SUB1 / Activated RNA polymerase II transcriptional coactivator p15 · IHC design guide

Design Immunohistochemistry for SUB1

Plan chromogenic SUB1 IHC in paraffin sections around widespread nuclear staining (HPA tissue IHC). The IHC-validated antibody M02698-3 has a 2–5 μg/ml IHC-P range, with 2 μg/ml used overnight at 4°C in its tissue images (datasheet M02698-3).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
Immunohistochemistry protocol sheet for SUB1 (IHC for SUB1): expected localisation Nuclear staining across tissues (HPA tissue IHC), antibody M02698-3, validated IHC image, and IHC protocol steps
Printable SUB1 IHC protocol sheet — expected localisation Nuclear staining across tissues (HPA tissue IHC), antibody M02698-3, controls and protocol steps. Open the full SUB1 IHC guide →

SUB1 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 M02698-3)
Positive control ⓘ Appendix+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 consistent across sections (standard IHC practice; not target-specific)
Caveat Smooth muscle and fibroblasts show low staining (HPA tissue IHC)
Regulation Expression regulation not annotated (UniProt)
Isoform / epitope No isoforms; chain 2–127, no membrane sidedness (UniProt)
Section 1

Recommended SUB1 IHC & IF Protocols

The catalog antibody’s IHC-P protocol is paired with one published SUB1 tissue-staining protocol (datasheet M02698-3; PMC5140777 methods).

Recommended immunohistochemistry (IHC-P) protocol parameters
SampleParaffin-embedded human colorectal adenocarcinoma tissue; fixative not specified (datasheet M02698-3)
FixationImage fixative and duration unreported (datasheet M02698-3); verify before use.
Sectioning4–5 µm sections on charged slides (standard)
DeparaffinisationXylene, graded ethanol series to water (standard)
Antigen retrievalHeat retrieval: EDTA pH 8.0 (datasheet M02698-3); 20 min, 95–100 °C (standard)
Peroxidase block3% H2O2, 10 min, room temperature (standard)
Blocking10% goat serum (datasheet M02698-3)
Primary antibodyMouse monoclonal (clone 8D9D1) anti-SUB1, 2-5 μg/ml (datasheet M02698-3)
Primary incubationOvernight at 4 °C (datasheet M02698-3)
DetectionHRP-conjugated secondary, DAB chromogen (datasheet M02698-3)
CounterstainHematoxylin, blue, dehydrate and mount (standard)
Expected resultSUB1-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control.
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet M02698-3); the published protocol does not specify retrieval (PMC5140777 methods).
Section 2

What Is the Expected SUB1 Staining Pattern?

SUB1 should stain predominantly in nuclei across many cell types (UniProt P53999: nucleus; HPA tissue IHC: ubiquitous nuclear expression). Strong examples include colon glandular cells, liver hepatocytes and bone marrow hematopoietic cells (HPA tissue IHC: High). SUB1 has no transmembrane segment (UniProt P53999 topology). HPA rates the tissue IHC pattern Supported, with medium consistency between staining and RNA expression (HPA tissue IHC reliability).

What am I looking at on my slide?
Distinct nuclear chromogen in colon glandular cells, hepatocytes or bone marrow hematopoietic cells.This matches the reported compartment and High staining in those cells (UniProt P53999: nucleus; HPA tissue IHC: High). Judge intensity against adjacent cells on the same section; a darker nucleus alone does not establish a different cell type or a tissue-specific pattern (HPA tissue IHC: ubiquitous nuclear expression; low tissue specificity).
Dominant membrane or cytoplasmic staining, with little nuclear signal.That distribution conflicts with the expected nuclear pattern and should be treated as suspect (UniProt P53999: nucleus; HPA tissue IHC: ubiquitous nuclear expression). Check morphology and the staining controls before scoring it as SUB1; the record supplies no membrane localisation or transmembrane segment (UniProt P53999 topology).
A cell population stains strongly while expected nuclei nearby remain unstained.Consider cross-reactivity or endogenous detection activity, then check cell identity and controls (standard IHC practice). SUB1 has low tissue specificity, so staining in a different cell type is not, by itself, evidence of cross-reactivity (HPA tissue IHC: low tissue specificity).
Broad chromogen haze obscures nuclear borders or covers tissue spaces.The result cannot be scored confidently as nuclear SUB1 (HPA tissue IHC: ubiquitous nuclear expression). Background may arise from nonspecific binding or detection chemistry; inspect the reagent control and reduce background using standard IHC practice before interpreting cell-to-cell differences.
No nuclear staining in a well-preserved colon, liver or bone marrow section.Investigate assay performance before calling the sample SUB1 negative: HPA reports High staining in colon glandular cells, hepatocytes and hematopoietic cells (HPA tissue IHC: High). Confirm the relevant cells are present and review the positive control, antibody and detection run (standard IHC practice).
💡Expected SUB1 appearanceCall a result positive when chromogen is concentrated in identifiable nuclei, especially in HPA High cell populations; dominant extranuclear colour or featureless haze is suspect (UniProt P53999: nucleus; HPA tissue IHC: ubiquitous nuclear expression, High in listed cells).
How each factor affects the staining
Which tissue gives a useful positive reference?HPA reports High staining in appendix, breast, cervix and colon glandular cells; liver hepatocytes; bone marrow hematopoietic cells; cerebellar molecular-layer cells; and gallbladder glandular cells (HPA tissue IHC). Choose a section in which the named cell population is identifiable.
Can a low-staining cell population serve as a negative reference?HPA reports Low staining in ovarian follicle cells, smooth muscle cells, skeletal muscle myocytes and soft-tissue fibroblasts, and lists no negative tissue (HPA tissue IHC). Low means a weak signal may be expected; it does not make these populations validated negative controls.
How certain is the tissue pattern?The overall IHC profile is Supported, with medium consistency between staining and RNA expression (HPA tissue IHC reliability). HPA lists HPA001311 and CAB015351 as IHC Supported; neither is labelled IHC Enhanced in the supplied antibody record (HPA antibodies). Interpret unexpected patterns with this evidence limit in mind.
What should IF/ICC show?HPA reports supported nucleoplasmic and nucleolar localisation in ICC-IF and lists images from A-431, U-251MG and U2OS (HPA subcellular ICC-IF). This is a compartment reference for the separate IF/ICC guide; it does not establish how distinctly nucleoli will appear in chromogenic tissue IHC.
Do topology or processing predict an extracellular pattern?UniProt places SUB1 in the nucleus, annotates no transmembrane segment and lists no signal peptide or propeptide (UniProt P53999). Those annotations support a nuclear expectation; they supply no basis for interpreting surface staining as the expected SUB1 pattern.
Why is my staining missing, weak or wrong?
SituationLikely causeNext action
Positive-control tissue has no nuclear signal.The relevant HPA High cell population may be absent from the viewed area, or the IHC run may have failed (HPA tissue IHC: High; standard IHC practice).Locate the named cells, inspect section preservation and review the antibody, retrieval and detection records with the run controls (standard IHC practice). No SUB1-specific retrieval condition is supplied.
Signal is mainly cytoplasmic or membranous.The compartment disagrees with nuclear SUB1 and may reflect nonspecific staining or misread morphology (UniProt P53999: nucleus; HPA tissue IHC: ubiquitous nuclear expression).Compare with a clearly identifiable HPA High cell population and the reagent control; score SUB1 only where nuclear localisation is convincing (HPA tissue IHC: High; standard IHC practice).
Chromogen appears in many cells but nuclei cannot be distinguished.Diffuse background can obscure the HPA nuclear pattern (HPA tissue IHC: ubiquitous nuclear expression; standard IHC practice).Review blocking, antibody dilution, washes and detection background using the run controls, then reassess nuclear boundaries (standard IHC practice).
Unexpected cells stain much more strongly than nearby expected cells.Cell identification, cross-reactivity or endogenous detection activity may explain the contrast (standard IHC practice); low tissue specificity limits conclusions from cell type alone (HPA tissue IHC).Check morphology, reagent controls and a named HPA High population before assigning the contrast to SUB1 biology (HPA tissue IHC: High; standard IHC practice).
Smooth muscle or fibroblasts show weak nuclear colour.Weak staining can agree with HPA Low calls for smooth muscle cells and soft-tissue fibroblasts (HPA tissue IHC: Low).Compare nuclear localisation and intensity with an HPA High population on a comparable run; do not treat Low as an absolute negative (HPA tissue IHC: Low and High).
A repeat section gives a different intensity ranking.HPA reports only medium consistency between antibody staining and RNA expression, so small differences need cautious interpretation (HPA tissue IHC reliability).Compare the same cell populations under consistent staining and scoring conditions, and verify the positive and reagent controls before reporting a biological difference (standard IHC practice).

Sample controls for SUB1 IHC & IF

🧪Run appendix first and score its glandular cells, which have High SUB1 staining (HPA: Appendix, glandular cells, High). HPA detects SUB1 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and treat cytoplasm and areas without cells on the positive slide as background checks rather than assuming any cell population is SUB1-negative (HPA: no negative rows; UniProt P53999: nucleus).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: None in HPA: SUB1 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 SUB1 in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported), Nucleoli (supported) (HPA subcellular).
Technical controls: Include a secondary-only slide, a concentration- and isotype-matched mouse IgG control for the caption’s mouse primary, and identically processed SUB1-knockout material as a biological negative (selected-SKU caption: mouse primary; standard IHC practice). Block endogenous peroxidase and inspect leukocyte-rich areas for residual chromogen background in appendix before scoring glandular nuclei (standard chromogenic IHC practice; HPA: Appendix, glandular cells, High).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected M02698-3 paraffin-section caption does not state its fixative (selected-SKU tissue-IHC caption). The caption documents heat retrieval in EDTA at pH 8.0 and DAB detection, but does not establish that retrieval is required or that frozen sections are easier (selected-SKU tissue-IHC caption). IF/ICC may make nucleoplasm and nucleoli easier to distinguish, while appendix leukocytes warrant attention to peroxidase background in chromogenic IHC (HPA: supported nucleoplasm and nucleoli; standard IHC/IF practice).

HPA tissue IHC evidence for SUB1

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

TissueCell typeLevelEvidenceSource
Appendix Glandular cells High Protein (IHC) HPA →
Bone marrow Hematopoietic cells High Protein (IHC) HPA →
Breast Glandular cells High Protein (IHC) HPA →
Cerebellum Cells in molecular layer High Protein (IHC) HPA →
Cervix Glandular cells High Protein (IHC) HPA →

Undetected expression · recommended negative controls

TissueCell typeLevelEvidenceSource
None in HPA: SUB1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
Section 3

Advanced SUB1 IHC Tips

Troubleshoot SUB1 staining in paraffin sections by checking retrieval, nuclear localisation, controls, and cell-level scoring before interpreting differences in signal.

What should I change when SUB1 nuclear staining is weak after retrieval?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 (datasheet M02698-3). The selected antibody stained a paraffin section after that retrieval, but its caption does not specify heating time or temperature (datasheet M02698-3). If nuclear signal is weak, compare a modestly longer heating period with the original condition on adjacent sections while keeping antibody concentration and detection constant (general IHC practice). Check whether stronger nuclear staining comes with tissue damage or diffuse background, and include a section processed without primary antibody to identify retrieval-related background (general IHC practice).
Could fixation explain weak or patchy SUB1 staining?
SUB1-specific sensitivity to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (datasheet M02698-3). Record the fixative, duration, tissue thickness, and delay before fixation for every specimen before comparing staining intensity (general IHC practice). When staining is patchy, compare well-preserved regions and adjacent sections processed together, then inspect morphology for uneven preservation or processing (general IHC practice). Keep EDTA retrieval at pH 8.0 and the primary concentration at 2 μg/ml constant during that comparison, because both are documented for the selected antibody (datasheet M02698-3).
Where should convincing SUB1 staining appear in a tissue section?
Prioritise staining within nuclei: SUB1 is annotated as nuclear, and tissue IHC shows ubiquitous nuclear expression (UniProt P53999; HPA tissue IHC). Nucleoplasmic signal, with possible nucleolar enrichment, is consistent with the supported cellular locations reported by IF imaging (HPA subcellular). Compare the DAB pattern with a nuclear counterstain at the level of individual cells, especially where crowded nuclei can make signal appear cytoplasmic (general IHC practice). Predominantly extracellular or membrane-rim staining needs further control checks because SUB1 has no annotated transmembrane segment and no signal peptide (UniProt P53999 topology and processing).
How can epitope uncertainty affect interpretation of uneven SUB1 staining?
The supplied record lists 0 isoforms and no annotated domains, so it offers no isoform-specific explanation for divergent staining (UniProt P53999). It does list modified residues, including phosphoserines at positions 4, 9, 10, 11, and 13 (UniProt P53999). Without a mapped epitope for M02698-3, whether those modifications affect antibody binding remains unknown (datasheet M02698-3; UniProt P53999). If sections disagree, first standardise retrieval and detection, then compare an independently validated SUB1 reagent or an orthogonal assay before attributing the difference to modification or epitope masking (general IHC practice).
How should I check SUB1 localisation by multiplex IF alongside tissue IHC?
Choose a marker for the expected cell population, such as a validated marker for glandular cells when examining colon, where glandular SUB1 staining is reported as high (HPA tissue IHC). Assign SUB1 a far-red fluorophore if shorter wavelengths show tissue autofluorescence, and compare single-stain controls before interpreting overlap (general IF practice). Use a nuclear counterstain and assess nucleoplasmic or nucleolar signal, the supported SUB1 locations in cellular imaging (HPA subcellular). Because SUB1 is nuclear and has no transmembrane segment, check that permeabilisation permits antibody access to nuclear epitopes, while optimising IF conditions separately from the paraffin-section IHC method (UniProt P53999; general IF practice).
How do I reduce diffuse brown signal while preserving SUB1-positive nuclei?
First compare the stained section with a no-primary control and inspect whether brown signal follows cells, tissue edges, or pigment (general IHC practice). The selected paraffin-section assay used 10% goat serum block, 2 μg/ml primary antibody overnight at 4°C, and an HRP/DAB readout (datasheet M02698-3). If background persists, check peroxidase blocking, washing, secondary-antibody compatibility, and DAB development time one variable at a time (general IHC practice). Score improvement against crisp nuclear staining, since SUB1 is nuclear and tissue IHC reports a ubiquitous nuclear pattern (UniProt P53999; HPA tissue IHC).
How should I score SUB1 staining across sections with different cell mixtures? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and nuclear region before scoring, because tissue IHC describes ubiquitous nuclear SUB1 expression (HPA tissue IHC). Report the percentage of positive nuclei and an H-score based on nuclear intensity, using the same thresholds and imaging settings across sections (general IHC practice). If cell abundance varies, normalise positive-cell counts to all evaluable nuclei in the specified compartment; for spatial counts, report nuclei per mm² of viable tissue (general IHC practice). Exclude folds, damaged edges, and necrotic areas using a prespecified rule, and compare sections processed in the same staining run when possible (general IHC practice).
When is a SUB1-positive DAB pattern credible rather than artefactual?
A credible pattern places DAB in intact nuclei, consistent with the nuclear SUB1 annotation and ubiquitous nuclear tissue profile (UniProt P53999; HPA tissue IHC). Apparent positivity confined to edges, folds, or necrotic regions warrants repeat assessment in intact tissue and review of the no-primary control (general IHC practice). Check the stained cell population against tissue architecture: HPA reports high staining in colon glandular cells but low staining in smooth muscle cells (HPA tissue IHC). Cytoplasmic, extracellular, or broadly diffuse brown signal should prompt checks for counterstain alignment, endogenous peroxidase, and detection background before it is called SUB1 expression (UniProt P53999; general IHC practice).
Boster reagents

Best SUB1 / Activated RNA polymerase II transcriptional coactivator p15 IHC Antibodies

Four anti-SUB1 antibodies have human paraffin-section IHC images; three also have human IF images. The catalog lists human, mouse and rat reactivity for all four (catalog applications, reactivity and image captions).

Real IHC data IHC analysis of PC4/SUB1 using anti-PC4/SUB1 antibody (M02698-3). PC4/SUB1 was detected in a paraffin-embedded section of human colorectal adenocarcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml mouse anti-PC4/SUB1 Antibody (M02698-3) overnight at 4°C. Peroxidase Conjugated Goat Anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Mouse IgG Super Vision Assay Kit (Catalog # SV0001) with DAB as the chromogen.
Anti-PC4/SUB1 Antibody ® (monoclonal, 8D9D1)
Cat # M02698-3
Real IHC data IHC analysis of PC4/SUB1 using anti-PC4/SUB1 antibody (M02698-2). PC4/SUB1 was detected in a paraffin-embedded section of human colonic adenoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml mouse anti-PC4/SUB1 Antibody (M02698-2) overnight at 4°C. Peroxidase Conjugated Goat Anti-mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Mouse IgG Super Vision Assay Kit (Catalog # SV0001) with DAB as the chromogen.
Anti-PC4/SUB1 Antibody ® (monoclonal, 6B5B10)
Cat # M02698-2
Real IHC data IHC analysis of PC4/SUB1 using anti-PC4/SUB1 antibody (A02698-1). PC4/SUB1 was detected in a paraffin-embedded section of human bladder epithelial carcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-PC4/SUB1 Antibody (A02698-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.
Anti-PC4/SUB1 Antibody ®
Cat # A02698-1
Real IHC data IHC analysis of PC4/SUB1 using anti-PC4/SUB1 antibody (PB10098). PC4/SUB1 was detected in a paraffin-embedded section of human esophageal squamous carcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-PC4/SUB1 Antibody (PB10098) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.
Anti-PC4/SUB1 Antibody ®
Cat # PB10098

M02698-3 has human colorectal adenocarcinoma IHC and intestine cancer IF images (M02698-3 IHC/IF captions); M02698-2 has a human colonic adenoma IHC image (M02698-2 IHC caption). A02698-1 has human bladder epithelial carcinoma IHC and cervical carcinoma IF images (A02698-1 IHC/IF captions); PB10098 has human esophageal squamous carcinoma IHC and intestinal cancer IF images (PB10098 IHC/IF captions).

Which to pick: For tissue IHC, M02698-2 is a mouse monoclonal with a human paraffin-section DAB image after EDTA pH 8 retrieval; the fixative is unreported (M02698-2 catalog and IHC caption). For IF, M02698-3, A02698-1 and PB10098 each have a human paraffin-section fluorescence image; ICC validation is unreported (respective catalog applications and IF captions). For mouse or rat samples, all four list reactivity, but their supplied IHC images show human tissue; M02698-3 also lists IF (catalog reactivity/applications; respective IHC captions).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

  1. UniProt Consortium. UniProt entry P53999 (TCP4_HUMAN, Activated RNA polymerase II transcriptional coactivator p15).
  2. Human Protein Atlas. SUB1 tissue IHC expression (reliability: Supported).
  3. Human Protein Atlas. SUB1 subcellular location (ICC-IF): Localized to the nucleoplasm and nucleoli..
  4. Human Protein Atlas. SUB1 antibody validation summary (2 antibodies).
  5. MicroRNA-101 regulated transcriptional modulator SUB1 plays a role in prostate cancer. Oncogene 2016 — PMC5140777.
  6. Proteogenomic characterization of molecular and cellular targets for treatment-resistant subtypes in locally advanced cervical cancers. Molecular cancer 2025 — PMC11908047.
  7. PubMed PMID:8062392 — UniProt-cited evidence.
  8. PubMed PMID:8062391 — UniProt-cited evidence.
  9. PubMed PMID:15489334 — UniProt-cited evidence.