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ESRP1 / Epithelial splicing regulatory protein 1 · IHC design guide
Design Immunohistochemistry for ESRP1
Plan chromogenic ESRP1 IHC around the expected nuclear staining pattern (HPA tissue IHC). Start with the IHC-validated antibody A06068 at 2.5 μg/mL (datasheet A06068), using gallbladder glandular cells as a positive reference (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ESRP1 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 in several tissues (HPA tissue IHC) | |
| Staining pattern | Nuclear signal in epithelial and other tissue cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A06068-1) | |
| Positive control | Gallbladder+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Antibody staining has medium consistency with RNA data (HPA tissue IHC) | |
| Regulation | Epithelial cell-specific expression (UniProt) | |
| Isoform / epitope | 5 isoforms; confirm antibody epitope coverage (UniProt) |
Section 1
Recommended ESRP1 IHC & IF Protocols
The catalog antibody protocol and 3 published ESRP1 IHC protocols cover prostate and ovarian tissue sections (PMC7986212; PMC7749503; PMC5668885).
| Sample | Paraffin-embedded human bladder tissue; fixative not specified (datasheet A06068-1) |
| Fixation | Image fixative and duration unreported (datasheet A06068-1); 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 A06068-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A06068-1) |
| Primary antibody | Rabbit anti-ESRP1, 1:100 (datasheet A06068-1) |
| Primary incubation | Overnight at 4 °C (datasheet A06068-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A06068-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ESRP1-positive staining in glandular cells of gallbladder (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in several different tissues, including epithelial cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet A06068-1); the published citrate and Tris-EDTA conditions offer tissue-specific starting points (PMC7986212; PMC7749503).
Section 2
What Is the Expected ESRP1 Staining Pattern?
ESRP1 is a nuclear protein without a transmembrane segment (UniProt Q6NXG1: location and topology). In paraffin section IHC, expect staining mainly in epithelial nuclei; HPA reports nuclear expression across several tissues, including epithelial cells (HPA: tissue IHC profile). HPA rates its tissue IHC evidence Enhanced, with medium consistency between staining and RNA expression (HPA: reliability).
| Gallbladder glandular cells show strong nuclear chromogen, while surrounding cells remain comparatively pale. | This matches a useful positive pattern: HPA reports High staining in gallbladder glandular cells and describes ESRP1 expression as nuclear (HPA: gallbladder; tissue IHC profile). Judge intensity within identified cells, since a whole-section average can obscure a convincing nuclear result (general IHC practice). |
| Prominent cytoplasmic or membranous staining appears without a convincing nuclear component. | Treat this as discordant with UniProt’s nuclear location and HPA’s nuclear tissue pattern (UniProt Q6NXG1: location; HPA: tissue IHC profile). Consider nonspecific binding, detection artefact, or misread counterstain; compartment alone cannot identify the cause (general IHC practice). |
| Cells expected to be unstained show strong chromogen, especially alongside weakly stained target cells. | Compare cell identity with HPA observations: adipocytes and smooth muscle cells are listed as Not detected (HPA: adipose tissue; smooth muscle). Unexpected staining may reflect cross-reactivity or endogenous detection activity (general IHC practice). HPA observations are comparison points, not proof that every such cell must be negative. |
| The section has diffuse haze that obscures nuclei and tissue boundaries. | Background across multiple cell types is difficult to interpret as ESRP1-specific, because the expected tissue pattern is nuclear (HPA: tissue IHC profile). Review blocking, washes, chromogen development, and the no-primary control as general IHC checks (general IHC practice). |
| A gallbladder section has no convincing signal in glandular nuclei. | This conflicts with the reported High staining in gallbladder glandular cells (HPA: gallbladder). First verify tissue identity and control performance; then review retrieval, primary-antibody use, and detection as general IHC workflow checks (general IHC practice). A failed run does not establish biological absence. |
💡Expected ESRP1 appearanceCall the IHC result positive when glandular or other identified cells show distinct nuclear chromogen, preferably strong in gallbladder glandular cells (HPA: tissue IHC profile; gallbladder); predominantly cytoplasmic staining or widespread nuclear haze without cell-type contrast is suspect (UniProt Q6NXG1: location; general IHC practice).
| Cell and tissue context | Gallbladder glandular cells are High; adrenal, appendix, colon, duodenum, and epididymis glandular cells are Medium (HPA: tissue IHC). HPA also reports Medium staining in cerebellar Purkinje cells, so its observations should not be reduced to an absolute epithelial-only rule (HPA: cerebellum; UniProt Q6NXG1: tissue specificity). |
| Low and undetected comparison cells | HPA lists lung alveolar cells and pancreatic exocrine glandular cells as Low, while adipocytes, cholangiocytes, and smooth muscle cells are Not detected (HPA: tissue IHC). These are relative staining expectations within HPA’s data, not guaranteed universal negative controls (HPA: reliability). |
| Antibody-specific validation | HPA023719 carries Enhanced IHC validation; HPA023720 and HPA067104 carry Enhanced ICC validation, with no IHC status listed for either (HPA: antibody validation). Do not transfer an ICC validation label to paraffin section IHC (HPA: antibody validation). |
| Subnuclear IF/ICC appearance — Q: Where should signal lie? | A: HPA reports ESRP1 mainly in nuclear bodies, with additional nucleoplasmic signal in ICC-IF (HPA: subcellular). This informs localisation checks in IF/ICC but does not establish a required punctate chromogenic IHC appearance (HPA: subcellular; tissue IHC profile). |
| Molecular features | UniProt lists five ESRP1 isoforms, three RNA recognition motifs, and no signal peptide or transmembrane segment (UniProt Q6NXG1: isoforms, domains, processing, topology). With no antibody epitope supplied, these facts cannot predict which isoforms a given stain detects or justify a membrane pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| Gallbladder positive-control glands are blank. | The run may have failed, or the expected cells may be absent from the examined area; HPA reports High gallbladder glandular staining (HPA: gallbladder). | Confirm glandular tissue on the counterstain, then check primary-antibody application, retrieval, and detection controls using the assay’s established IHC workflow (general IHC practice). |
| Nuclei look weak in a tissue expected to stain at a Medium level. | HPA’s Medium category describes observed staining, not a guaranteed intensity for every section or assay (HPA: tissue IHC; reliability). | Compare the section with a concurrently processed gallbladder control and score identifiable nuclei rather than the section-wide color level (HPA: gallbladder; general IHC practice). |
| Most cells show brown cytoplasm but little nuclear signal. | The compartment conflicts with ESRP1’s reported nuclear location (UniProt Q6NXG1: location; HPA: tissue IHC profile). | Check the no-primary control and inspect whether precipitate, endogenous activity, or nonspecific binding explains the color before calling the result positive (general IHC practice). |
| A nominally negative cell population appears strongly stained. | Cross-reactivity or endogenous detection activity is possible; HPA lists adipocytes and smooth muscle cells as Not detected (HPA: tissue IHC; general IHC practice). | Verify cell identity and nuclear localisation, compare with adjacent tissue and a no-primary control, and report discordance rather than assuming target expression (general IHC practice). |
| Diffuse background makes epithelial nuclei hard to distinguish. | Broad color deposition does not match the reported nuclear tissue pattern and may arise during blocking, washing, or chromogen detection (HPA: tissue IHC profile; general IHC practice). | Review no-primary background, washing, blocking, and development time under the established assay conditions; reassess only when nuclear boundaries can be scored (general IHC practice). |
| An IF/ICC image shows nuclear puncta that differ from a smooth IHC nuclear stain. | HPA reports nuclear bodies plus nucleoplasm in ICC-IF, while its tissue IHC profile is described at the nuclear level (HPA: subcellular; tissue IHC profile). | Interpret each application against its own reported localisation and validation; do not require the ICC-IF subnuclear pattern in chromogenic paraffin section IHC (HPA: subcellular; antibody validation). |
Experiment evidence
IHC & IF images showing ESRP1 staining
A06068-1 · IHCIHC analysis of ESRP1 using anti-ESRP1 antibody (A06068-1). ESRP1 was detected in a paraffin-embedded section of human bladder tissue. Heat
A06068-1 · IHCIHC analysis of ESRP1 using anti-ESRP1 antibody (A06068-1). ESRP1 was detected in a paraffin-embedded section of human lung tissue. Heat med
A06068 · IHCImmunohistochemistry of RBM35A in human colon tissue with RBM35A antibody at 2.5 μg/mL.
A06068 · IFImmunofluorescence of RBM35A in human colon tissue cells with RBM35A antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000104413-ESRP1/tissue/cerebral+cortex#imid_6071240
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000104413-ESRP1/tissue/colon#imid_6063038
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000104413-ESRP1/tissue/liver#imid_6063039
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000104413-ESRP1/tissue/kidney#imid_6063040
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA023720; green = ESRP1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA023720; green = ESRP1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ESRP1 IHC & IF
🧪Run gallbladder first; its glandular cells should show nuclear ESRP1 staining (HPA: High in gallbladder glandular cells; UniProt: nucleus). Use smooth muscle as the negative tissue (HPA: not detected in smooth muscle cells); within the gallbladder slide, assess nonepithelial stromal nuclei as internal negative cells, consistent with epithelial cell-specific expression (UniProt: epithelial cell-specific).
Positive control tissue: Gallbladder (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ESRP1 in A-431, RT-4, U2OS, with annotated localisation: Nuclear bodies (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a concentration-matched rabbit IgG isotype control matched to the primary antibody’s clonality; and, if available, an ESRP1 knockout specimen or validated peptide-block control (caption: rabbit anti-ESRP1; standard IHC practice). Block endogenous peroxidase before HRP/DAB detection, and distinguish staining from luminal bile pigment in gallbladder sections (caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A06068-1 tissue-IHC caption does not state a fixative (selected SKU caption: fixative unreported). The demonstrated paraffin-section procedure used heat retrieval in EDTA at pH 8.0, followed by primary antibody at 1:100 overnight at 4°C; retrieval dependence has not been established by a comparison without retrieval (selected SKU caption). The supplied evidence does not establish whether frozen sections or IF are easier; ICC-IF images are available for A-431, RT-4 and U2OS (HPA: subcellular images), while luminal bile pigment warrants separate assessment when scoring gallbladder DAB staining (standard IHC practice).
HPA tissue IHC evidence for ESRP1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 |
|---|---|---|---|---|
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Salivary gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | Chondrocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ESRP1 IHC Tips
Troubleshoot ESRP1 chromogenic IHC by checking retrieval, nuclear localisation and cell type before comparing staining intensity across sections.
How should I retrieve ESRP1 in paraffin sections when nuclear staining is weak?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 (datasheet A06068-1). The selected paraffin-section image used this retrieval, followed by 10% goat serum block and antibody at 1:100 overnight at 4°C (datasheet A06068-1). If staining remains weak, compare a small set of retrieval heating times on adjacent sections while keeping detection and exposure to DAB constant (standard IHC practice). Judge improvement in intact epithelial nuclei, because ESRP1 is nuclear and epithelial cell-specific, rather than by stronger diffuse tissue colour (UniProt Q6NXG1). Include the same positive control in each run (standard IHC practice).
Could fixation explain loss of ESRP1 staining in my paraffin sections?
Target-specific ESRP1 sensitivity to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (datasheet A06068-1). Record the fixative, fixation duration and processing history for each specimen before attributing weak staining to antigen loss (standard IHC practice). Compare sections processed together using the documented EDTA pH 8.0 retrieval and 1:100 primary dilution (datasheet A06068-1). If only older or differently processed blocks fail, test matched controls before changing retrieval or antibody concentration (standard IHC practice). Nuclear epithelial staining in a concurrently processed control supports an interpretable run (UniProt Q6NXG1; standard IHC practice).
Should ESRP1 stain nuclei, cytoplasm or both in tissue IHC?
Expect nuclear staining: ESRP1 is annotated in the nucleus, and tissue IHC reports nuclear expression in several tissues, including epithelial cells (UniProt Q6NXG1; HPA tissue IHC). Nuclear bodies and nucleoplasm are reported in subcellular imaging, although chromogenic tissue sections may not resolve those patterns distinctly (HPA subcellular; standard IHC practice). Score nuclei within the relevant cell population and compare them with nearby cells on the same section (standard IHC practice). Predominantly cytoplasmic or uniform stromal colour warrants review of counterstain, DAB development and background controls before calling ESRP1 positive (UniProt Q6NXG1; standard IHC practice).
Can ESRP1 isoforms or epitope masking change the IHC pattern?
ESRP1 has 5 annotated isoforms and 3 RNA recognition motifs at residues 225–302, 326–406 and 445–525 (UniProt Q6NXG1). The supplied caption does not identify the antibody epitope, so its isoform coverage cannot be inferred from the staining image (datasheet A06068-1). If staining differs between specimens, first repeat the documented EDTA pH 8.0 retrieval with a matched positive control to assess processing effects (datasheet A06068-1; standard IHC practice). For an isoform-specific claim, obtain epitope mapping or independent isoform evidence; chromogenic nuclear staining alone cannot assign an ESRP1 isoform (UniProt Q6NXG1; standard IHC practice).
How can I check an ESRP1 IHC finding by multiplex IF?
Pair ESRP1 with a marker that identifies the epithelial cells of interest, since ESRP1 is described as epithelial cell-specific (UniProt Q6NXG1). Choose spectrally separated fluorophores after checking tissue autofluorescence, and include single-colour and no-primary controls when assessing overlap (standard IF practice). Permeabilise sufficiently to expose the nuclear epitope, then compare nuclear signal with a nuclear counterstain; ESRP1 is nuclear and has no annotated transmembrane segment (UniProt Q6NXG1; standard IF practice). Optimise IF fixation, permeabilisation and antibody concentration independently, because the supplied 1:100 overnight conditions describe paraffin-section chromogenic IHC, with fixation unreported (datasheet A06068-1).
What causes widespread brown staining that obscures ESRP1-positive nuclei?
First inspect a no-primary control for signal from the detection system and review endogenous peroxidase blocking before DAB development (standard chromogenic IHC practice). The selected paraffin-section image used a peroxidase-conjugated secondary and DAB, so those steps should be checked when diffuse brown colour appears (datasheet A06068-1). Compare the documented 10% goat serum block and 1:100 primary dilution with the conditions actually used, then assess washing and development time on matched sections (datasheet A06068-1; standard IHC practice). True ESRP1 interpretation should favour defined nuclear staining over diffuse background, consistent with its nuclear annotation (UniProt Q6NXG1).
How should I quantify ESRP1 staining across heterogeneous tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and analysis regions before scoring, then report the percentage of positive nuclei and a nuclear intensity score or H-score (standard IHC practice). Normalise counts to the number of evaluable nuclei in that population, or report positive-cell density per mm² when tissue area is the comparison unit (standard IHC practice). Exclude folds, damaged edges and necrotic regions using the same rules for every section (standard IHC practice). Keep retrieval at EDTA pH 8.0 and use a shared control across staining runs before comparing scores (datasheet A06068-1; standard IHC practice). Report cell type because HPA staining levels vary among annotated populations (HPA tissue IHC).
How can I distinguish genuine ESRP1 staining from artefact?
Look for staining in intact nuclei of the expected epithelial population, consistent with ESRP1 nuclear localisation and epithelial cell specificity (UniProt Q6NXG1). Gallbladder glandular cells are reported as high, whereas smooth muscle cells are reported as not detected, providing cell-level context for control selection (HPA tissue IHC). Treat staining confined to section edges, necrotic areas or the wrong compartment as suspect and inspect matched controls before assigning positivity (standard IHC practice; UniProt Q6NXG1). A no-primary control helps identify detection background, including endogenous enzyme signal in chromogenic workflows (standard IHC practice). HPA rates tissue-IHC reliability as enhanced but describes only medium consistency with RNA expression, so interpret discordant samples with independent evidence (HPA tissue IHC).
Boster reagents
Best ESRP1 / Epithelial splicing regulatory protein 1 IHC Antibodies
Anti-ESRP1 antibodies have IHC images from human colon and paraffin-embedded human bladder, lung, liver, and colon cancer sections; IF imagery covers human colon tissue cells (catalog image captions).
Real IHC data
Anti-ESRP1 Antibody
Cat # A06068-1
Real IHC data
Anti-RBM35A ESRP1 Antibody
Cat # A06068
A06068 has an IHC-P application listing and human colon IHC at 2.5 μg/mL, plus IF imaging of human colon tissue cells at 20 μg/mL (catalog applications; catalog image captions). A06068-1 has IHC images from paraffin-embedded human bladder, lung, liver, and colon cancer sections using 1:100, while its listed applications include IF and ICC, but not IHC-P (catalog image captions; catalog applications).
Which to pick: For tissue IHC, start with A06068 because IHC-P is listed and human colon IHC is shown (catalog applications; catalog image caption); A06068-1 has paraffin-section IHC images but no listed IHC-P application, and its fixative is unreported (catalog applications; catalog image captions). For IF/ICC, consider A06068-1 because both applications are listed and it is described as polyclonal (catalog applications; catalog dilution data); A06068 also has an IF image, but its clonality is unreported (catalog image caption; catalog clone field). For broader stated species reactivity, A06068 includes human, mouse, and rat, whereas A06068-1 includes human and mouse; the supplied IHC images show human samples only (catalog reactivity; catalog image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q6NXG1 (ESRP1_HUMAN, Epithelial splicing regulatory protein 1).
- Human Protein Atlas. ESRP1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ESRP1 subcellular location (ICC-IF): Mainly localized to the nuclear bodies. In addition localized to the nucleoplasm..
- Human Protein Atlas. ESRP1 antibody validation summary (3 antibodies).
- Prognostic role of TSPAN1, KIAA1324 and ESRP1 in prostate cancer. APMIS : acta pathologica, microbiologica, et immunologica Scandinavica 2021 — PMC7986212.
- Epithelial splicing regulatory protein 1 and 2 (ESRP1 and ESRP2) upregulation predicts poor prognosis in prostate cancer. BMC cancer 2020 — PMC7749503.
- The ESRP1 promoter reporter can function as an in vivo sensor of DNA methyltransferase inhibition. BMC biotechnology 2025 — PMC12382134.
- ESRP1 is overexpressed in ovarian cancer and promotes switching from mesenchymal to epithelial phenotype in ovarian cancer cells. Oncogenesis 2017 — PMC5668885.
- PubMed PMID:17974005 — UniProt-cited evidence.
- PubMed PMID:16421571 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ESRP1 (Epithelial splicing regulatory protein 1). Expected localisation Nuclear staining in several tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A06068-1). Validated IHC antibody: A06068-1 at 1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Gallbladder (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human bladder tissue; fixative not specified (datasheet A06068-1); 2. Fixation: Image fixative and duration unreported (datasheet A06068-1); 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 A06068-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A06068-1); 8. Primary antibody: Rabbit anti-ESRP1, 1:100 (datasheet A06068-1); 9. Primary incubation: Overnight at 4 °C (datasheet A06068-1). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Antibody staining has medium consistency with RNA data (HPA tissue IHC); Isoform / epitope: 5 isoforms; confirm antibody epitope coverage (UniProt); Regulation: Epithelial cell-specific expression (UniProt). Evidence: UniProt Q6NXG1, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nuclear bodies. In addition localized to the…, Datasheet A06068-1, PMC7986212, PMC7749503. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ESRP1/immunohistochemistry-ihc-guide.