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ESPL1 / Separin · IHC design guide
Design Immunohistochemistry for ESPL1
Plan ESPL1 chromogenic IHC around nuclear staining in several tissues (HPA tissue IHC). Seminal vesicle glandular cells show high staining, but low consistency between staining and RNA levels warrants verification (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ESPL1 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 tissue staining; cytoplasm annotated (HPA tissue IHC; UniProt) | |
| Staining pattern | Nuclear staining in several tissues (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Seminal vesicle+1 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA levels show low consistency (HPA tissue IHC) | |
| Regulation | Expression regulation unreported (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope effects undetermined (UniProt) |
Section 1
Recommended ESPL1 IHC & IF Protocols
The catalog antibody protocol is followed by published ESPL1 IHC methods for leiomyosarcoma (PMC11829311) and bladder cancer (PMC11133711).
| Sample | Paraffin-embedded human colon carcinoma tissue; fixative not specified (datasheet A04364) |
| Fixation | Image fixative and duration unreported (datasheet A04364); 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 anti-ESPL1, 1:100-1:300 (datasheet A04364) |
| 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 | ESPL1-positive staining in glandular cells of seminal vesicle (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 HIER at 95–98 °C for 20 min (page retrieval rule: nuclear antigen); the published methods used different retrieval solutions (PMC11829311; PMC11133711).
Section 2
What Is the Expected ESPL1 Staining Pattern?
ESPL1 is reported in the nucleus and cytoplasm (UniProt Q14674: subcellular location), while tissue IHC shows nuclear expression in several tissues (HPA: tissue IHC). Expect the clearest reported staining in seminal vesicle glandular cells and weaker staining in lung macrophages (HPA: High; Medium). ESPL1 has no transmembrane segment (UniProt Q14674: topology). Treat these patterns as provisional: HPA rates its tissue IHC Approved, with low agreement between staining and RNA data pending external verification (HPA: reliability).
| Distinct nuclear staining in seminal vesicle glandular cells, with stronger signal than nearby unstained cells (HPA: High). | This matches the strongest supplied tissue IHC observation and the reported nuclear tissue pattern (HPA: tissue IHC). Assess the named cell population rather than calling the entire section positive; HPA's Approved rating carries a low staining–RNA consistency caveat (HPA: reliability). |
| Nuclear staining in lung macrophages at a lower intensity than the seminal vesicle glandular signal (HPA: Medium; High). | This is compatible with the reported cell-specific tissue pattern (HPA: lung macrophages Medium). Check morphology and nuclear counterstain before assigning the stained cells; the HPA tissue record does not establish that every lung cell should stain (HPA: tissue IHC). |
| Predominantly membrane-bound or extracellular chromogen, without a convincing nuclear pattern. | This conflicts with the reported nuclear tissue IHC pattern and the absence of a transmembrane segment (HPA: tissue IHC; UniProt Q14674: topology). Review morphology and staining controls for artefact. Some cytoplasmic signal can be biologically compatible with UniProt's location annotation (UniProt Q14674: subcellular location). |
| Strong staining in an unexpected cell population, especially where the specified HPA cell type is unstained. | Consider antibody cross-reactivity or endogenous detection activity, then compare with a reagent-omission control (general IHC practice). HPA reports several cell-specific negative observations; these do not prove that every other cell in those tissues lacks ESPL1 (HPA: tissue IHC). |
| Diffuse chromogen across nuclei, cytoplasm and tissue edges, or no signal in seminal vesicle glandular cells. | Diffuse staining limits compartment scoring; inspect blocking, washing and detection controls (general IHC practice). An absent signal in a reported High cell population warrants a run-control review, but HPA's low staining–RNA consistency prevents treating one negative section as decisive (HPA: High; reliability). |
💡Expected ESPL1 appearanceCall positive when seminal vesicle glandular cells show clear nuclear chromogen at the reported High level, or lung macrophages show the reported Medium pattern (HPA: tissue IHC); broad membrane, extracellular or cell-independent deposit is suspect (HPA: nuclear tissue pattern; UniProt Q14674: topology; general IHC practice).
| Compartment and detection method | Tissue IHC is described as nuclear, while UniProt also lists cytoplasm (HPA: tissue IHC; UniProt Q14674: subcellular location). Score a nuclear pattern first; do not automatically reject a limited cytoplasmic component solely from the location record. |
| Cell population and tissue context | Seminal vesicle glandular cells are High and lung macrophages Medium; adipocytes and several other specified cell populations are Not detected (HPA: tissue IHC). These observations support cell-level comparisons, not a whole-organ positive or negative designation. |
| Evidence confidence | HPA labels tissue IHC Approved yet notes low consistency with RNA expression and pending external verification (HPA: reliability). RNA is tissue enhanced in bone marrow, esophagus and lymphoid tissue, while the listed bone marrow hematopoietic and esophageal squamous cells are Not detected by IHC (HPA: RNA specificity; tissue IHC). |
| Protein forms and antibody interpretation | UniProt lists two isoforms and one annotated 1–2120 Separin chain, with no signal peptide or propeptide (UniProt Q14674: isoforms; processing). Without an epitope map in the supplied record, staining cannot be assigned to one isoform or to a specific processed form. |
| IF/ICC Q&A: where should fluorescence appear? | HPA reports mainly nucleoplasmic signal, with additional vesicle localization, in its ICC-IF subcellular record (HPA: nucleoplasm supported; vesicles approved). This provides an IF localization comparison; the supplied record provides no IF/ICC protocol option. |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in seminal vesicle glandular cells expected to be High (HPA: tissue IHC). | The run may have weak detection, or the sampled section may differ from the HPA observation (general IHC practice; HPA: reliability caveat). | Check section identity, nuclear counterstain and a working positive run control; review the antibody's IHC validation before changing conditions (general IHC practice; HPA: antibody IHC Approved). |
| Lung section appears negative despite a reported Medium macrophage signal (HPA: tissue IHC). | Macrophages may be sparse in the examined field, and Medium is a cell-specific observation (HPA: lung macrophages Medium). | Find morphologically identifiable macrophages and score them separately from other lung cells; compare a suitable control section within the same run (HPA: tissue IHC; general IHC practice). |
| Bone marrow or esophagus stains strongly because its RNA is tissue enhanced (HPA: RNA specificity). | RNA enrichment does not predict staining in every cell: listed bone marrow hematopoietic and esophageal squamous cells are Not detected by IHC (HPA: tissue IHC). | Identify the stained cell type, inspect detection controls and avoid using tissue-level RNA alone to validate the IHC signal (HPA: tissue IHC; general IHC practice). |
| Chromogen is widespread across unrelated structures or follows tissue edges. | Nonspecific reagent binding, endogenous detection activity or uneven processing can produce background (general IHC practice). | Inspect reagent-omission controls, blocking, wash steps and chromogen development; score ESPL1 only where cell morphology and compartment are interpretable (general IHC practice; HPA: nuclear tissue pattern). |
| Signal is chiefly membranous or extracellular, with little nuclear staining. | That distribution conflicts with the observed nuclear tissue pattern and lacks support from ESPL1 topology (HPA: tissue IHC; UniProt Q14674: no transmembrane segment). | Check the counterstain and section morphology, then compare controls and the expected glandular or macrophage cell populations before calling the stain specific (HPA: tissue IHC; general IHC practice). |
| A positive call depends on faint cytoplasmic staining alone. | UniProt includes cytoplasm, but HPA describes tissue IHC as nuclear; the supplied records do not establish a cytoplasmic IHC scoring threshold (UniProt Q14674: subcellular location; HPA: tissue IHC). | Record the compartment separately, seek a convincing nuclear pattern in the reported cell types, and qualify any cytoplasm-only interpretation (HPA: tissue IHC; reliability caveat). |
Experiment evidence
IHC & IF images showing ESPL1 staining
A04364 · IHCImmunohistochemistry analysis of paraffin-embedded human colon carcinoma tissue, using SEPARASE Antibody. The picture on the right is blocke
A04364 · IFImmunofluorescence analysis of HUVEC cells, using SEPARASE Antibody. The picture on the right is blocked with the synthesized peptide.
A04364-1 · IHCImmunohistochemistry (IHC) analyzes of Separase (A795) pAb in paraffin-embedded human breast carcinoma tissue at 1:100.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000135476-ESPL1/tissue/cerebral+cortex#imid_20575363
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000135476-ESPL1/tissue/colon#imid_20575305
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000135476-ESPL1/tissue/liver#imid_20575306
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000135476-ESPL1/tissue/kidney#imid_20575299
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB012185; green = ESPL1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB012185; green = ESPL1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ESPL1 IHC & IF
🧪Run seminal vesicle first; its glandular cells should stain (HPA: High in seminal-vesicle glandular cells). Run adipose tissue as the negative comparator (HPA: Not detected in adipocytes); on the positive slide, use morphologically distinct cells without specific staining as an internal background reference, without assuming every nonglandular cell is ESPL1-negative.
Positive control tissue: Seminal vesicle (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ESPL1 in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host- and immunoglobulin-matched isotype controls; for a polyclonal primary, use matched nonimmune IgG. Include an ESPL1 knockout specimen or a peptide-block control (selected SKU A04364 caption: peptide block shown). Quench endogenous peroxidase for chromogenic detection and check seminal-vesicle luminal material for background staining (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and antigen-retrieval dependence are unreported in the supplied evidence; the paraffin-section caption does not state a fixative (selected SKU A04364 caption: fixative unreported). The evidence does not establish that frozen sections or IF are easier; ICC-IF offers a separate localisation check (HPA: nucleoplasm supported; vesicles approved). Score seminal-vesicle glandular cells separately from luminal material to avoid counting background as cellular staining (standard IHC scoring practice).
HPA tissue IHC evidence for ESPL1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data. 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 |
|---|---|---|---|---|
| Seminal vesicle | Glandular cells | High | Protein (IHC) | HPA → |
| Lung | Macrophages | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ESPL1 IHC Tips
Troubleshoot chromogenic ESPL1 staining in paraffin sections by checking retrieval, compartment, controls and scoring before interpreting tissue differences.
What retrieval should I start with for weak ESPL1 staining in paraffin sections?
Start with Tris-EDTA, pH 9.0, heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval rule: nuclear antigen). Keep section thickness, cooling time and detection conditions constant while comparing retrieval runs, so changes in nuclear signal can be attributed to retrieval (standard IHC practice; HPA: nuclear expression in several tissues). Include a known positive section and a no-primary control in each run to distinguish improved antigen exposure from stronger background (standard IHC practice). If staining remains weak, test a milder retrieval condition as a fallback and assess tissue damage alongside signal; the selected A04364 caption reports paraffin sections but no retrieval method (selected A04364 caption).
How should I troubleshoot fixation-related loss of ESPL1 staining?
Target-specific fixation sensitivity for ESPL1 is unknown from the supplied evidence, so treat fixation as an experimental variable rather than an established cause of weak staining (selected A04364 caption: fixative unreported). The A04364 image documents staining in paraffin-embedded human colon carcinoma and peptide blocking, but does not identify the fixative or fixation duration (selected A04364 caption). Compare sections with documented processing histories using the same Tris-EDTA pH 9.0 retrieval, 20 min heating and detection run (page retrieval rule; standard IHC practice). Record morphology and nuclear signal together, because poorly preserved tissue can make compartment-level interpretation unreliable (standard IHC practice; HPA: nuclear expression in several tissues).
Should ESPL1 staining be nuclear, cytoplasmic, or both?
Assess nuclear staining first: tissue IHC reports nuclear expression in several tissues, while subcellular IF supports nucleoplasmic localisation (HPA: tissue IHC; HPA: subcellular). Cytoplasmic staining can be biologically plausible because ESPL1 is annotated in both cytoplasm and nucleus, but diffuse chromogen alone is insufficient to establish specificity (UniProt Q14674: subcellular location; standard IHC practice). Score nuclear and cytoplasmic compartments separately, using a hematoxylin counterstain to identify nuclei and preserve cell-level context (standard IHC practice). Compare the compartment pattern with the peptide-blocked A04364 image and a no-primary control; the caption documents peptide blocking but does not specify which compartment was blocked (selected A04364 caption; standard IHC practice).
Could isoforms or epitope accessibility explain discordant ESPL1 staining?
ESPL1 has 2 annotated isoforms, so determine which sequence the antibody recognises before interpreting a negative section as absence of the protein (UniProt Q14674: isoforms). The supplied caption does not map the A04364 epitope or establish isoform coverage; peptide blocking supports recognition of the immunising peptide under that image condition (selected A04364 caption). ESPL1 has no annotated transmembrane segment and has a Peptidase C50 domain at residues 1945–2040, but these annotations do not locate the antibody epitope (UniProt Q14674: topology and domains). Document retrieval and compartment-specific staining consistently across samples, then seek epitope documentation before making isoform-specific claims (standard IHC practice; UniProt Q14674: isoforms).
How can IF help resolve an ambiguous chromogenic ESPL1 pattern?
Use IF/ICC as a separate validation experiment, since the selected A04364 evidence describes paraffin-section IHC rather than IF performance (selected A04364 caption). Multiplex ESPL1 with an independently validated marker for the cell type being examined, and choose spectrally separated fluorophores after checking the tissue’s autofluorescence in unstained controls (standard IF practice). ESPL1 lacks a transmembrane segment and is annotated in nucleus and cytoplasm, so use permeabilisation appropriate for access to an intracellular epitope, then confirm its location from antibody documentation (UniProt Q14674: topology and localisation; standard IF practice). Compare nuclear and cytoplasmic signal with single-stain and no-primary controls; HPA reports supported nucleoplasmic localisation in its subcellular IF data (HPA: subcellular; standard IF practice).
What should I check when ESPL1 chromogen obscures nuclear signal?
First compare the stained section with a no-primary control to identify detection-system background before changing the IHC-validated antibody concentration (standard IHC practice). For peroxidase-based chromogenic detection, check the peroxidase block and inspect whether pigment or endogenous enzyme activity contributes to the apparent DAB signal (standard IHC practice). Titrate the primary antibody and detection conditions while holding Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min constant (page retrieval rule; standard IHC practice). Judge improvements by clearer nuclear boundaries and preserved morphology, because HPA reports nuclear tissue expression and also cautions that its tissue staining has low consistency with RNA data (HPA: tissue IHC).
How should I quantify ESPL1 IHC across sections with different cellularity? ⚠ ANSWER MARKED FOR VERIFICATION
Define the analysed cell population and score nuclear staining separately from cytoplasmic staining before comparing sections (standard IHC practice; HPA: nuclear tissue expression; UniProt Q14674: cytoplasm and nucleus). For nuclear signal, report the percentage of positive eligible cells and an H-score based on intensity categories; keep thresholds and imaging settings fixed across the comparison (standard IHC practice). Normalise counts to the number of eligible cells, or report positive-cell density per mm² of viable analysed tissue when spatial density is the question (standard IHC practice). Exclude folds and necrotic areas using a predefined rule, and report the denominator so tissue composition does not masquerade as an ESPL1 change (standard IHC practice).
How do I distinguish true ESPL1 positivity from staining artefact?
Give greatest weight to reproducible cellular staining with a nuclear component, because HPA reports nuclear tissue expression and supported nucleoplasmic localisation; cytoplasmic signal also merits evaluation because UniProt annotates that compartment (HPA: tissue IHC and subcellular; UniProt Q14674: localisation). Check that the signal belongs to the cells being scored, then compare it with a no-primary control and the peptide-blocked A04364 image where relevant (standard IHC practice; selected A04364 caption). Treat section-edge accentuation, necrotic staining and peroxidase-associated DAB deposits as potential artefacts requiring control review (standard IHC practice). Interpret tissue contrasts cautiously: HPA labels its tissue IHC Approved but reports low consistency with RNA expression and pending external verification (HPA: tissue IHC reliability).
Boster reagents
Best ESPL1 / Separin IHC Antibodies
Two anti-ESPL1 antibodies have human paraffin-section IHC images (catalog IHC captions). A04364 also has a HUVEC IF image (catalog IF caption); listed reactivity covers human, mouse, and, for A04364-1, rat (catalog reactivity).
Real IHC data
Anti-Separase Antibody
Cat # A04364
Real IHC data
Anti-Separase (A795) ESPL1 Antibody
Cat # A04364-1
A04364 is listed for IHC and IF in human and mouse, with a paraffin-embedded human colon carcinoma IHC image and a HUVEC IF image (catalog applications/reactivity; A04364 image captions). A04364-1 is listed for IHC and IF in human, mouse, and rat, with a paraffin-embedded human breast carcinoma IHC image (catalog applications/reactivity; A04364-1 IHC caption).
Which to pick: For tissue IHC, choose A04364 for its peptide-blocked colon carcinoma image or A04364-1 for its breast carcinoma image; both captions specify paraffin embedding, and neither reports the fixative (catalog IHC captions). For cell IF, A04364 has a HUVEC image with peptide blocking; A04364-1 lists IF but has no IF image in the payload (catalog IF captions; catalog applications). For rat samples, A04364-1 lists rat reactivity, while A04364 lists human and mouse; A04364-1’s IHC caption calls it a pAb, while clonality is unreported for A04364 (catalog reactivity; A04364-1 IHC caption; catalog clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q14674 (ESPL1_HUMAN, Separin).
- Human Protein Atlas. ESPL1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ESPL1 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to vesicles..
- Human Protein Atlas. ESPL1 antibody validation summary (2 antibodies).
- ESPL1 Is a Novel Prognostic Biomarker Associated With the Malignant Features of Glioma. Frontiers in genetics 2021 — PMC8428966.
- E2F1-mediated ESPL1 transcriptional activation predicts poor prognosis and promotes the proliferation of leiomyosarcoma. CytoJournal 2025 — PMC11829311.
- The upregulation and transcriptional regulatory mechanisms of Extra spindle pole bodies like 1 in bladder cancer: An immunohistochemistry and high-throughput screening Evaluation. Heliyon 2024 — PMC11133711.
- Genome-wide CRISPR screen identifies ESPL1 limits the response of gastric cancer cells to apatinib. Cancer cell international 2024 — PMC10893712.
- PubMed PMID:12194817 — UniProt-cited evidence.
- PubMed PMID:8724849 — UniProt-cited evidence.
- PubMed PMID:16541075 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ESPL1 (Separin). Expected localisation Nuclear tissue staining; cytoplasm annotated (HPA tissue IHC; UniProt). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A04364 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Seminal vesicle (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon carcinoma tissue; fixative not specified (datasheet A04364); 2. Fixation: Image fixative and duration unreported (datasheet A04364); 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 anti-ESPL1, 1:100-1:300 (datasheet A04364); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining and RNA levels show low consistency (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope effects undetermined (UniProt); Regulation: Expression regulation unreported (UniProt). Evidence: UniProt Q14674, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to…, Datasheet A04364, PMC11829311, PMC11133711. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ESPL1/immunohistochemistry-ihc-guide.