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STAT5B / Signal transducer and activator of transcription 5B · IHC design guide
Design Immunohistochemistry for STAT5B
Plan STAT5B paraffin IHC around cytoplasmic and nuclear tissue staining (HPA tissue IHC). Compare high staining in testis Leydig cells with undetected staining in adipocytes, and interpret the HPA pattern cautiously because its antibody may detect more than one gene (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 tissue 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 | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M00681) | |
| Caveat | HPA antibody may detect more than one gene (HPA tissue IHC) | |
| Regulation | Activation shifts STAT5B to nuclei (UniProt) | |
| Isoform / epitope | No isoforms; one unprocessed, intracellular chain (UniProt) |
Section 1
Recommended STAT5B IHC & IF Protocols
The catalog antibody's IHC-P protocol is followed by published STAT5B chromogenic IHC methods for pancreatic cancer, lymphoma, breast cancer, and placental tissue (PMC4869940; PMC7012494; PMC4053108; 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 retrieval for the catalog antibody (page retrieval); published protocols also used citrate retrieval (PMC4869940; PMC4053108; PMC9131539).
Section 2
What Is the Expected STAT5B Staining Pattern?
STAT5B is found in the cytoplasm and nucleus, with nuclear translocation after phosphorylation (UniProt P51692 subcellular location). In paraffin-section IHC, expect staining in Leydig cells and several glandular, tubular, and other listed cell populations; staining is not uniform across tissues (HPA tissue IHC). HPA calls its tissue profile Approved but cautions that it may detect protein from more than one gene and awaits external verification (HPA tissue IHC). STAT5B has no transmembrane segment (UniProt P51692 topology).
| Leydig cells stain strongly; hepatocytes or kidney tubule cells stain more moderately. | This follows the reported High testis and Medium liver and kidney patterns (HPA tissue IHC). Compare cell types within the section; those levels describe HPA observations, not a guaranteed intensity for every specimen or IHC run (HPA tissue IHC; general IHC practice). |
| Staining is cytoplasmic, nuclear, or both in an expected cell population. | Both compartments are compatible with STAT5B; phosphorylation can drive nuclear translocation (UniProt P51692 subcellular location). Nuclear staining alone does not establish pathway activation without an activation-specific assay (UniProt P51692 modified residues; general IHC interpretation). |
| A crisp membrane rim dominates the staining. | Membrane localisation is inconsistent with the supplied cytoplasm/nucleus annotation and absence of a transmembrane segment (UniProt P51692 subcellular location and topology). Review morphology, antibody specificity, and detection controls before assigning that rim to STAT5B (general IHC practice). |
| Adipocytes or skeletal-muscle myocytes stain as strongly as the expected positive cells. | HPA reports STAT5B as Not detected in those cell types (HPA tissue IHC). Unexpected staining warrants checks for cross-reactivity or endogenous detection activity; HPA itself cautions that its tissue profile may detect protein from more than one gene (HPA tissue IHC; general IHC practice). |
| Color covers most of the section, including areas without expected positive cells. | Diffuse deposit obscures the cell-level pattern and can reflect nonspecific binding or detection background (general IHC practice). It cannot be scored as STAT5B solely because HPA reports general cytoplasmic and nuclear expression (HPA tissue IHC; general IHC interpretation). |
💡Expected STAT5B appearanceCall the IHC result positive when expected cells show interpretable cytoplasmic and/or nuclear staining, such as High Leydig-cell or Medium hepatocyte staining; widespread featureless deposit or a dominant membrane rim is suspect (HPA tissue IHC; UniProt P51692 subcellular location and topology; general IHC interpretation).
| Tissue and cell selection | Testis Leydig cells offer a reported High reference; liver hepatocytes and kidney tubule cells are Medium references, while adipocytes are Not detected (HPA tissue IHC). Score the specified cells, since a tissue name alone does not identify the stained population (general IHC practice). |
| Activation and compartment | STAT5B is annotated in cytoplasm and nucleus and moves into the nucleus in response to phosphorylation (UniProt P51692 subcellular location). A change in nuclear fraction may merit follow-up, but total-STAT5B IHC does not identify which modified residue is present (UniProt P51692 modified residues; general IHC interpretation). |
| Specificity of the tissue reference | HPA assigns Approved reliability while warning that the profile may target protein from more than one gene and remains pending external verification (HPA tissue IHC). Treat a match to its cell pattern as supporting evidence, not proof that every colored cell contains STAT5B (HPA tissue IHC; general IHC interpretation). |
| IF/ICC: Where should the signal appear? | The HPA ICC/IF summary identifies cytosol as the approved main location (HPA subcellular ICC/IF). UniProt also allows nuclear localisation following phosphorylation, so assess the compartment in the experiment's biological context (UniProt P51692 subcellular location). |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain appears in Leydig cells of an otherwise intact testis section. | HPA reports High Leydig-cell staining, making this a useful positive-reference failure; weak detection, retrieval, or staining workflow may be involved (HPA tissue IHC; general IHC practice). | Check section integrity, primary-antibody application, detection reagents, and chromogen development; then review retrieval against the antibody's IHC instructions (general IHC practice). Do not infer a STAT5B-specific fixation effect from this result. |
| A membrane-like outline is stronger than cytoplasmic or nuclear staining. | A dominant membrane pattern conflicts with the annotated compartments and no-transmembrane topology (UniProt P51692 subcellular location and topology). | Inspect adjacent morphology and a primary-omission control, then compare expected positive and Not detected cell types on the same run (general IHC practice; HPA tissue IHC). |
| Adipocytes show strong brown deposit. | Adipocytes are reported Not detected; cross-reactivity or endogenous detection activity may explain the discordance (HPA tissue IHC; general IHC practice). | Check a primary-omission control and the relevant endogenous-activity blocking step; compare the pattern with Leydig cells or another reported positive population (general IHC practice; HPA tissue IHC). |
| All cells have hazy color that hides their boundaries. | The pattern is consistent with detection background or nonspecific binding, rather than a scorable cell-specific result (general IHC practice). | Review blocking, washing, antibody concentration, and detection-control staining; rescore only when cell boundaries and compartments are visible (general IHC practice). |
| Nuclear staining appears, but the study expects mostly cytoplasmic staining. | Nuclear STAT5B can occur after phosphorylation; total staining alone does not identify the activation state (UniProt P51692 subcellular location and modified residues; general IHC interpretation). | Confirm nuclear location against the counterstain, compare matched biological conditions, and use an activation-specific assay if phosphorylation is the question (general IHC practice; UniProt P51692 subcellular location). |
| The HPA-like tissue pattern is present, but target identity remains uncertain. | HPA marks the tissue profile Approved yet cautions that it may detect protein from more than one gene and awaits external verification (HPA tissue IHC). | Interpret the pattern alongside independent specificity evidence before making a STAT5B-specific claim; an HPA pattern match alone does not resolve the stated caveat (HPA tissue IHC; general IHC interpretation). |
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, where adipocytes are not detected (HPA: Not detected in adipocytes); on the testis slide, use neighboring cells that lack staining as internal negative comparators without assigning them a cell type (standard IHC practice).
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 no-primary (secondary-only) and isotype controls matched to the primary antibody’s host species and clonality, plus STAT5B-knockout tissue as a biological negative if available (standard IHC practice). In testis, check endogenous peroxidase background before interpreting chromogenic signal (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the fixative is unreported for the selected M00681 paraffin-section caption (M00681 caption). Retrieval dependency is also unreported; the supplied evidence does not establish whether frozen sections or IF are easier (M00681 caption; HPA: ICC-IF images available). When scoring testis, distinguish Leydig-cell pigment from chromogen (standard histology practice).
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-section chromogenic IHC by checking retrieval, compartment, cellular context and controls before interpreting signal.
What should I change when STAT5B staining is weak after antigen retrieval?
Start with Tris-EDTA pH 9.0 heat-induced retrieval at 95–98 °C for 20 min (page retrieval setting: nuclear antigen). If staining remains weak, compare a shorter heating interval on matched sections while holding antibody and detection conditions constant (standard IHC practice). Include a control section with a known staining pattern, such as testis Leydig cells, which have high reported staining (HPA: High in Leydig cells). Check whether the loss affects nuclear and cytoplasmic signal alike, since STAT5B occupies both compartments (UniProt P51692: subcellular location). Record the retrieval run and score comparable cell populations before changing another variable (standard IHC practice).
Can I infer the best fixative from the available STAT5B tissue staining?
The selected antibody caption identifies paraffin-embedded human breast but does not report a fixative (M00681 tissue-IHC caption). Target-specific sensitivity to fixation is therefore unknown from the supplied evidence (M00681 tissue-IHC caption). For a fixation comparison, process matched specimens with documented fixation conditions, then apply the same pH 9.0 retrieval, staining and scoring workflow to every section (page retrieval setting; standard IHC practice). Assess morphology and nuclear versus cytoplasmic staining separately, because STAT5B can occupy either compartment (UniProt P51692: subcellular location). Treat any apparent fixation effect as a result of that comparison, rather than inferring it from tissue-expression patterns or phosphorylation sites (standard IHC interpretation practice).
Should STAT5B stain nuclei, cytoplasm, or both in paraffin sections?
Both compartments can be relevant: STAT5B is cytoplasmic and can move into nuclei after phosphorylation (UniProt P51692: subcellular location). General tissue IHC reports cytoplasmic and nuclear expression, whereas the approved subcellular ICC/IF location is cytosol (HPA: tissue profile; HPA: subcellular location). In chromogenic sections, score nuclear and cytoplasmic staining separately within the same defined cell type, using a consistent counterstain and comparable exposure to detection reagent (standard IHC practice). Compare stimulated and unstimulated material only when their handling is documented, because location can change with activation (UniProt P51692: subcellular location). Diffuse staining outside recognizable cells warrants review of morphology and controls before assigning a compartment (standard IHC practice).
How do I evaluate whether my antibody detects the intended STAT5B epitope?
The supplied protein record describes a 787-residue STAT5B chain and lists 0 isoforms, so this record supplies no isoform-specific epitope explanation (UniProt P51692: processing and isoforms). Its SH2 domain spans residues 589–686, and reported modified residues include phosphotyrosines at 90, 682 and 699 (UniProt P51692: domains and modified residues). Obtain the antibody's stated immunogen or epitope before interpreting epitope accessibility or phosphorylation dependence (standard antibody-validation practice). Compare staining with an independently validated reagent or an appropriate loss-of-target control when available (standard IHC validation practice). Do not label a chromogenic signal phosphorylation-specific unless the antibody's specificity supports that claim (standard antibody-validation practice).
How can I investigate STAT5B localisation by IF alongside this IHC guide?
For the separate IF/ICC workflow, pair STAT5B with a validated marker for the cell population being examined, such as a Leydig-cell marker when examining testis (HPA: High in Leydig cells; standard multiplex IF practice). Choose spectrally separated fluorophores and consider a far-red channel when tissue autofluorescence obscures shorter wavelengths (standard IF practice). STAT5B has no transmembrane segment and occurs in cytoplasm and nucleus, so assess permeabilisation that permits antibody access to intracellular epitopes (UniProt P51692: topology and subcellular location; standard IF practice). Include single-label controls to assess bleed-through and a no-primary control for background (standard IF practice). Compare compartment patterns without assuming that the paraffin-section caption establishes IF fixation conditions (M00681 tissue-IHC caption).
What should I check when chromogenic STAT5B staining appears diffuse or patchy?
First inspect a no-primary control and the counterstained morphology to separate nonspecific deposit from staining in intact cells (standard IHC practice). Block endogenous peroxidase for an enzyme-based chromogenic workflow, and check whether signal persists without primary antibody (standard IHC practice). Titrate the primary antibody and detection exposure using matched sections, changing one variable at a time (standard IHC practice). Compare putative positive cells with a reported high-expression reference, such as testis Leydig cells, while recognizing that the HPA tissue antibody carries a cross-gene caution (HPA: High in Leydig cells; HPA: reliability description). Document edge-associated or necrotic staining separately from evaluable tissue (standard IHC practice).
How should I quantify STAT5B chromogenic staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring; report nuclear and cytoplasmic STAT5B separately because both locations are documented (UniProt P51692: subcellular location). An H-score can combine staining intensity and percentage of positive cells, while percentage positive alone is useful when intensity is unreliable (standard IHC scoring practice). For spatial analyses, report positive-cell density per mm² of evaluable tissue, excluding folds, necrosis and section edges (standard IHC quantification practice). Normalize counts to the number of eligible cells or measured tissue area, and use the same threshold and counterstain criteria across sections (standard IHC practice). Keep cell types separate because HPA reports differing levels across tissue compartments (HPA: tissue IHC profile).
Which observations support a true STAT5B signal rather than an artefact?
A plausible signal lies within intact cells and can appear in cytoplasm or nucleus, with nuclear accumulation consistent with activation-dependent translocation (UniProt P51692: subcellular location). Check cell identity against the section's morphology: HPA reports high staining in Leydig cells and no detected staining in adipocytes, but cautions that its tissue antibody may recognize more than one gene (HPA: tissue IHC profile and reliability description). Staining concentrated at cut edges, necrotic areas or damaged cells needs separate review (standard IHC interpretation practice). Persistent chromogen in a no-primary control suggests detection background, including endogenous enzyme activity (standard IHC practice). Nuclear staining alone does not establish phosphorylation-specific STAT5B detection without antibody-specific validation (standard antibody-validation practice).
Boster reagents
Best STAT5B / Signal transducer and activator of transcription 5B IHC Antibodies
Anti-STAT5B antibodies have IHC images from human breast and human mammary cancer tissue (image captions), and an IF/ICC image from U20S cells (image caption).
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 on paraffin-embedded human breast (image caption); PA1841 shows IHC(P) on human mammary cancer tissue (image caption). PB9512 shows IF/ICC in U20S cells (image caption) and lists Human, Mouse and Rat reactivity (catalog reactivity).
Which to pick: For tissue IHC, choose M00681 for paraffin-embedded human breast (image caption) or PA1841 for IHC(P) of human mammary cancer tissue (image caption); fixation is unreported in both captions. For IF/ICC, PB9512 has an IF image from U20S cells (image caption). For work spanning Human, Mouse and Rat, M00681 lists all three species and both IHC and IF/ICC applications, and is rabbit monoclonal clone HDF-19 (catalog reactivity, applications and clone).
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 tissue 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: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M00681); Caveat: HPA antibody may detect more than one gene (HPA tissue IHC); Isoform / epitope: No isoforms; one unprocessed, intracellular chain (UniProt); Regulation: Activation shifts STAT5B to nuclei (UniProt). Evidence: UniProt P51692, HPA tissue IHC · Approved, HPA ICC-IF · cytosol., Datasheet M00681, PMC4869940, PMC7012494. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/STAT5B/immunohistochemistry-ihc-guide.