross-check of the IHC result and ask whether PYGO2 signal overlaps nuclei in the expected cell population (UniProt Q9BRQ0: nucleus; HPA tissue IHC: general nuclear expression). For example, multiplex with a validated cytotrophoblast marker when assessing placenta, since HPA reports high PYGO2 staining in cytotrophoblasts (HPA tissue IHC: high in cytotrophoblasts; general IF practice). Choose a far-red channel where tissue autofluorescence is lower, and include single-label and no-primary controls for bleed-through and background (general IF practice). Because the expected epitope is intracellular and nuclear, use permeabilisation appropriate for nuclear access; PYGO2 has no annotated transmembrane segment (UniProt Q9BRQ0: nucleus and topology; general IF practice).
What should I check when PYGO2 chromogenic staining looks diffuse?
First distinguish diffuse chromogen from nuclear signal by inspecting morphology and a no-primary control on comparable sections (UniProt Q9BRQ0: nucleus; general IHC practice). In a peroxidase-based workflow, a 3% hydrogen-peroxide block for about 10 min and subsequent DAB development are general IHC steps, not evidence of PYGO2-specific performance (general IHC practice). Reduce nonspecific staining by checking blocking, primary-antibody concentration, wash completeness, and DAB development time together with the control (general IHC practice). Record whether colour concentrates at folds, tissue edges, or damaged areas before calling it expression (general IHC practice). Confirm any scored signal is predominantly nuclear (HPA tissue IHC: general nuclear expression).
How should I quantify PYGO2 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and nuclear scoring rules before reading slides, because HPA reports general nuclear expression with cell-type differences across tissues (HPA tissue IHC: profile and positive cells). An H-score combines nuclear intensity categories 0–3 with the percentage of cells in each category, producing 0–300; alternatively report percentage of positive nuclei (general IHC practice). For spatial analyses, report positive nuclei per mm² of viable tissue and the total nuclei or viable area used as the denominator (general IHC practice). Apply the same threshold, section selection, and imaging conditions across samples, and report excluded necrotic or folded regions (general IHC practice).
What separates convincing PYGO2 positivity from staining artefact?
A convincing result places signal in nuclei of intact cells and follows an interpretable cell pattern, consistent with PYGO2’s nuclear assignment and HPA’s general nuclear staining profile (UniProt Q9BRQ0: nucleus; HPA tissue IHC: profile). High nuclear staining in placental cytotrophoblasts or cervical squamous epithelial cells is compatible with reported patterns, while isolated strong cardiomyocyte staining warrants closer review (HPA tissue IHC: high in cytotrophoblasts and cervical squamous epithelial cells; not detected in cardiomyocytes). Inspect edges, folds, necrosis, and no-primary controls for artefact or endogenous peroxidase activity (general IHC practice). Treat isolated membrane signal cautiously because HPA marks that location uncertain (HPA subcellular: plasma membrane uncertain).
Boster reagents
Best PYGO2 / Pygopus homolog 2 IHC Antibodies
M05260 has a real IHC image from human paraffin-embedded uterus (catalog IHC image caption) and lists IF/ICC and human/mouse reactivity (catalog applications/reactivity).
Real IHC data
Anti-Pygopus 2 Rabbit Monoclonal Antibody
Cat # M05260
M05260 will render with its own human uterus paraffin-section IHC image (catalog IHC image caption). It lists IHC, IF and ICC for human and mouse (catalog applications/reactivity); no IF image is supplied (catalog IF image alts).
Which to pick: Choose M05260 for tissue IHC when working with human paraffin sections: its own caption documents human uterus staining, but does not report the fixative (catalog IHC image caption). For IF/ICC, M05260 is listed for both applications and is a rabbit monoclonal, although no IF image is supplied (catalog applications/host/clone/IF image alts). For work across human and mouse, M05260 lists reactivity with both species; its supplied IHC image documents human tissue only (catalog reactivity/IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9BRQ0 (PYGO2_HUMAN, Pygopus homolog 2).
- Human Protein Atlas. PYGO2 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. PYGO2 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the plasma membrane..
- Human Protein Atlas. PYGO2 antibody validation summary (2 antibodies).
- Nuclear Expression of Pygo2 Correlates with Poorly Differentiated State Involving c-Myc, PCNA and Bcl9 in Myanmar Hepatocellular Carcinoma. Acta histochemica et cytochemica 2021 — PMC8727843.
- Pygopus 2 promotes kidney cancer OS-RC-2 cells proliferation and invasion in vitro and in vivo. Asian journal of urology 2015 — PMC5730714.
- Chromatin effector Pygo2 regulates mammary tumor initiation and heterogeneity in MMTV-Wnt1 mice. Oncogene 2014 — PMC3913260.
- Histopathological Evaluation of PYGO2 Expression in Esophageal Squamous Cell Carcinoma. Iranian journal of pathology 2024 — PMC11872025.
- PubMed PMID:11955446 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:15498874 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for PYGO2 (Pygopus homolog 2). Expected localisation General nuclear staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: M05260 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Heart muscle. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human uterus tissue; fixative not specified (datasheet M05260); 2. Fixation: Image fixative and duration unreported (datasheet M05260); 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-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 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 monoclonal (clone ABI-16) anti-PYGO2, 1:50 (datasheet M05260); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep formalin fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA expression have medium consistency (HPA tissue IHC); Isoform / epitope: No isoforms annotated; mature chain spans residues 2–406 (UniProt); Regulation: Expression regulation is not annotated (UniProt). Evidence: UniProt Q9BRQ0, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet M05260, PMC5730714, PMC3913260. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PYGO2/immunohistochemistry-ihc-guide.