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SUPT5H / Transcription elongation factor SPT5 · IHC design guide
Design Immunohistochemistry for SUPT5H
Plan chromogenic IHC for SUPT5H using its widespread nuclear tissue pattern (HPA tissue IHC). Compare nuclear staining across sections with consistent fixation, and interpret intensity in light of the medium consistency between antibody staining and RNA data (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SUPT5H 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 cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03426-2) | |
| Positive control | Placenta+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Antibody staining has medium consistency with RNA data (HPA tissue IHC) | |
| Regulation | Ubiquitously expressed (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope differences are unspecified (UniProt) |
Section 1
Recommended SUPT5H IHC & IF Protocols
The catalog antibody's IHC-P protocol (datasheet A03426-2) and one published SUPT5H IHC protocol (PMC4741733) provide starting conditions for paraffin sections.
| Sample | Paraffin-embedded human ovarian serous carcinoma tissue; fixative not specified (datasheet A03426-2) |
| Fixation | Image fixative and duration unreported (datasheet A03426-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A03426-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03426-2) |
| Primary antibody | Rabbit anti-SUPT5H, 2-5 μg/ml (datasheet A03426-2) |
| Primary incubation | Overnight at 4 °C (datasheet A03426-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A03426-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SUPT5H-positive staining in cytotrophoblasts of placenta (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet A03426-2); the published protocol used pressure-cooker citrate pH 6.0 (PMC4741733).
Section 2
What Is the Expected SUPT5H Staining Pattern?
SUPT5H is a nuclear transcription elongation factor with no transmembrane segment (UniProt O00267: location, topology). Expect nuclear staining across many cell types, with prominent staining in placental cytotrophoblasts (HPA: ubiquitous nuclear expression; cytotrophoblasts High). HPA rates the tissue staining pattern Supported, with medium consistency between antibody staining and RNA expression; interpret intensity differences with that qualification (HPA: tissue IHC reliability).
| Nuclear staining in placental cytotrophoblasts, with weaker or variable nuclear staining elsewhere (HPA: cytotrophoblasts High; ubiquitous nuclear profile). | This matches the expected compartment and a documented strong tissue example. Compare nuclei within each cell population; broad expression does not require every nucleus to have identical intensity (UniProt O00267: nucleus, ubiquitous expression; HPA: tissue IHC profile). |
| Predominantly cytoplasmic, membranous, or extracellular color while nuclei remain pale (UniProt O00267: nucleus, no transmembrane segment). | Treat this as a discordant pattern requiring controls, rather than evidence of SUPT5H redistribution. Check whether staining persists without primary antibody and review the detection system (UniProt O00267: location, topology; general IHC practice). |
| Intense signal in a cell population expected to stain only weakly, especially with little corresponding nuclear signal (HPA: glial cells in hippocampus and caudate Low). | Consider cross-reactivity or endogenous detection activity; low HPA staining is not proof that these cells lack SUPT5H. Resolve the signal's compartment and control dependence before calling it specific (HPA: Low, not negative; general IHC practice). |
| Uniform color over cells and tissue spaces that obscures nuclear boundaries (HPA: expected nuclear profile). | This is background rather than an interpretable nuclear pattern. Examine a no-primary control and review blocking, washing, and chromogen development for the chosen detection system (HPA: tissue IHC profile; general IHC practice). |
| No nuclear signal in placental cytotrophoblasts on an otherwise interpretable section (HPA: cytotrophoblasts High). | A false negative is plausible. Confirm that the antibody and detection reagents worked, then review the antibody's IHC-P instructions and the laboratory's retrieval settings; HPA does not establish SUPT5H-specific fixation sensitivity (HPA: cytotrophoblasts High; general IHC practice). |
💡Expected SUPT5H appearanceCall a positive result when nuclei stain in expected cell populations, prominently in placental cytotrophoblasts; predominantly nonnuclear color is suspect (HPA: ubiquitous nuclear profile; cytotrophoblasts High; UniProt O00267: nucleus).
| Compartment | SUPT5H is nuclear and lacks a transmembrane segment; score nuclear color, and investigate dominant membrane or cytoplasmic color before assigning specificity (UniProt O00267: location, topology). |
| Tissue and cell intensity | HPA reports High staining in placental cytotrophoblasts, Medium in several other listed populations, and Low in some glial and other cells. These are observed levels, not guaranteed results for every section (HPA: tissue IHC). |
| IHC antibody evidence | HPA lists IHC as Supported for HPA029273 and CAB034370, and rates the tissue profile Supported with medium RNA–staining consistency. Check the validation status of the antibody actually used (HPA: antibodies; tissue IHC reliability). |
| Isoforms and modifications | UniProt lists two isoforms and multiple modified residues. Their effect on staining cannot be assigned without an antibody epitope and recognition data; avoid interpreting intensity as an isoform or modification readout (UniProt O00267: isoforms, modified residues). |
| IF/ICC question: where should signal appear? | In the nucleoplasm (HPA: subcellular ICC-IF, supported). HPA shows ICC-IF images in A-431, U-251MG, and U2OS and lists ICC validation as Enhanced for HPA029273 and Supported for CAB034370 (HPA: subcellular; antibodies). |
| Situation | Likely cause | Next action |
|---|---|---|
| Placental cytotrophoblast nuclei have no visible signal (HPA: cytotrophoblasts High). | The run may have failed, or the chosen conditions may not yield detectable staining (general IHC practice). | Verify detection reagents and a run control; then check the catalog antibody's IHC-P instructions and the laboratory's retrieval settings. Do not infer SUPT5H fixation sensitivity from this result (general IHC practice; HPA: tissue IHC scope). |
| Cytoplasm or cell borders stain more strongly than nuclei (UniProt O00267: nucleus). | The distribution conflicts with SUPT5H's reported location and may reflect nonspecific binding or detection activity (UniProt O00267: location; general IHC practice). | Compare a no-primary control and inspect nuclear counterstain alignment; assess whether the color truly lies within nuclei before scoring (general IHC practice). |
| Weakly profiled cells stain as intensely as placental cytotrophoblasts (HPA: selected cells Low; cytotrophoblasts High). | Cross-reactivity or endogenous activity is possible, but HPA intensity levels do not define a negative cell population (HPA: tissue IHC; general IHC practice). | Check compartment and no-primary staining, and compare matched regions under the same detection conditions; report the discrepancy without declaring those cells SUPT5H-negative (HPA: ubiquitous nuclear profile; general IHC practice). |
| Brown or other chromogenic color spreads across tissue spaces and masks nuclei (HPA: expected nuclear profile). | Excess background may arise from detection activity, inadequate washing, or excessive chromogen development (general IHC practice). | Review the no-primary control, blocking and washes; adjust development within the detection system's instructions, then reassess nuclear contrast (general IHC practice). |
| Staining appears only in a few cell types despite an adequate run control (HPA: ubiquitous nuclear expression). | Technical loss of detectable signal is possible; HPA's Supported profile has only medium consistency with RNA expression (HPA: reliability; general IHC practice). | Compare expected nuclear populations in the same section and check antibody and detection conditions before treating the restricted pattern as biological (HPA: tissue IHC profile; general IHC practice). |
| A low-staining HPA tissue is proposed as a negative control (HPA: listed Low populations; negative list empty). | Low staining means weak observed signal, not established absence of SUPT5H (HPA: tissue IHC). | Use a no-primary control to assess detection background, and interpret low-staining populations as relative intensity comparisons only (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing SUPT5H staining
A03426-2 · IHCIHC analysis of SPT5/SUPT5H using anti-SPT5/SUPT5H antibody (A03426-2). SPT5/SUPT5H was detected in a paraffin-embedded section of human ova
A03426-2 · IHCIHC analysis of SPT5/SUPT5H using anti-SPT5/SUPT5H antibody (A03426-2). SPT5/SUPT5H was detected in a paraffin-embedded section of human thy
A03426-2 · IFIF analysis of SPT5/SUPT5H using anti-SPT5/SUPT5H antibody (A03426-2) and anti-Tubulin beta antibody (M05613-4). SPT5/SUPT5H was detected in
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000196235-SUPT5H/tissue/cerebral+cortex#imid_8226805
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000196235-SUPT5H/tissue/colon#imid_8241172
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000196235-SUPT5H/tissue/liver#imid_8241171
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000196235-SUPT5H/tissue/kidney#imid_8241154
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA029273; green = SUPT5H, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA029273; green = SUPT5H, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SUPT5H IHC & IF
🧪Run placenta first: cytotrophoblasts should show nuclear staining (HPA: High in placental cytotrophoblasts; UniProt O00267: Nucleus). HPA detects SUPT5H in all 45 scored tissues, so there is no validated negative tissue or negative cell population on the positive slide; use no-primary and isotype controls to define background, and assess cytoplasmic staining against that background (HPA: no negative tissue rows; standard IHC practice).
Positive control tissue: Placenta (Cytotrophoblasts, HPA High)
Negative control tissue: None in HPA: SUPT5H is detected in all 45 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 SUPT5H in A-431, U-251MG, U2OS, U2OS, siRNA 1 (10x), U2OS, siRNA 1 (40x), U2OS, siRNA 2 (10x), U2OS, siRNA 2 (40x), U2OS, scrambled (10x), U2OS, scrambled (40x), with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a secondary-only slide, a concentration-matched nonimmune rabbit IgG isotype control, and a matched SUPT5H-depleted or knockout specimen to test specificity (selected A03426-2 tissue-IHC caption: rabbit primary antibody; standard IHC practice). For placental chromogenic IHC, block endogenous peroxidase and inspect the controls for background staining (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A03426-2 tissue-IHC caption does not state a fixative. That paraffin-section example uses heat retrieval in EDTA at pH 8.0, so retrieval is a practical starting point, though its necessity has not been established (selected A03426-2 tissue-IHC caption). Frozen sections cannot be judged easier from the supplied evidence; IF has supported nucleoplasmic localization, but placental autofluorescence should be checked if that application is pursued (HPA: Nucleoplasm supported; standard IF practice).
HPA tissue IHC evidence for SUPT5H
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 → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: SUPT5H is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced SUPT5H IHC Tips
Troubleshoot SUPT5H staining in paraffin sections by prioritising nuclear signal, matched controls and the conditions documented for the IHC-validated antibody.
How should I adjust retrieval when SUPT5H nuclear staining is weak?
Start with heat-mediated antigen retrieval in EDTA pH 8.0 for paraffin sections (datasheet A03426-2). The selected image used 2 μg/ml primary antibody overnight at 4°C after retrieval, providing a reference while changing heat exposure (datasheet A03426-2). If nuclear signal is weak, verify deparaffinisation and complete immersion in hot buffer, then compare heating durations on serial sections (standard IHC practice). Try another buffer only as a fallback after optimising EDTA, comparing nuclear contrast, morphology and edge staining (standard IHC practice). SUPT5H is nuclear, with supported nucleoplasmic localisation (UniProt O00267; HPA subcellular).
Could fixation explain weak or uneven SUPT5H staining?
The selected paraffin-section caption does not report a fixative, so target-specific fixation sensitivity is unknown (datasheet A03426-2). Record the fixative, fixation duration and processing history for each specimen before attributing weak staining to SUPT5H biology (standard IHC practice). Compare similarly processed sections while holding EDTA pH 8.0 retrieval and 2 μg/ml overnight primary incubation constant (datasheet A03426-2). If nuclear staining varies alongside tissue morphology, examine processing and retrieval with matched control sections (standard IHC practice). Ubiquitous expression and reported modifications do not establish tolerance to any particular fixative (UniProt O00267; HPA tissue IHC).
What should I check if staining appears predominantly cytoplasmic?
Expect nuclear staining: UniProt places SUPT5H in the nucleus, and HPA supports nucleoplasmic localisation (UniProt O00267; HPA subcellular). Predominantly cytoplasmic DAB therefore warrants a review of primary concentration, secondary detection and the no-primary control before biological interpretation (standard IHC practice). The selected paraffin image used 2 μg/ml primary antibody, 10% goat serum block and DAB detection (datasheet A03426-2). Compare nuclei and cytoplasm within intact cells, avoiding damaged edges where staining can mislead (standard IHC practice). SUPT5H has no annotated transmembrane segment, which provides no basis for expecting membrane staining (UniProt O00267 topology).
Can isoforms or modifications account for inconsistent nuclear staining?
SUPT5H has 2 annotated isoforms and several modified residues, including phosphorylation and arginine methylation (UniProt O00267). Its annotated KOW domains span several regions, but the supplied IHC caption does not identify the antibody epitope (UniProt O00267; datasheet A03426-2). Obtain the immunogen or epitope information before claiming isoform coverage or modification sensitivity, then check it against both isoform sequences (standard antibody validation practice). Compare matched sections under EDTA pH 8.0 retrieval and the documented 2 μg/ml primary concentration (datasheet A03426-2). An intensity difference alone cannot establish an isoform switch or a phosphorylation change (standard IHC interpretation practice).
How can IF help assess a questionable SUPT5H IHC pattern?
Use IF as a separate localisation check: HPA supports nucleoplasmic SUPT5H staining in ICC/IF images (HPA subcellular). In multiplex images, pair SUPT5H with a marker for the expected cell type and a nuclear counterstain; placenta cytotrophoblasts have high tissue-IHC signal (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and assess tissue autofluorescence in each channel before interpreting overlap (standard IF practice). Because the target is nuclear and has no transmembrane segment, permeabilisation must permit antibody access to the nuclear epitope (UniProt O00267 topology; standard IF practice). Do not transfer the paraffin-section caption's unstated fixation conditions to IF (datasheet A03426-2).
How do I reduce diffuse brown background without losing nuclear signal?
First inspect a no-primary section for signal from the secondary detection system or endogenous peroxidase (standard chromogenic IHC practice). Include a peroxidase block before HRP detection and control DAB development time on matched sections (standard chromogenic IHC practice). The selected image used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and a 30-minute secondary incubation at 37°C (datasheet A03426-2). If diffuse colour persists, compare primary concentration and washing on serial sections while preserving identifiable nuclear signal (standard IHC practice). Score intact nuclei, since SUPT5H is nuclear (UniProt O00267).
How should I quantify SUPT5H across sections with different staining intensity? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and score nuclear DAB within that population, consistent with SUPT5H's supported nucleoplasmic location (HPA subcellular; standard IHC practice). Report the percentage of positive nuclei and an H-score from 0–300 using nuclear intensity categories, with thresholds fixed before comparison (standard IHC scoring practice). Normalise counts to all assessable nuclei in the same cell compartment; if reporting positive-cell density, state the sampled area in mm² (standard histology practice). Use matched processing and EDTA pH 8.0 retrieval when comparing specimens (datasheet A03426-2; standard IHC practice). Exclude necrosis, folds and damaged edges using consistent rules (standard histology practice).
How can I distinguish genuine SUPT5H staining from artefact?
A credible pattern is predominantly nuclear, consistent with UniProt's nuclear assignment and HPA's supported nucleoplasmic localisation (UniProt O00267; HPA subcellular). Compare the annotated cell population: HPA reports high cytotrophoblast staining and low staining in several listed cell types, while describing ubiquitous nuclear expression overall (HPA tissue IHC). Treat isolated cytoplasmic colour, tissue-edge accentuation and staining in necrotic regions as reasons to inspect controls and morphology (standard IHC practice). Check a no-primary section and peroxidase blocking when endogenous enzyme activity might produce DAB colour (standard chromogenic IHC practice). The HPA tissue-IHC profile has Supported reliability with medium RNA–protein consistency, so intensity alone is insufficient for a mechanistic claim (HPA tissue IHC).
Boster reagents
Best SUPT5H / Transcription elongation factor SPT5 IHC Antibodies
A03426-2 has paraffin-section IHC images from human tissue and an IF image from human cells (catalog image captions); M03426 lists IHC and IF applications with human, mouse and rat reactivity (catalog applications/reactivity).
Real IHC data
Anti-SPT5/SUPT5H Antibody ®
Cat # A03426-2
A03426-2 shows IHC in paraffin sections of human ovarian serous carcinoma, thyroiditis and tonsil, plus IF in A549 cells (catalog IHC/IF image captions). M03426 lists IHC and ICC/IF applications and human, mouse and rat reactivity; no IHC or IF image is supplied (catalog applications/reactivity; catalog image listings).
Which to pick: Choose A03426-2 for tissue IHC because its own images document paraffin sections and EDTA retrieval at pH 8.0; the fixative is unreported (A03426-2 IHC image captions). For IF/ICC, A03426-2 has an A549 cell image, while M03426 lists ICC/IF without an image (catalog IF image caption; catalog applications/image listings). Consider M03426 when rat reactivity matters because it lists rat and is monoclonal; establish performance in the intended specimen before use (catalog reactivity/clone; catalog image listings).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O00267 (SPT5H_HUMAN, Transcription elongation factor SPT5).
- Human Protein Atlas. SUPT5H tissue IHC expression (reliability: Supported).
- Human Protein Atlas. SUPT5H subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. SUPT5H antibody validation summary (2 antibodies).
- Suppressor of Ty homolog-5, a novel tumor-specific human telomerase reverse transcriptase promoter-binding protein and activator in colon cancer cells. Oncotarget 2015 — PMC4741733.
- PubMed PMID:8975720 — UniProt-cited evidence.
- PubMed PMID:9199507 — UniProt-cited evidence.
- PubMed PMID:9450929 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SUPT5H (Transcription elongation factor SPT5). Expected localisation Nuclear staining across tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03426-2). Validated IHC antibody: A03426-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Placenta (Cytotrophoblasts); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human ovarian serous carcinoma tissue; fixative not specified (datasheet A03426-2); 2. Fixation: Image fixative and duration unreported (datasheet A03426-2); 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: EDTA pH 8.0 (datasheet A03426-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03426-2); 8. Primary antibody: Rabbit anti-SUPT5H, 2-5 μg/ml (datasheet A03426-2); 9. Primary incubation: Overnight at 4 °C (datasheet A03426-2). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Antibody staining has medium consistency with RNA data (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope differences are unspecified (UniProt); Regulation: Ubiquitously expressed (UniProt). Evidence: UniProt O00267, HPA tissue IHC · Supported, HPA ICC-IF · nucleoplasm., Datasheet A03426-2, PMC4741733. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SUPT5H/immunohistochemistry-ihc-guide.