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STAT5B / Signal transducer and activator of transcription 5B · IHC design guide
Design Immunohistochemistry for STAT5B
Plan STAT5B paraffin-section IHC using the reported cytoplasmic and nuclear tissue pattern (HPA tissue IHC). Compare staining with Leydig cells, where signal is high, and account for the tissue antibody’s possible detection of proteins from multiple genes (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
STAT5B 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 | Cytoplasmic and nuclear staining (HPA tissue IHC) | |
| Staining pattern | General cytoplasmic and nuclear staining (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Testis+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific) | |
| Caveat | Antibody may detect proteins from multiple genes (HPA tissue IHC) | |
| Regulation | Phosphorylation promotes nuclear entry (UniProt) | |
| Isoform / epitope | 0 annotated isoforms; one 1–787 chain (UniProt) |
Section 1
Recommended STAT5B IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published STAT5B protocols for pancreatic cancer sections (PMC4869940) and human placental villi (PMC9131539).
| Sample | Paraffin-embedded human breast tissue; fixative not specified (datasheet M00681) |
| Fixation | Image fixative and duration unreported (datasheet M00681); 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 HDF-19) anti-STAT5B, 1:50 (datasheet M00681) |
| 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 | STAT5B-positive staining in leydig cells of testis (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic and nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 HIER at 95–98 °C for 20 min (page antigen retrieval); compare the published citrate conditions during optimization (PMC4869940; PMC9131539).
Section 2
What Is the Expected STAT5B Staining Pattern?
STAT5B can appear in the cytoplasm and nucleus; phosphorylation promotes nuclear translocation (UniProt P51692: subcellular location). In paraffin sections, expect cytoplasmic and nuclear staining across several cell types, with the strongest listed staining in testicular Leyig cells (HPA: tissue IHC). Interpret this pattern cautiously: HPA rates its tissue profile Approved but warns that the staining may represent proteins from more than one gene and awaits external verification (HPA: tissue IHC). STAT5B has no transmembrane segment (UniProt P51692: topology).
| Cytoplasmic staining, with nuclear staining in some cells. | This fits HPA’s general cytoplasmic and nuclear IHC profile (HPA: tissue IHC). Nuclear localisation is biologically plausible because STAT5B translocates to the nucleus after phosphorylation (UniProt P51692: subcellular location). Staining alone does not establish phosphorylation or pathway activation (general IHC interpretation). |
| Strong staining in testicular Leydig cells, or moderate staining in listed positive cells. | Leydig cells are High; hepatocytes, kidney tubular cells and lung alveolar cells are among the Medium examples (HPA: tissue IHC). Compare the compartment and cell type, not just overall slide darkness (general IHC interpretation). HPA’s multi-gene warning limits target attribution (HPA: tissue IHC reliability). |
| Predominantly membranous or extracellular staining. | That distribution conflicts with the reported cytoplasmic and nuclear IHC pattern (HPA: tissue IHC) and with STAT5B’s lack of a transmembrane segment (UniProt P51692: topology). Treat it as suspect and check controls and antibody specificity; appearance alone cannot identify the source (general IHC practice). |
| Strong staining in a cell population HPA lists as not detected. | For example, HPA reports no staining in adipocytes or skeletal-muscle myocytes (HPA: tissue IHC). Review tissue identification, detection controls and antibody specificity before calling it STAT5B; HPA’s tissue profile is antibody based and carries a multi-gene warning (HPA: tissue IHC reliability). |
| Diffuse colour across cells and surrounding tissue, or no signal in a known-positive section. | Diffuse colour obscures the expected cell and compartment pattern (HPA: tissue IHC); assess background controls and detection conditions (general IHC practice). A blank Leydig-cell positive control conflicts with HPA’s High result (HPA: tissue IHC); check the staining run before interpreting the sample. |
💡Expected STAT5B appearanceCall a section positive when staining is cytoplasmic and/or nuclear in an expected population, particularly High Leydig cells; diffuse extracellular or predominantly membranous colour is suspect (HPA: tissue IHC; UniProt P51692: topology).
| Location and activation state | UniProt places STAT5B in cytoplasm and nucleus and reports nuclear translocation after phosphorylation (UniProt P51692: subcellular location). Nuclear staining can therefore vary with cell state; localisation alone cannot confirm activation (general IHC interpretation). |
| Cell-specific reference pattern | HPA reports High Leydig-cell staining and Medium staining in several other populations, including hepatocytes and kidney tubular cells (HPA: tissue IHC). Its Low and Not detected categories refer to specified cell populations, not every cell in an organ (HPA: tissue IHC). |
| Specificity limit | The HPA tissue profile is Approved but cautions that the staining targets protein from more than one gene and awaits external verification (HPA: tissue IHC reliability). An expected pattern supports interpretation without proving that every stained cell contains STAT5B. |
| Topology and processing | STAT5B has no transmembrane segment, signal peptide, propeptide or annotated glycosylation site; its listed chain spans residues 1–787 (UniProt P51692: topology and processing). These annotations support an intracellular expectation but provide no target-specific fixation or antigen-retrieval rule. |
| IF/ICC: What pattern should I expect? | HPA reports approved cytosolic localisation in ICC-IF (HPA: subcellular). UniProt also permits nuclear localisation after phosphorylation (UniProt P51692: subcellular location). The ICC-IF observation is a comparison point, not a paraffin-section protocol or proof that every cell should show nuclear signal. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive testis section has no Leydig-cell signal. | HPA reports High Leydig-cell staining (HPA: tissue IHC); a blank control suggests a staining-run or reagent problem (general IHC practice). | Confirm tissue identity, counterstain and reagent delivery; review antigen retrieval and detection steps against the antibody’s IHC-P instructions (general IHC practice). |
| Colour is diffuse across the section. | Diffuse colour cannot be assigned confidently to the cytoplasmic or nuclear pattern (HPA: tissue IHC); detection background is possible (general IHC practice). | Examine a primary-antibody-omission control and review blocking, washing and chromogen development (general chromogenic IHC practice). |
| Signal is predominantly membranous or extracellular. | That pattern conflicts with HPA’s cytoplasmic/nuclear profile (HPA: tissue IHC) and STAT5B topology (UniProt P51692). | Compare with a positive tissue and appropriate detection control; reassess antibody specificity before scoring the signal as STAT5B (general IHC practice). |
| Unexpected strong staining appears in HPA-negative cells. | HPA lists adipocytes and skeletal-muscle myocytes as Not detected, while warning of possible multi-gene staining (HPA: tissue IHC). | Verify the cell identity, inspect controls and seek independent target-specific confirmation before assigning the staining to STAT5B (general IHC practice). |
| A positive cell shows cytoplasm but little nuclear signal. | Cytoplasmic localisation is expected; nuclear translocation follows phosphorylation (UniProt P51692: subcellular location). | Score the observed compartment separately from intensity. Do not infer pathway inactivity from a single stain without an activation-specific assay (general IHC interpretation). |
| A listed low-staining tissue looks blank. | HPA labels certain specified cells Low, including cerebral-cortex endothelial cells; weak staining may be hard to distinguish from background (HPA: tissue IHC; general IHC practice). | Check a High Leydig-cell control first, then assess the correct cell population and detection background before calling the run failed (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing STAT5B staining
M00681 · IHCImmunohistochemical analysis of paraffin-embedded human breast, using STAT5b Antibody.
PA1841 · IHCAnti-STAT5b antibody, PA1841, IHC(P) IHC(P): Human Mammary Cancer Tissue
PB9512 · IFIF analysis of STAT5b using anti-STAT5b antibody (PB9512). STAT5b was detected in immunocytochemical section of U20S cells. Enzyme antigen r
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000173757-STAT5B/tissue/cerebral+cortex#imid_14861584
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000173757-STAT5B/tissue/colon#imid_14861518
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000173757-STAT5B/tissue/liver#imid_14861511
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000173757-STAT5B/tissue/kidney#imid_14861504
HPA · ICC-IF HeLaHuman Protein Atlas immunofluorescence, HeLa, antibody CAB005418; green = STAT5B, blue = nucleus, red = microtubules.
HPA · ICC-IF HeLaHuman Protein Atlas immunofluorescence, HeLa, antibody CAB005418; green = STAT5B, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for STAT5B IHC & IF
🧪Run testis first: Leydig cells should stain (HPA: High in Leydig cells). Use adipose tissue as the negative tissue, focusing on adipocytes (HPA: Not detected in adipocytes); on the testis slide, assess morphologically distinct cells that remain at background, without assuming every non-Leydig cell is negative (HPA: High in Leydig cells).
Positive control tissue: Testis (Leydig cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show STAT5B in HeLa, K-562, U2OS, hTERT-RPE1 (serum starved), with annotated localisation: Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and clonality; use STAT5B knockout tissue or a validated peptide block as a biological specificity control (standard IHC practice). On testis sections, check for endogenous peroxidase signal with the no-primary control and block it if present (standard chromogenic IHC practice).
⚠️Feasibility: A STAT5B-specific fixation window and the effect of antigen retrieval are unreported in the supplied evidence; optimize retrieval empirically for paraffin sections (standard IHC practice). The selected M00681 paraffin-section breast caption does not report a fixative, so it does not establish fixation (selected M00681 tissue-IHC caption). Frozen-section suitability and whether IF is easier are unreported; assess testis background during optimization rather than assuming a tissue-specific artefact (HPA: High in Leydig cells). The selected M00681 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image M00681).
HPA tissue IHC evidence for STAT5B
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Caution, targets protein from more than one gene. 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 |
|---|---|---|---|---|
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | Medium | Protein (IHC) | HPA → |
| Liver | Hepatocytes | Medium | Protein (IHC) | HPA → |
| Lung | Alveolar cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
| Ovary | Follicle cells | Not detected | Protein (IHC) | HPA → |
| Pancreas | Exocrine glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced STAT5B IHC Tips
Troubleshoot STAT5B staining in paraffin sections by checking retrieval, compartment, controls, and scoring before interpreting nuclear signal as pathway activity.
What retrieval conditions should I use when STAT5B staining is weak?
Use Tris-EDTA pH 9.0 heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval setting). Allow sections to cool consistently, then compare a retrieval-treated section with a matched control processed in the same run (standard IHC practice). If staining remains weak, vary retrieval time cautiously while holding antibody concentration and detection conditions constant; excessive heating can damage morphology and increase background (standard IHC practice). Assess cytoplasmic and nuclear staining separately, because STAT5B is reported in both compartments and can translocate to the nucleus after phosphorylation (UniProt P51692 subcellular annotation).
Could fixation explain weak or uneven STAT5B staining in paraffin sections?
Target-specific sensitivity to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (caption: M00681). Record each specimen’s fixative and fixation duration, then compare sections processed with the same retrieval and detection conditions (standard IHC practice). Include a consistently processed reference section in each run so changes in staining can be separated from specimen preparation differences (standard IHC practice). If staining is uneven, inspect tissue preservation and section adhesion before changing retrieval or antibody concentration; neither the HPA tissue pattern nor STAT5B topology establishes a fixation effect (standard IHC practice; UniProt P51692 topology).
How should I assess cytoplasmic versus nuclear STAT5B staining?
Score cytoplasmic and nuclear chromogen separately within defined cell populations, using the counterstain to identify nuclei (standard IHC practice). STAT5B is annotated in cytoplasm and nucleus, with nuclear translocation in response to phosphorylation (UniProt P51692 subcellular annotation). The HPA tissue-IHC profile reports general cytoplasmic and nuclear expression, whereas its approved subcellular assessment identifies cytosol as the main location (HPA tissue IHC; HPA subcellular). Nuclear staining alone does not establish current signaling activity in a fixed section; compare matched conditions and confirm activation with a separately validated phospho-specific assay if that is the biological question (UniProt P51692 subcellular annotation; standard IHC practice).
How can epitope choice affect what my STAT5B stain means?
Check the catalog antibody’s stated immunogen and epitope before interpreting a compartment difference; neither epitope position nor phospho-specificity is supplied here (standard IHC practice). The supplied UniProt record lists no alternative isoforms and places the SH2 domain at residues 589–686 (UniProt P51692 domains and isoforms). It also lists phosphorylation at Tyr682 and Tyr699, among other modified residues, so total STAT5B staining should not be described as a readout of either modification without antibody-specific evidence (UniProt P51692 modified residues). If results conflict across antibodies, compare their documented epitopes and matched controls before assigning a biological explanation (standard IHC practice).
How should I follow up a STAT5B IHC pattern with multiplex IF?
For multiplex IF, pair STAT5B with a validated marker for the cell population being assessed and include single-stain controls to check spectral spillover (standard IF practice). Choose fluorophores after examining unstained tissue autofluorescence; a spectrally separated, brighter channel can help distinguish weak specific signal from autofluorescence (standard IF practice). STAT5B has no transmembrane segment and is annotated in cytoplasm and nucleus, so use controlled permeabilisation to reach intracellular epitopes, while checking that nuclear morphology remains intact (UniProt P51692 topology and subcellular annotation; standard IF practice). Interpret IF localisation alongside matched IHC sections, because the HPA approved subcellular assessment identifies cytosol as its main location (HPA subcellular).
What should I change when STAT5B chromogen appears diffuse or nonspecific?
Run a no-primary control and inspect whether diffuse color persists; this helps identify detection-system or endogenous-enzyme background (standard IHC practice). For peroxidase-based chromogenic detection, apply a peroxidase block before DAB development and check that washing and blocking are consistent across sections (standard IHC practice). Titrate the primary antibody using the same retrieval setting, Tris-EDTA pH 9.0 at 95–98 °C for 20 min, so a concentration change can be judged independently (page retrieval setting; standard IHC practice). Compare staining in the intended cells with surrounding compartments, and treat edge-heavy or necrotic staining cautiously (standard IHC practice).
How should I quantify STAT5B staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and scoring region before reviewing treatment groups, then score nuclear and cytoplasmic chromogen separately (standard IHC practice; UniProt P51692 subcellular annotation). An H-score can combine the percentage of cells at each intensity level; alternatively, report the percentage of positive cells or positive-cell density per mm² (standard IHC practice). Normalise counts to the number of eligible cells or measured viable tissue area, and apply one positivity threshold across matched sections (standard IHC practice). Report sampling rules and excluded damaged regions, because variable cell composition can change a section-level score without changing staining within the target cells (standard IHC practice).
How can I distinguish credible STAT5B staining from artefact?
Look for reproducible signal within intact cells in a plausible compartment: STAT5B is annotated in cytoplasm and nucleus, with phosphorylation-associated nuclear translocation (UniProt P51692 subcellular annotation). HPA reports high staining in testicular Leydig cells and no detection in adipocytes, but its tissue-IHC entry cautions that the antibody targets protein from more than one gene (HPA tissue IHC). Treat staining confined to tissue edges, necrosis, or a no-primary control as suspect, and check endogenous peroxidase when DAB is used (standard IHC practice). Confirm a disputed cell identity with morphology or a separately validated marker before attributing its signal to STAT5B (standard IHC practice).
Boster reagents
Best STAT5B / Signal transducer and activator of transcription 5B IHC Antibodies
Anti-STAT5B antibodies have IHC images from human breast and mammary cancer tissue, plus IF data from U20S cells (image captions). All three list human, mouse, and rat reactivity (catalog reactivity).
Real IHC data
Anti-STAT5b Rabbit Monoclonal Antibody
Cat # M00681
Real IHC data
Anti-STAT5b Antibody ®
Cat # PA1841
Real IF data
Anti-STAT5b Antibody ®
Cat # PB9512
M00681 shows IHC staining in paraffin-embedded human breast, while PA1841 shows IHC(P) staining in human mammary cancer tissue (M00681 and PA1841 image captions). PB9512 shows IF staining in U20S cells and lists IF and ICC applications (PB9512 image caption; catalog applications).
Which to pick: For tissue IHC, choose M00681 for a rabbit monoclonal with a paraffin-embedded human breast image, or PA1841 for its human mammary cancer IHC(P) image; neither caption reports the fixative (catalog host/clone; M00681 and PA1841 image captions). For IF/ICC, PB9512 has a U20S IF image and a listed dilution of 2 μg/ml for human cells (PB9512 image caption; catalog dilution). For human, mouse, or rat IHC work, M00681 lists all three species and an IHC dilution of 1:50, though its tissue image demonstrates human staining only (M00681 catalog reactivity/dilution; image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P51692 (STA5B_HUMAN, Signal transducer and activator of transcription 5B).
- Human Protein Atlas. STAT5B tissue IHC expression (reliability: Approved).
- Human Protein Atlas. STAT5B subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. STAT5B antibody validation summary (5 antibodies).
- Suppression of STAT5b in pancreatic cancer cells leads to attenuated gemcitabine chemoresistance, adhesion and invasion. Oncology reports 2016 — PMC4869940.
- High activation of STAT5A drives peripheral T-cell lymphoma and leukemia. Haematologica 2020 — PMC7012494.
- Low levels of Stat5a protein in breast cancer are associated with tumor progression and unfavorable clinical outcomes. Breast cancer research : BCR 2012 — PMC4053108.
- Role of EZH2-mediated H3K27me3 in placental ADAM12-S expression: implications for fetoplacental growth. BMC medicine 2022 — PMC9131539.
- PubMed PMID:8732682 — UniProt-cited evidence.
- PubMed PMID:8631883 — UniProt-cited evidence.
- PubMed PMID:12039059 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for STAT5B (Signal transducer and activator of transcription 5B). Expected localisation 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: M00681 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Leydig cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast tissue; fixative not specified (datasheet M00681); 2. Fixation: Image fixative and duration unreported (datasheet M00681); 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 HDF-19) anti-STAT5B, 1:50 (datasheet M00681); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific); Caveat: Antibody may detect proteins from multiple genes (HPA tissue IHC); Isoform / epitope: 0 annotated isoforms; one 1–787 chain (UniProt); Regulation: Phosphorylation promotes nuclear entry (UniProt). Evidence: UniProt P51692, HPA tissue IHC · Approved, HPA ICC-IF · cytosol., Datasheet M00681, PMC4869940, PMC9131539. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/STAT5B/immunohistochemistry-ihc-guide.