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STAT3 / Signal transducer and activator of transcription 3 · IHC design guide
Design Immunohistochemistry for STAT3
Plan chromogenic STAT3 IHC in paraffin sections using its variable cytoplasmic and nuclear tissue staining as a reference (HPA tissue IHC). Compare compartments across conditions because LIF can increase nuclear STAT3 (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
STAT3 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 | Variable cytoplasmic and nuclear staining (HPA tissue IHC) | |
| Staining pattern | Variable cytoplasmic/nuclear signal; high in cytotrophoblasts (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Placenta+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 sections. (standard IHC practice; not target-specific) | |
| Caveat | Nuclear staining alone does not prove Tyr705 phosphorylation (UniProt) | |
| Regulation | LIF increases nuclear STAT3 (UniProt) | |
| Isoform / epitope | Three isoforms; verify antibody epitope coverage (UniProt) |
Section 1
Recommended STAT3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by four published phospho-STAT3 IHC workflows (PMC7114280; PMC5374098; PMC6829927; PMC4287356).
| Sample | Paraffin-embedded tissue sections; fixative not specified (datasheet M00007-1; sample unspecified) |
| Fixation | Image fixative and duration unreported (datasheet M00007-1); 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 CAB-19) anti-STAT3, 1:50-1:200 (datasheet M00007-1) |
| 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 | STAT3-positive staining in cytotrophoblasts of placenta (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic and nuclear expression at variable levels. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min (page retrieval rule); adjust for phospho-STAT3 using the published conditions (PMC5374098; PMC6829927; PMC4287356).
Section 2
What Is the Expected STAT3 Staining Pattern?
STAT3 is a cytoplasmic and nuclear protein that shuttles between compartments; it has no transmembrane segment (UniProt P40763). In paraffin-section IHC, expect variable staining across cells, including high staining in placental cytotrophoblasts and medium staining in bronchial respiratory epithelial cells (HPA: tissue IHC). HPA rates the tissue pattern Supported, with medium consistency between antibody staining and RNA expression (HPA: tissue IHC).
| Placental cytotrophoblasts stain strongly, with cytoplasmic and/or nuclear signal. | This fits the reported high cytotrophoblast staining and general cytoplasmic and nuclear pattern (HPA: tissue IHC). Score the two compartments separately: nuclear signal can reflect STAT3 localisation, but total STAT3 staining alone does not establish pathway activation (UniProt P40763). |
| Some cells show stronger nuclear staining than nearby cells. | STAT3 shuttles, is predominantly cytoplasmic without stimuli, and can accumulate in the nucleus after signalling (UniProt P40763). Constitutive nuclear presence can also occur independently of tyrosine phosphorylation, so nuclear staining alone cannot identify the stimulus or prove phosphorylation (UniProt P40763). |
| Signal is confined to an extracellular rim or a crisp plasma-membrane outline. | An exclusively extracellular or membrane-bound pattern is inconsistent with the reported cytoplasmic and nuclear localisation and absence of a transmembrane segment (UniProt P40763; HPA: tissue IHC). Check whether the pattern persists in controls before assigning it to STAT3 (standard IHC practice). |
| Strong staining appears in a cell population reported as not detected by HPA. | For example, HPA reports no staining in heart-muscle cardiomyocytes or prostate glandular cells (HPA: tissue IHC). Cross-reactivity or endogenous detection activity is possible; HPA’s Supported rating has only medium staining-to-RNA consistency, so investigate the discrepancy rather than declaring the cells STAT3-negative (HPA: tissue IHC; standard IHC practice). |
| Color covers tissue broadly, obscuring cell borders and nuclei. | This diffuse pattern cannot support a compartment or cell-type call (standard IHC practice). Compare a primary-omission control and the expected distribution before interpreting any remaining cellular signal; nonspecific binding or detection background may be responsible (standard IHC practice; HPA: tissue IHC). |
💡Expected STAT3 appearanceA convincing positive is cell-resolved cytoplasmic and/or nuclear staining, potentially high in placental cytotrophoblasts, whereas broad haze or an exclusively extracellular rim is suspect (HPA: tissue IHC; UniProt P40763; standard IHC practice).
| Cell and tissue context | HPA reports high cytotrophoblast staining, medium bronchial respiratory epithelial and adipocyte staining, and low hepatocyte staining (HPA: tissue IHC). Use the named cell population when comparing sections; a tissue-level positive does not imply uniform intensity in every cell (HPA: tissue IHC). |
| Activation and compartment | STAT3 is predominantly cytoplasmic without stimuli and can move into the nucleus after signalling, including LIF stimulation (UniProt P40763). Nuclear presence can also be constitutive and independent of tyrosine phosphorylation; record localisation without calling activation from total STAT3 staining alone (UniProt P40763). |
| Antibody evidence | HPA lists five antibodies with Supported IHC status, while its overall tissue-IHC reliability is Supported with medium staining-to-RNA consistency (HPA: antibodies; HPA: tissue IHC). This supports comparison with the atlas pattern, but does not validate every antibody or every cell-level result (HPA: antibodies; HPA: tissue IHC). |
| Protein forms and epitope coverage | UniProt lists three STAT3 isoforms and no signal peptide or propeptide (UniProt P40763). Without an antibody epitope in the supplied evidence, isoform coverage cannot be predicted; do not assign a staining difference to a particular isoform from morphology alone (UniProt P40763). |
| IF/ICC Q: where should STAT3 appear? | HPA reports enhanced nucleoplasm and cytosol localisation in ICC-IF (HPA: subcellular). That supports the compartment expectation, but the ICC-IF observation does not establish a paraffin-section intensity or an IHC protocol (HPA: subcellular; HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in placental cytotrophoblasts. | The reported high-staining cell population is absent from the section, or the IHC run has inadequate detection (HPA: tissue IHC; standard IHC practice). | Confirm cytotrophoblasts are present, then review the catalog antibody’s IHC-P instructions and run controls for primary-antibody and detection steps (HPA: tissue IHC; standard IHC practice). |
| Only background color appears in a primary-omission control. | Detection chemistry or endogenous tissue activity may contribute signal without the primary antibody (standard IHC practice). | Review detection blocking and the chromogen-development step, then compare the corrected control with cellular staining (standard IHC practice). |
| Strong color appears in HPA not-detected cells. | Cross-reactivity or endogenous detection activity is possible; HPA reports cardiomyocytes and prostate glandular cells as not detected (HPA: tissue IHC; standard IHC practice). | Check cell identity and a primary-omission control, then compare the same pattern with another IHC-Supported antibody if available (HPA: antibodies; standard IHC practice). |
| Signal appears only as a membrane-like outline. | The pattern does not match the reported cytoplasmic and nuclear localisation or lack of a transmembrane segment (UniProt P40763; HPA: tissue IHC). | Inspect the primary-omission control and section morphology; report the pattern as discordant until cell-resolved cytoplasmic or nuclear staining is shown (standard IHC practice; HPA: tissue IHC). |
| Nuclear staining is interpreted as proof of STAT3 activation. | STAT3 can be constitutively nuclear independently of tyrosine phosphorylation (UniProt P40763). | Score nuclear and cytoplasmic staining separately, and reserve phosphorylation claims for a separately validated phosphorylation-specific assay (UniProt P40763; standard IHC practice). |
| A low-signal tissue is treated as a failed run. | HPA reports low staining in hepatocytes and no staining in some named cell populations despite broad tissue expression (HPA: tissue IHC; UniProt P40763). | Compare the exact cell population with HPA and assess an appropriate positive-control section before changing the IHC workflow (HPA: tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing STAT3 staining
M00007-1 · IHCImmunohistochemical analysis of paraffin-embedded Human Hodgkin's lymphoma, using the Antibody at 1:50 dilution.
M00007-1 · IHCImmunohistochemical analysis of paraffin-embedded Human astrocytoma, using the Antibody at 1:50 dilution.
M00007-1 · IFImmunofluorescent analysis of Hela cells treated with IFNalpha, using STAT3 Antibody.
A00007-1 · IHCImmunohistochemistry of STAT3 in human small intestine tissue with STAT3 antibody at 5 μg/ml.
A00007-1 · IFImmunofluorescence of STAT3 in human small intestine tissue with STAT3 antibody at 20 μg/ml.
M00007 · IHCImmunohistochemical analysis of paraffin-embedded human thyroid carcinoma, using STAT3 Antibody.
M00007 · IFImmunofluorescent analysis of HeLa cells, using STAT3 Antibody.
M00007-2 · IHCImmunohistochemical analysis of paraffin-embedded mouse liver, using STAT3 Antibody.
M00007-2 · IFImmunofluorescent analysis of Hela cells, using STAT3 Antibody .
M00007-4 · IHCHuman pancreas was stained with anti-STAT3 rabbit antibody
P00007-1 · IHCImmunohistochemical analysis of paraffin-embedded rat brain, using Phospho-STAT3 (S727) Antibody.
P00007-1 · IFIF analysis of STAT3 using anti-STAT3 antibody (P00007-1). STAT3 was detected in immunocytochemical section of Hela cell. Enzyme antigen ret
PB9511 · IHC
PB9511 · IFIF analysis of STAT3 using anti-STAT3 antibody (PB9511) and anti-Beta Tubulin antibody (M01857-3). STAT3 was detected in immunocytochemical
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000168610-STAT3/tissue/cerebral+cortex#imid_18953034
HPA · LungHuman Protein Atlas IHC, Lung. Source: https://www.proteinatlas.org/ENSG00000168610-STAT3/tissue/lung#imid_18914996
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000168610-STAT3/tissue/colon#imid_18915027
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000168610-STAT3/tissue/liver#imid_18915842
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001671; green = STAT3, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001671; green = STAT3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for STAT3 IHC & IF
🧪Run placenta first; cytotrophoblasts should stain strongly (HPA: High in placental cytotrophoblasts). Use heart muscle cardiomyocytes as the tissue negative (HPA: Not detected in cardiomyocytes); on the placenta slide, internal negative cells should lack specific chromogen staining, without assuming a particular cell type is negative.
Positive control tissue: Placenta (Cytotrophoblasts, HPA High)
Negative control tissue: Heart muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show STAT3 in A-431, U-251MG, U2OS, SK-MEL-30, SiHa, with annotated localisation: Nucleoplasm (enhanced), Cytosol (enhanced) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and concentration-matched isotype controls using the primary antibody’s host species and clonality, plus STAT3-knockout tissue or cells as a biological negative (standard IHC practice). In placenta, block endogenous peroxidase for chromogenic detection and check for endogenous biotin if using avidin–biotin detection (standard IHC practice).
⚠️Feasibility: A STAT3-specific fixation window or fixation effect is unreported in the supplied evidence; the fixative for the selected M00007-1 paraffin-section caption is also unreported (selected-SKU caption). Retrieval dependency is unreported, so optimize antigen retrieval empirically for paraffin sections (standard IHC practice). Neither frozen sections nor IF/ICC can be judged easier from these data; placental endogenous peroxidase or biotin may complicate chromogenic interpretation (standard IHC practice).
HPA tissue IHC evidence for STAT3
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 |
|---|---|---|---|---|
| Placenta | Cytotrophoblasts | High | Protein (IHC) | HPA → |
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Adipocytes | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Prostate | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced STAT3 IHC Tips
Use compartment aware controls and matched processing to troubleshoot STAT3 staining in paraffin section IHC (UniProt P40763; HPA tissue IHC).
How should I adjust retrieval when STAT3 nuclear staining is weak?
For STAT3 paraffin IHC, start with Tris-EDTA pH 9.0 HIER at 95–98 °C for 20 min (page retrieval rule: nuclear antigen). If nuclear staining remains weak, compare a shorter heating interval on adjacent sections while keeping the buffer and detection conditions constant (standard IHC practice). If that fails, test citrate pH 6.0 as a fallback, using matched control sections to assess signal and tissue preservation (standard IHC practice). Check that sections remain attached and morphology is intact before increasing primary antibody concentration, because damaged tissue can make staining difficult to interpret (standard IHC practice). Score nuclear and cytoplasmic signal separately, since STAT3 occupies both compartments (UniProt P40763; HPA tissue IHC).
Can fixation explain inconsistent STAT3 staining across paraffin sections?
Target specific fixation sensitivity is unknown from the supplied evidence, and the selected M00007-1 tissue caption reports paraffin embedding without stating a fixative (selected IHC caption). For a controlled comparison, process matched specimens with a consistent fixation and embedding schedule, then stain them in the same run (standard IHC practice). Record fixation duration, section age, retrieval conditions, and detection batch so a change in staining can be traced to processing (standard IHC practice). Avoid attributing a weak nucleus specifically to STAT3 epitope damage without a matched comparison, because retrieval and detection also affect nuclear signal (standard IHC practice). Compare morphology and compartment specific staining before changing antibody dilution (standard IHC practice).
Is cytoplasmic STAT3 staining a failure of nuclear detection?
Cytoplasmic staining can be plausible because STAT3 shuttles between cytoplasm and nucleus and is predominantly cytoplasmic without stimuli (UniProt P40763). Nuclear accumulation may follow signaling and tyrosine phosphorylation, while constitutive nuclear presence can also occur independently of tyrosine phosphorylation (UniProt P40763). Review adjacent sections stained in the same run and record nuclear and cytoplasmic signal separately within each cell population (standard IHC practice). The tissue IHC profile reports variable nuclear and cytoplasmic expression, so a nucleus only acceptance rule would discard plausible staining (HPA tissue IHC). If staining is confined to tissue edges or lacks intact cellular boundaries, examine section quality and detection controls before calling it localisation (standard IHC practice).
Can STAT3 isoforms or modifications change what this antibody detects?
STAT3 has 3 listed isoforms, including Del-701, and its SH2 domain spans residues 580–670 (UniProt P40763). Multiple modified residues are also listed, including acetylated and methylated lysines, but the supplied caption does not identify the M00007-1 binding epitope (UniProt P40763; selected IHC caption). Check the antibody's documented immunogen or epitope before claiming equal recognition of every isoform or modification state (standard IHC practice). Compare staining with an independently validated antibody against a different region if an isoform dependent result matters (standard IHC practice). Do not interpret total STAT3 chromogenic staining as a direct readout of Tyr-705 phosphorylation without phospho specific validation (UniProt P40763; standard IHC practice).
How can I check STAT3 localisation by IF alongside this IHC result?
On a separate IF or ICC preparation, pair STAT3 with a marker for the cell population being evaluated and a nuclear counterstain to assign signal to intact cells (standard IF practice). Choose spectrally separated fluorophores and inspect an unstained preparation for tissue autofluorescence before interpreting a weak STAT3 channel (standard IF practice). STAT3 has no transmembrane segment and occupies cytoplasm and nucleus, so use controlled permeabilisation to expose intracellular epitopes while preserving cellular boundaries (UniProt P40763; standard IF practice). Include single stain and secondary only controls when multiplexing, and assess bleed through before comparing compartments (standard IF practice). Treat the paraffin tissue caption as IHC evidence only; it does not establish IF fixation or staining conditions (selected IHC caption).
How do I distinguish diffuse STAT3 staining from chromogenic background?
STAT3 can produce genuine cytoplasmic and nuclear signal, and the tissue profile describes variable expression across cells (UniProt P40763; HPA tissue IHC). Inspect a no primary control and the section's morphology to identify secondary reagent signal, pigment, folds, and tissue edge deposits (standard IHC practice). Use a peroxidase block before DAB detection and assess whether residual endogenous activity persists in the control (standard chromogenic IHC practice). If diffuse signal obscures nuclei, titrate the primary around the caption's 1:50 condition on matched sections and keep DAB development consistent (selected IHC caption; standard IHC practice). Judge each trial against an expected positive compartment and a low staining compartment on the same run (HPA tissue IHC; standard IHC practice).
What should I measure when STAT3 spans nuclei and cytoplasm? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, then record nuclear and cytoplasmic STAT3 separately because both locations are documented (UniProt P40763; HPA tissue IHC). For each compartment, report the percentage of positive cells and, when intensity is reliable, an H-score calculated as percentage at each intensity multiplied by its intensity grade (standard IHC scoring practice). Normalise positive cell counts to all evaluable cells of that population, or report positive cell density per mm² of viable tissue (standard IHC scoring practice). Exclude folds, necrosis, and edge artefact using the same rules across sections (standard IHC practice). Keep retrieval, antibody dilution, DAB development, and image thresholds consistent across compared specimens (standard IHC practice).
When is a STAT3 positive cell convincing rather than artefactual?
A convincing result follows intact cellular boundaries and a plausible nuclear or cytoplasmic pattern, since STAT3 is reported in both compartments (UniProt P40763; HPA tissue IHC). Assess the identity of stained cells: cytotrophoblasts are reported high, whereas cardiomyocytes in heart muscle are reported not detected in the supplied tissue profile (HPA tissue IHC). Those reference patterns are context for review, rather than universal pass or fail controls across differently processed specimens (HPA tissue IHC; standard IHC practice). Discount signal concentrated at section edges, folds, or necrotic areas, and check a no primary control for endogenous enzyme or detection reagent staining (standard chromogenic IHC practice). Nuclear total STAT3 alone does not prove phosphorylation dependent activation, because constitutive nuclear presence is reported (UniProt P40763).
Boster reagents
Best STAT3 / Signal transducer and activator of transcription 3 IHC Antibodies
Catalog IHC images show STAT3 staining in human tissues, mouse liver, and rat brain; IF images include human cells (catalog IHC/IF captions).
Real IHC data
Anti-STAT3 Rabbit Monoclonal Antibody
Cat # M00007-1
Real IHC data
Anti-STAT3 Antibody
Cat # A00007-1
Real IHC data
Anti-STAT3 Rabbit Monoclonal Antibody
Cat # M00007
Real IHC data
Anti-STAT3 Rabbit Monoclonal Antibody
Cat # M00007-2
Real IHC data
Anti-STAT3 Rabbit Monoclonal Antibody
Cat # M00007-4
Real IHC data
Anti-Phospho-STAT3 (S727) Rabbit Monoclonal Antibody
Cat # P00007-1
M00007-1 shows paraffin-embedded human Hodgkin’s lymphoma (catalog IHC caption); A00007-1 shows human small intestine (catalog IHC caption); M00007 shows paraffin-embedded human thyroid carcinoma (catalog IHC caption). M00007-2 shows paraffin-embedded mouse liver despite listing human-only reactivity (catalog IHC caption; catalog reactivity); M00007-4 shows human pancreas (catalog IHC caption); P00007-1 shows paraffin-embedded rat brain with phospho-STAT3 (S727) staining (catalog IHC caption).
Which to pick: For tissue IHC, start with M00007-1: its image documents a paraffin-embedded human Hodgkin’s lymphoma section at 1:50, but does not report the fixative (catalog IHC caption). For IF/ICC, M00007 is a rabbit monoclonal listed for both applications at 1:50, with a HeLa IF image (catalog applications, dilution, clone and IF caption). For work across species, M00007-1 lists Human, Mouse and Rat reactivity and has a paraffin-embedded rat brain IHC image; check performance in each intended tissue (catalog reactivity and IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P40763 (STAT3_HUMAN, Signal transducer and activator of transcription 3).
- Human Protein Atlas. STAT3 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. STAT3 subcellular location (ICC-IF): Localized to the nucleoplasm and cytosol..
- Human Protein Atlas. STAT3 antibody validation summary (6 antibodies).
- Saturated hydrogen alleviates CCl(4)-induced acute kidney injury via JAK2/STAT3/p65 signaling. The Journal of international medical research 2020 — PMC7114280.
- Significance of Stat3 Signaling in Epithelial Cell Differentiation of Fetal Mouse Lungs. Acta histochemica et cytochemica 2017 — PMC5374098.
- The role of p-Stat3 Y705 immunohistochemistry in glioblastoma prognosis. Diagnostic pathology 2019 — PMC6829927.
- Immunohistochemical expression of the oncogenic molecules active Stat3 and survivin in benign and malignant salivary gland tumors. Oral surgery, oral medicine, oral pathology, oral radiology, and endodontics 2009 — PMC4287356.
- PubMed PMID:7512451 — UniProt-cited evidence.
- PubMed PMID:9630560 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for STAT3 (Signal transducer and activator of transcription 3). Expected localisation Variable cytoplasmic and nuclear staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: M00007-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Placenta (Cytotrophoblasts); negative Heart muscle. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded tissue sections; fixative not specified (datasheet M00007-1; sample unspecified); 2. Fixation: Image fixative and duration unreported (datasheet M00007-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-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 CAB-19) anti-STAT3, 1:50-1:200 (datasheet M00007-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep formalin fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Nuclear staining alone does not prove Tyr705 phosphorylation (UniProt); Isoform / epitope: Three isoforms; verify antibody epitope coverage (UniProt); Regulation: LIF increases nuclear STAT3 (UniProt). Evidence: UniProt P40763, HPA tissue IHC · Supported, HPA ICC-IF · nucleoplasm and cytosol., Datasheet M00007-1, PMC7114280, PMC5374098. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/STAT3/immunohistochemistry-ihc-guide.