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GLI2 / Transcription activator GLI2 · IHC design guide
Design Immunohistochemistry for GLI2
Plan paraffin-section GLI2 IHC around widespread nuclear staining (HPA tissue IHC). Interpret the signal with the reported low consistency between antibody staining and RNA expression in mind (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
GLI2 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 | Nuclear staining across tissues (HPA tissue IHC) | |
| Staining pattern | Widespread nuclear staining across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A00701-5) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | None in HPA (detected in all 43 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet A00701-5); verify before use. | |
| Caveat | Staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | SUFU sequesters GLI2 without SMO signaling (UniProt) | |
| Isoform / epitope | Five isoforms plus GLI2R; epitope coverage is unknown (UniProt) |
Section 1
Recommended GLI2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet A00701-5) is accompanied by a published chromogenic GLI2 protocol for paraffin-embedded liver tissue (PMC3565946).
| Sample | Paraffin-embedded human mammary cancer tissues; fixative not specified (datasheet A00701-5) |
| Fixation | Image fixative and duration unreported (datasheet A00701-5); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet A00701-5) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A00701-5) |
| Primary antibody | Rabbit anti-GLI2, 0.5-1μg/ml (datasheet A00701-5) |
| Primary incubation | Overnight at 4 °C (datasheet A00701-5) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A00701-5) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | GLI2-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate retrieval at pH 6 (datasheet A00701-5); the published protocol also used citrate at pH 6.0 (PMC3565946).
Section 2
What Is the Expected GLI2 Staining Pattern?
GLI2 should show predominantly nuclear staining across tissues, with strong signal reported in several cell populations, including adipocytes, hematopoietic cells and respiratory epithelial cells (HPA: ubiquitous nuclear expression; High in listed cells). Cytoplasmic localisation is also biologically plausible when full-length GLI2 is sequestered by SUFU (UniProt P10070). GLI2 has no transmembrane segment (UniProt P10070 topology). HPA rates its tissue IHC profile Approved, while noting low consistency with RNA expression (HPA: reliability description).
| Distinct nuclear chromogen in adipocytes, marrow hematopoietic cells or bronchial respiratory epithelium. | This fits the reported nuclear profile and High staining in those populations (HPA: tissue IHC). Assess nuclei against the counterstain and compare signal across cells on the same section (standard IHC practice). HPA's low antibody–RNA consistency limits conclusions drawn from staining intensity alone (HPA: reliability description). |
| Cytoplasmic staining accompanies, or appears stronger than, nuclear staining. | Cytoplasmic GLI2 can be plausible because SUFU sequesters full-length GLI2 there without smoothened signaling (UniProt P10070). Record nuclear and cytoplasmic signal separately; compartment alone does not establish pathway activity (UniProt P10070 function and subcellular location). A mainly membranous outline would be unexpected because GLI2 lacks a transmembrane segment (UniProt P10070 topology). |
| Strong signal appears in a cell population outside the listed High examples. | The supplied HPA list names positive examples but provides no negative cell populations (HPA: tissue IHC). This finding cannot by itself establish cross-reactivity. Check cell identity, morphology and control staining; residual endogenous detection activity or antibody binding may account for an unexplained pattern (standard IHC practice). |
| Weak, diffuse chromogen obscures nuclei throughout the section. | This does not permit a confident nuclear GLI2 call against the HPA profile (HPA: ubiquitous nuclear expression). Diffuse signal may reflect background from detection reagents, incomplete blocking or excessive antibody concentration (standard IHC practice). Interpret only signal that can be assigned to individual cells and compartments. |
| No nuclear signal appears in a section containing an HPA-listed High cell population. | A negative result conflicts with the reported staining level for that population, but does not prove GLI2 is absent (HPA: tissue IHC). Check the section's cell identity, control performance and assay conditions before making a biological interpretation (standard IHC practice). HPA reports low consistency between antibody staining and RNA data (HPA: reliability description). |
💡Expected GLI2 appearanceCall a result positive when discrete, predominantly nuclear staining is visible in identifiable cells, including an HPA-listed High population; diffuse section-wide color or a membrane-only outline is suspect (HPA: tissue IHC; UniProt P10070 topology; standard IHC practice).
| Localisation and signaling | GLI2 is annotated in the nucleus, cytoplasm and cilium; SUFU can retain full-length GLI2 in cytoplasm without smoothened signaling (UniProt P10070). Score nuclear and cytoplasmic patterns separately. Staining location alone cannot establish whether GLI2 is acting as an activator or repressor (UniProt P10070 function). |
| Isoforms and processing | UniProt lists five isoforms and a full-length activator plus a repressor form (UniProt P10070). The supplied sources do not map either antibody's epitope to these forms, so an IHC pattern cannot identify which form was detected. Do not infer processing or isoform abundance from chromogenic intensity alone (UniProt P10070 processing and isoforms). |
| Antibody evidence | One listed antibody has Approved IHC status; a different antibody has Supported ICC status (HPA: CAB008558 IHC Approved; HPA074275 ICC Supported). These ratings are application specific. HPA also describes low consistency between tissue staining and RNA expression, so compare observed patterns with controls before interpreting expression differences (HPA: reliability description). |
| IF/ICC Q&A | Q: Should nucleoli be required in paraffin IHC? A: No. HPA's ICC-IF summary reports mainly nucleolar localisation, with additional nucleoplasm, centrosome and basal body signals (HPA: subcellular ICC-IF). The tissue IHC summary describes ubiquitous nuclear expression (HPA: tissue IHC); use that tissue pattern for this IHC call. |
| Detection chemistry | Broad chromogen can arise from endogenous enzyme activity or nonspecific detection reagent binding (standard IHC practice). Evaluate the detection-only control and the location of staining before attributing such signal to GLI2. This is general assay guidance, not a GLI2-specific effect reported by HPA or UniProt. |
| Situation | Likely cause | Next action |
|---|---|---|
| A listed High cell population lacks nuclear staining. | The expected tissue pattern is missing, or the assay failed to reveal it (HPA: tissue IHC; standard IHC practice). | Confirm the relevant cells are present, then review positive-control staining, reagent performance and the chosen IHC conditions (standard IHC practice). Treat the negative call as provisional while controls fail. |
| Color is confined to cell borders or luminal surfaces. | A membrane-only pattern conflicts with GLI2's lack of a transmembrane segment (UniProt P10070 topology). | Check morphology and a detection-only control; investigate nonspecific chromogen deposition before scoring the signal as GLI2 (standard IHC practice). |
| Cytoplasmic staining is prominent. | SUFU-associated cytoplasmic sequestration is plausible (UniProt P10070 subcellular location). | Record cytoplasmic and nuclear staining separately, compare identifiable cells and controls, and avoid calling the cytoplasmic pattern an artefact solely because it is extranuclear (UniProt P10070; standard IHC practice). |
| Most cells show diffuse low-level color. | Background from blocking, detection reagents or antibody concentration may obscure localisation (standard IHC practice). | Review the detection-only control and blocking step, then optimise assay conditions using a known-positive section; score only cell-resolved signal (standard IHC practice). |
| An unexpected cell population stains strongly. | The supplied HPA tissue record contains no negative populations, so its positive list cannot exclude staining elsewhere (HPA: tissue IHC). | Verify cell identity and morphology, inspect controls for endogenous activity, and seek independent confirmation before assigning the signal to GLI2 (standard IHC practice). |
| IHC lacks the nucleolar detail seen in ICC-IF images. | The supplied application summaries differ: tissue IHC reports nuclear expression, while ICC-IF reports mainly nucleolar localisation (HPA: tissue IHC; subcellular ICC-IF). | Judge paraffin IHC against its nuclear tissue pattern and the IHC control. Record nucleolar detail if visible, without requiring it for a positive IHC call (HPA: tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing GLI2 staining
A00701-5 · IHCIHC analysis of GLI2 using anti-GLI2 antibody (A00701-5). GLI2 was detected in paraffin-embedded section of human mammary cancer tissues. He
A00701-5 · IHCIHC analysis of GLI2 using anti-GLI2 antibody (A00701-5). GLI2 was detected in paraffin-embedded section of mouse brain tissues. Heat mediat
A00701-6 · IHCIHC analysis of GLI2 using anti-GLI2 antibody (A00701-6). GLI2 was detected in a paraffin-embedded section of human esophageal squamous carc
A00701-6 · IHCIHC analysis of GLI2 using anti-GLI2 antibody (A00701-6). GLI2 was detected in a paraffin-embedded section of human testicular germ cell tum
A00701-6 · IFIF analysis of GLI2 using anti-GLI2 antibody (A00701-6) and anti-Beta Tubulin antibody (M01857-3). GLI2 was detected in immunocytochemical s
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000074047-GLI2/tissue/cerebral+cortex#imid_2344424
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000074047-GLI2/tissue/colon#imid_2328734
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000074047-GLI2/tissue/liver#imid_2328735
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000074047-GLI2/tissue/kidney#imid_2328720
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA074275; green = GLI2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA074275; green = GLI2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for GLI2 IHC & IF
🧪Run breast tissue first and assess its adipocytes, which have High GLI2 staining (HPA: High in breast adipocytes). HPA detects GLI2 in all 43 scored tissues, so there is no documented negative tissue; use no-primary and isotype controls, and treat any unstained cells within the positive section as a background reference without assuming their cell type is GLI2-negative (HPA: no negative rows; GLI2 detected in all 43 scored tissues).
Positive control tissue: Adipose tissue (Adipocytes, HPA High)
Negative control tissue: None in HPA: GLI2 is detected in all 43 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show GLI2 in A-549, BJ [Human fibroblast], U2OS, ASC52telo, RPTEC/TERT1, Sperm, with annotated localisation: Nucleoli (supported) (HPA subcellular).
Technical controls: Run a secondary-only slide and a concentration-matched rabbit IgG isotype control alongside the rabbit catalog antibody, and use GLI2 knockout material or a validated peptide-block control to assess target specificity (selected-SKU caption: rabbit anti-GLI2; standard IHC practice). For the biotin-based DAB method, quench endogenous peroxidase and assess endogenous biotin background in breast tissue (selected-SKU caption: biotinylated secondary, SABC and DAB; standard IHC practice).
⚠️Feasibility: The selected-SKU paraffin-section caption reports citrate retrieval at pH 6 for 20 minutes and primary antibody at 1 μg/ml overnight at 4°C; its fixative is unreported, and no target-specific fixation window or fixation effect is supplied (selected-SKU tissue-IHC caption). No supplied evidence establishes that frozen sections or IF/ICC are easier than paraffin IHC; HPA lists ICC-IF images but provides no comparative preparation result (HPA: subcellular record; selected-SKU tissue-IHC caption). In adipocyte-rich breast tissue, distinguish cellular staining from empty lipid spaces and section-edge artefact (HPA: High in breast adipocytes; standard IHC practice).
HPA tissue IHC evidence for GLI2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Adipocytes | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: GLI2 is detected in all 43 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced GLI2 IHC Tips
Troubleshoot GLI2 chromogenic IHC by checking retrieval, intracellular localisation, antibody specificity and tissue controls before interpreting staining intensity.
Which retrieval conditions should I start with for GLI2 in paraffin sections?
Start with heat-mediated citrate retrieval at pH 6 for 20 minutes (datasheet A00701-5). The catalog antibody's paraffin-section image used those conditions in human mammary cancer tissue, followed by 1 μg/ml primary antibody overnight at 4°C (datasheet A00701-5). If staining is weak, compare modest changes in heating time on adjacent sections while keeping antibody concentration and detection constant (standard IHC practice). Include an unheated section and a no-primary control to distinguish retrieval-dependent signal from background, and compare nuclear staining rather than total brown area because GLI2 has a nuclear role (UniProt P10070 function and subcellular location; standard IHC practice).
How should I troubleshoot variable GLI2 staining between paraffin blocks?
The selected image documents paraffin sections but does not report their fixative, so GLI2-specific fixation sensitivity is unknown (datasheet A00701-5 caption). Record each block's fixative and fixation duration, then compare sections processed with the same citrate pH 6, 20-minute retrieval and detection conditions (datasheet A00701-5; standard IHC practice). If signal varies, examine morphology and staining in matched control sections before changing retrieval or antibody concentration (standard IHC practice). Do not infer a fixation effect from GLI2 phosphorylation, intracellular location or tissue staining patterns; those observations do not establish how this antibody performs after fixation (UniProt P10070 modified residues and subcellular location; HPA tissue IHC).
How do I assess predominantly cytoplasmic GLI2 staining in a chromogenic section?
Score nuclear and cytoplasmic staining separately because GLI2 can occupy both compartments, and SUFU can sequester full-length GLI2 in the cytoplasm when smoothened signaling is absent (UniProt P10070 subcellular location and subunit). Nuclear staining is relevant to its transcription factor function, but cytoplasmic signal alone does not establish inactivity or antibody specificity (UniProt P10070 function and subcellular location; standard IHC interpretation). Compare staining within intact cells across replicate sections, using the same hematoxylin counterstain to identify nuclei (standard IHC practice). HPA describes widespread nuclear tissue staining yet reports low consistency with RNA expression, so treat its pattern as context rather than a definitive reference (HPA tissue IHC).
Can this IHC assay distinguish GLI2 isoforms or the repressor form?
GLI2 has 5 listed isoforms, and its full-length activator and processed repressor forms have different functional roles (UniProt P10070 isoforms and processing). The supplied catalog image gives antibody concentration and retrieval conditions but no mapped epitope, so staining cannot be assigned to one isoform or form from this evidence alone (datasheet A00701-5; UniProt P10070 processing). Obtain epitope mapping or validation against the forms of interest before making an isoform-specific claim (standard IHC validation practice). Meanwhile, report the fraction and intensity of stained nuclei as GLI2 immunoreactivity, with any inferred activator or repressor status clearly separated from the staining observation (standard IHC interpretation).
How should I relate multiplex IF results to this chromogenic GLI2 assay?
Use the chromogenic section to define tissue architecture, then pair GLI2 IF with a validated marker for the cell population being evaluated; concordant cell identity helps interpret nuclear signal (standard IHC/IF practice). Choose fluorophores in spectral regions with low measured tissue autofluorescence and include single-stain and no-primary controls for multiplex interpretation (standard IF practice). GLI2 lacks a transmembrane segment and has intracellular locations, so assess permeabilisation for access to nuclear and cytoplasmic epitopes, adjusting it to the marker panel (UniProt P10070 topology and subcellular location; standard IF practice). Do not transfer the chromogenic assay's 1 μg/ml primary concentration directly to IF without titration, because that value comes from the paraffin-section image (datasheet A00701-5; standard IF practice).
What should I change when GLI2 DAB staining appears diffuse?
Inspect a no-primary section first to identify signal from the secondary and chromogenic detection system (standard IHC practice). The catalog image used 10% goat serum, biotinylated goat anti-rabbit secondary antibody, streptavidin–biotin detection and DAB; background should be assessed against that workflow (datasheet A00701-5). Apply an endogenous peroxidase block and consider a biotin-control or alternative detection system if tissue-dependent background persists (standard IHC practice). Titrate the 1 μg/ml primary antibody around the documented condition and compare nuclear signal with surrounding tissue and the no-primary section before attributing diffuse cytoplasmic brown staining to GLI2 (datasheet A00701-5; UniProt P10070 subcellular location; standard IHC practice).
How should I quantify heterogeneous GLI2 staining across a tissue section? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region of interest before scoring, excluding folds, damaged edges and necrotic areas (standard IHC practice). For each region, report the percentage of positive nuclei and an H-score using nuclear intensity categories 0–3; record cytoplasmic staining separately (standard IHC scoring practice; UniProt P10070 subcellular location). Normalise positive-cell counts to the number of intact, evaluable cells in that same region, or report positive-cell density per mm² when area is the relevant denominator (standard IHC quantification practice). Apply identical thresholds, counterstaining and imaging settings across sections, and retain no-primary controls because HPA reports low consistency between GLI2 staining and RNA expression (standard IHC practice; HPA tissue IHC).
How can I distinguish plausible GLI2 staining from section artefact?
Look for staining within intact cells and record its nuclear and cytoplasmic distribution; GLI2 has both locations, while its transcription factor role makes nuclear staining especially relevant (UniProt P10070 subcellular location and function). Compare the stained cell population with tissue morphology rather than assuming every brown area represents a GLI2-positive cell (standard IHC practice). Discount staining concentrated at cut edges, folds or necrotic regions, and investigate signal in the no-primary section as possible endogenous enzyme or detection background (standard IHC practice). The documented mammary cancer image supports use of this paraffin-section workflow, but HPA's low staining–RNA consistency limits claims based on staining alone (datasheet A00701-5; HPA tissue IHC).
Boster reagents
Best GLI2 / Transcription activator GLI2 IHC Antibodies
Anti-GLI2 antibodies have IHC images from human, mouse, and rat paraffin sections (A00701-5 and A00701-6 IHC captions), plus a human U2OS IF/ICC image (A00701-6 IF caption).
Real IHC data
Anti-Zinc finger protein GLI2 GLI2 Antibody ®
Cat # A00701-5
Real IHC data
Anti-GLI2 Antibody ®
Cat # A00701-6
A00701-5 has IHC images from human mammary and intestinal cancers and mouse and rat brain (A00701-5 IHC captions). A00701-6 has IHC images from human esophageal squamous carcinoma, testicular germ cell tumors, and tonsil, plus an IF/ICC image from human U2OS cells (A00701-6 IHC and IF captions).
Which to pick: For human tissue IHC, A00701-6 has paraffin-section examples using EDTA pH 8 retrieval and 2 μg/ml primary antibody; the fixative is unreported (A00701-6 IHC captions). For human IF/ICC, choose A00701-6, the only IF/ICC-listed SKU, with a U2OS image using 5 μg/ml primary antibody (catalog applications; A00701-6 IF caption). For IHC across human, mouse, and rat samples, choose A00701-5: its paraffin-section images use citrate pH 6 retrieval and 1 μg/ml primary antibody; the fixative is unreported (A00701-5 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P10070 (GLI2_HUMAN, Transcription activator GLI2).
- Human Protein Atlas. GLI2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. GLI2 subcellular location (ICC-IF): Mainly localized to the nucleoli. In addition localized to the nucleoplasm, centrosome and basal body..
- Human Protein Atlas. GLI2 antibody validation summary (2 antibodies).
- Expression of glioma-associated oncogene 2 (Gli 2) is correlated with poor prognosis in patients with hepatocellular carcinoma undergoing hepatectomy. World journal of surgical oncology 2013 — PMC3565946.
- Hedgehog pathway and pediatric nonalcoholic fatty liver disease. Hepatology (Baltimore, Md.) 2013 — PMC3637920.
- Signals from dying hepatocytes trigger growth of liver progenitors. Gut 2010 — PMC3632642.
- Arsenic inhibits hedgehog signaling during P19 cell differentiation. Toxicology and applied pharmacology 2014 — PMC4255151.
- PubMed PMID:9557682 — UniProt-cited evidence.
- PubMed PMID:15994174 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for GLI2 (Transcription activator GLI2). Expected localisation Nuclear staining across tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A00701-5). Validated IHC antibody: A00701-5 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative None in HPA (detected in all 43 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human mammary cancer tissues; fixative not specified (datasheet A00701-5); 2. Fixation: Image fixative and duration unreported (datasheet A00701-5); 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: Citrate pH 6 , 20 min (datasheet A00701-5); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A00701-5); 8. Primary antibody: Rabbit anti-GLI2, 0.5-1μg/ml (datasheet A00701-5); 9. Primary incubation: Overnight at 4 °C (datasheet A00701-5). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet A00701-5); verify before use.; Caveat: Staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: Five isoforms plus GLI2R; epitope coverage is unknown (UniProt); Regulation: SUFU sequesters GLI2 without SMO signaling (UniProt). Evidence: UniProt P10070, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoli. In addition localized to the…, Datasheet A00701-5, PMC3565946. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/GLI2/immunohistochemistry-ihc-guide.