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EN1 / Homeobox protein engrailed-1 · IHC design guide
Design Immunohistochemistry for EN1
Plan EN1 IHC-P around the nuclear signal reported in eccrine sweat glands (HPA tissue IHC). The catalog antibody has a listed IHC-P dilution of 1:25 (datasheet A05395).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
EN1 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 eccrine sweat glands (HPA tissue IHC) | |
| Staining pattern | Distinct nuclear signal in eccrine sweat glands (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A05395) | |
| Positive control | Skin | |
| 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 | Sections lacking eccrine glands may miss the signal (HPA tissue IHC) | |
| Regulation | Expression regulation unannotated (UniProt) | |
| Isoform / epitope | No isoforms or processing annotated; nuclear target (UniProt) |
Section 1
Recommended EN1 IHC & IF Protocols
The catalog antibody uses citrate pH 6 retrieval (datasheet A05395). Published chromogenic IHC protocols cover salivary adenoid cystic carcinoma and breast TMA or cell-block sections (PMC3208084; PMC10829235; PMC5902237).
| Sample | FFPE human lung carcinoma tissue (datasheet A05395) |
| Fixation | Image formalin-fixed; duration unreported (datasheet A05395); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6 (datasheet A05395); 20 min, 95–100 °C (standard) |
| 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-EN1, 1:25 (datasheet A05395) |
| Primary incubation | 1 hours at 37°C (datasheet A05395) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | EN1-positive staining in secretory cells of skin (HPA tissue IHC: Medium). HPA tissue profile: Distinct nuclear expression in eccrine sweat glands. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 heat retrieval for the catalog antibody (datasheet A05395). Tris-EDTA retrieval is a published alternative for salivary gland tumour sections (PMC3208084).
Section 2
What Is the Expected EN1 Staining Pattern?
EN1 is a nuclear protein with no transmembrane segment (UniProt Q05925). In tissue IHC, expect distinct nuclear staining in eccrine sweat gland secretory cells in skin; HPA reports medium staining in those cells (HPA tissue IHC). HPA rates the tissue IHC evidence Enhanced, while noting medium consistency between antibody staining and RNA expression data (HPA tissue IHC).
| Distinct nuclear chromogen in eccrine sweat gland secretory cells, with little staining elsewhere on the section. | This matches the reported skin pattern and medium staining level (HPA tissue IHC). Score the stained cells and their nuclei, since a positive gland does not establish uniform EN1 staining across skin (HPA tissue IHC). |
| Strong cytoplasmic or membrane staining dominates while gland cell nuclei remain unstained. | The compartment conflicts with nuclear EN1 localization (UniProt Q05925) and the nuclear tissue pattern (HPA tissue IHC). Treat it as suspect; inspect controls and detection background before assigning it to EN1 (standard IHC practice). |
| Prominent staining appears in adipocytes or other cells outside the reported positive gland population. | HPA reports EN1 as not detected in adipocytes in adipose tissue and breast, while its stated positive skin population is eccrine secretory cells (HPA tissue IHC). Check for antibody cross-reactivity or endogenous detection activity (standard IHC practice). |
| Diffuse chromogen covers tissue, including areas without a distinct nuclear pattern. | Diffuse background cannot establish the reported cell-specific nuclear pattern (HPA tissue IHC). Compare with a control omitting primary antibody, then review blocking, washing and detection conditions (standard IHC practice). |
| No gland cell nuclear signal appears in a skin section containing identifiable eccrine sweat glands. | This misses the reported positive population, whose staining level is medium (HPA tissue IHC). Check that the glands are present and that staining and detection controls worked before interpreting the result as absent EN1 (standard IHC practice). |
💡Expected EN1 appearanceCall a skin section positive when eccrine sweat gland secretory cells show distinct, approximately medium nuclear chromogen (HPA tissue IHC); dominant cytoplasmic or widespread diffuse staining is a suspect result (UniProt Q05925; HPA tissue IHC).
| Tissue and cell selection | Skin eccrine secretory cells provide the reported positive tissue IHC pattern; adipocytes in adipose tissue and breast are reported as not detected (HPA tissue IHC). Select comparison areas by their identified cells, since these observations do not assign one staining status to every cell in an organ (HPA tissue IHC). |
| IHC evidence and RNA agreement | HPA labels tissue IHC reliability Enhanced but describes only medium consistency between antibody staining and RNA expression data (HPA tissue IHC). Its group-enriched RNA tissues include adipose tissue, brain, skeletal muscle and skin; that RNA classification does not predict a positive chromogenic result in each sampled cell type (HPA tissue IHC). |
| Protein location and processing | UniProt places EN1 in the nucleus, records no transmembrane segment, signal peptide or propeptide, and lists one chain spanning residues 1–392 (UniProt Q05925). These annotations support scoring nuclear signal; they do not specify an IHC epitope or an antigen retrieval condition (UniProt Q05925). |
| Antibody validation scope | HPA lists HPA073141 as Enhanced for IHC and CAB014884 as Supported for ICC, with no IHC status supplied for CAB014884 (HPA antibodies). Interpret each validation label within its stated assay; the ICC status alone does not validate chromogenic paraffin-section staining (HPA antibodies). |
| IF/ICC Q&A: should its fine nuclear pattern set the IHC scoring rule? | HPA ICC-IF reports a mainly nucleoli-rim signal (approved) plus nucleoplasm (supported), with images in A-431, U-251MG and U2OS (HPA subcellular). Tissue IHC reports distinct nuclear staining in eccrine secretory cells (HPA tissue IHC); assess the finer ICC-IF pattern within that assay. |
| Situation | Likely cause | Next action |
|---|---|---|
| Eccrine glands are visible, but their secretory cell nuclei show no chromogen. | The expected tissue-positive population has been missed (HPA tissue IHC); the slide alone cannot distinguish absent signal from a failed staining run (standard IHC practice). | Confirm gland morphology and compare a successfully stained positive control processed in the same run; then review primary antibody application, retrieval and detection steps (standard IHC practice). |
| Cytoplasmic staining is stronger than nuclear staining in gland cells. | The dominant compartment disagrees with nuclear EN1 and HPA's nuclear tissue pattern (UniProt Q05925; HPA tissue IHC); nonspecific primary or detection staining is possible (standard IHC practice). | Compare a primary-omission control and assess whether any distinct nuclear signal remains after background is accounted for; interpret the compartment before scoring intensity (standard IHC practice). |
| Adipocytes stain prominently in a comparison section. | Adipocytes are reported as not detected in adipose tissue and breast (HPA tissue IHC). Cross-reactivity or endogenous chromogenic activity could produce an unexpected signal (standard IHC practice). | Verify cell identity, check a primary-omission control and review the detection system's endogenous-activity blocking steps; do not treat the signal as confirmed EN1 from color alone (standard IHC practice). |
| Nearly every structure carries weak, hazy chromogen. | Broad haze obscures the distinct gland cell nuclear pattern reported for EN1 (HPA tissue IHC); excess detection background or inadequate washing are general possibilities (standard IHC practice). | Compare background controls, inspect wash and blocking steps, and use the catalog antibody's IHC-P instructions for antibody working conditions (standard IHC practice). |
| The result varies between sections or batches. | Section content or run conditions may differ (standard IHC practice); HPA's Enhanced rating still comes with only medium staining-to-RNA consistency (HPA tissue IHC). | Compare sections with identifiable eccrine glands and the same control tissue, then document nuclear intensity and the proportion of stained secretory cells separately (standard IHC practice). |
| Fine nucleoli-rim staining is absent from an otherwise nuclear-positive IHC section. | The finer nucleoli-rim assignment comes from ICC-IF, whereas the tissue IHC description is distinct nuclear expression in eccrine glands (HPA subcellular; HPA tissue IHC). | Judge the paraffin-section IHC result against its tissue pattern and controls; use the separate IF/ICC guide when evaluating the ICC-IF subnuclear pattern (HPA tissue IHC; HPA subcellular). |
Experiment evidence
IHC & IF images showing EN1 staining
A05395 · IHCFormalin-fixed and paraffin-embedded human lung carcinoma tissue reacted with EN1 antibody (N-term) (Cat. #A05395), which was peroxidase-con
A05395 · IHCA05395 staining EN1 in Human tonsil tissue sections by Immunohistochemistry (IHC-P -paraformaldehyde-fixed, paraffin-embedded sections). Tis
A05395 · IFImmunofluorescent analysis of 4% paraformaldehyde-fixed, 0. 1% Triton X-100 permeabilized U-2 OS (Human Sarcoma cell line) cells labeling HM
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000163064-EN1/tissue/cerebral+cortex#imid_19745755
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000163064-EN1/tissue/colon#imid_19743529
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000163064-EN1/tissue/liver#imid_19743522
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000163064-EN1/tissue/kidney#imid_19743523
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB014884; green = EN1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB014884; green = EN1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for EN1 IHC & IF
🧪Run skin first: secretory cells should show EN1 staining (HPA: Medium in skin secretory cells). Run adipose tissue as a negative comparator (HPA: Not detected in adipocytes); on the skin slide, neighboring nonsecretory cells used as internal negatives should lack specific nuclear staining, but their EN1 status must be checked against the negative controls (UniProt Q05925: nucleus; HPA reports the skin result for secretory cells only).
Positive control tissue: Skin (Secretory cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show EN1 in A-431, U-251MG, U2OS, with annotated localisation: Nucleoli rim (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and immunoglobulin class; use EN1 knockout material or peptide blocking, if available, to assess target specificity (standard IHC practice). For chromogenic skin sections, quench endogenous peroxidase and check pigment-related background (standard IHC practice).
⚠️Feasibility: The selected A05395 IHC caption explicitly describes formalin-fixed, paraffin-embedded tissue with peroxidase/DAB detection, but reports no target-specific fixation window (A05395 caption). EN1-specific retrieval requirements and any fixation effect are unreported in the supplied evidence; optimize retrieval for the catalog antibody (standard IHC practice). HPA shows ICC-IF images in A-431, U-251MG and U2OS, but the supplied evidence does not establish whether IF or frozen sections are easier than paraffin IHC (HPA subcellular).
HPA tissue IHC evidence for EN1
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 |
|---|---|---|---|---|
| Skin | Secretory cells | 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 → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced EN1 IHC Tips
Troubleshoot EN1 staining by checking retrieval, nuclear localisation, cell identity and controls before interpreting signal intensity.
How should I retrieve EN1 in paraffin sections with weak nuclear staining?
Start with heat-mediated citrate retrieval at pH 6 for paraffin sections (datasheet A05395). Keep heating and cooling conditions consistent between slides, and compare a short and longer retrieval within the instrument’s recommended range while monitoring tissue integrity (standard IHC practice). If nuclear staining remains weak, test a second retrieval condition only as a documented fallback, alongside the pH 6 reference slide (standard IHC practice). Score signal in nuclei of eccrine sweat-gland secretory cells, a documented positive population, rather than judging retrieval from diffuse staining elsewhere (HPA: medium staining in skin secretory cells; UniProt Q05925: nucleus).
Could fixation explain weak EN1 staining in my IHC sections?
EN1-specific sensitivity to fixation is unknown from the supplied evidence, so do not assign weak staining to a particular fixation duration or reagent (supplied evidence: no target-specific fixation comparison). Record fixation and processing conditions for every block, then compare sections processed together using identical retrieval and detection settings (standard IHC practice). Examine morphology and nuclear counterstain before increasing antibody exposure, since damaged sections can make a nuclear result difficult to assess (standard IHC practice; UniProt Q05925: nucleus). Include a documented positive cell population and a no-primary control in each comparison so changes in antigen access can be distinguished from detection background (HPA: skin secretory cells, medium; standard IHC practice).
Where should convincing EN1 chromogenic staining appear?
Prioritise nuclear DAB staining because EN1 is annotated as nuclear and has no transmembrane segment (UniProt Q05925: nucleus; no transmembrane segment). In tissue, eccrine sweat-gland secretory cells provide a documented positive population with medium staining (HPA: skin secretory cells, medium). The reported nucleoli-rim and additional nucleoplasmic pattern comes from subcellular imaging; assess whether those details are resolvable in your chromogenic section before using them as criteria (HPA subcellular: nucleoli rim approved; nucleoplasm supported; standard IHC practice). Compare the nuclear signal with the counterstain and cell boundaries, and treat diffuse cytoplasmic colour without a matching nuclear pattern as unconfirmed (standard IHC practice; UniProt Q05925: nucleus).
How should I assess an uncertain EN1 epitope in paraffin sections?
The selected antibody is described as N-terminal, while the supplied EN1 record lists one 1–392 chain and no annotated isoforms (caption A05395: N-term; UniProt Q05925: chain 1–392; zero isoforms). No epitope coordinates or fixation-dependent epitope data are supplied, so avoid attributing a negative section to a specific masked sequence (supplied evidence: no epitope coordinates or fixation comparison). Compare retrieval conditions on adjacent sections and keep detection settings matched when assessing epitope accessibility (standard IHC practice). Interpret staining by cell identity and nuclear compartment, and use an independent specificity control when available before assigning a new pattern to EN1 (HPA: skin secretory cells, medium; UniProt Q05925: nucleus; standard IHC practice).
How can IF help check the EN1 pattern seen by chromogenic IHC?
On a separately validated IF preparation, multiplex EN1 with a validated marker for eccrine secretory cells to test whether the chromogenic nuclear pattern occurs in the expected population (HPA: skin secretory cells, medium; standard IF practice). Select spectrally separated fluorophores and inspect unstained tissue in each channel before choosing a longer-wavelength channel where autofluorescence is lower (standard IF practice). Permeabilise sufficiently for antibody access to the nuclear epitope, since EN1 is nuclear and has no transmembrane segment; optimise detergent exposure against nuclear morphology (UniProt Q05925: nucleus; no transmembrane segment; standard IF practice). Compare the result with the reported nucleoli-rim and nucleoplasmic locations, without assuming IF settings transfer to chromogenic paraffin sections (HPA subcellular: nucleoli rim approved; nucleoplasm supported; standard IHC/IF practice).
What should I check when EN1 DAB staining spreads beyond nuclei?
First compare the slide with a no-primary control to identify staining contributed by the detection system (standard IHC practice). Include a peroxidase-blocking step before DAB detection and check whether residual colour follows blood-rich or damaged regions rather than cell nuclei (standard chromogenic IHC practice; UniProt Q05925: nucleus). If the control is clean, titrate the primary antibody and adjust blocking and wash conditions while keeping retrieval constant (standard IHC practice). Evaluate the resulting pattern against medium nuclear staining in eccrine secretory cells; widespread cytoplasmic colour or staining in control-negative cell populations needs additional validation (HPA: skin secretory cells, medium; HPA subcellular: nucleoli rim and nucleoplasm; standard IHC practice).
How should I score EN1 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue compartment and count only evaluable nuclei within that compartment before scoring (UniProt Q05925: nucleus; standard IHC practice). Report the percentage of positive nuclei and, when intensity is reproducible, an H-score calculated as the sum of each intensity category multiplied by its percentage, using 0–3 intensity categories (standard IHC practice). Normalise counts or positive-cell density to the number of evaluable cells or measured tissue area, and exclude folds, necrosis and poorly preserved regions using the same rules for every slide (standard IHC practice). Keep retrieval, DAB development and image-analysis thresholds consistent, and identify eccrine secretory cells separately when assessing the documented skin pattern (standard IHC practice; HPA: skin secretory cells, medium).
When is a positive EN1 IHC result credible?
A credible result shows staining in the expected nuclear compartment and can be reproduced in an appropriate positive population under matched conditions (UniProt Q05925: nucleus; HPA: skin secretory cells, medium; standard IHC practice). Eccrine secretory-cell staining supports that pattern, whereas the selected lung-carcinoma image demonstrates antibody use for IHC without establishing clinical relevance (HPA: distinct nuclear expression in eccrine sweat glands; caption A05395: lung carcinoma IHC, clinical relevance not evaluated). Examine edge staining, necrosis and colour retained in the no-primary control before calling a new positive population (standard chromogenic IHC practice). Treat isolated cytoplasmic DAB or signal confined to damaged areas as unconfirmed until morphology and independent controls support it (UniProt Q05925: nucleus; standard IHC practice).
Boster reagents
Best EN1 / Homeobox protein engrailed-1 IHC Antibodies
The IHC-validated anti-EN1 antibody has human paraffin-section images and human cell IF images (catalog images: A05395); its listed reactivity also includes mouse (catalog: A05395).
Real IHC data
Anti-EN1 (Engrailed 1) Antibody (N-term)
Cat # A05395
A05395 will render with an IHC figure showing formalin-fixed, paraffin-embedded human lung carcinoma tissue stained by DAB detection (A05395 IHC image caption). Its application list includes IHC-P and IF, and its IF images show human U-2 OS and U251 cells (catalog: A05395 applications; A05395 IF image captions).
Which to pick: Choose A05395 for tissue IHC: its own images show human lung carcinoma after formalin fixation and paraffin embedding, plus human tonsil sections after formaldehyde fixation and paraffin embedding (A05395 IHC image captions). Choose the same rabbit polyclonal antibody for IF/ICC because IF is listed and human U-2 OS and U251 cell images are supplied (catalog: A05395 host, clonality and applications; A05395 IF image captions). For cross-species work, A05395 lists human and mouse reactivity, although the supplied IHC and IF images show human samples (catalog: A05395 reactivity; A05395 IHC and IF image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q05925 (HME1_HUMAN, Homeobox protein engrailed-1).
- Human Protein Atlas. EN1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. EN1 subcellular location (ICC-IF): Mainly localized to the nucleoli rim. In addition localized to the nucleoplasm..
- Human Protein Atlas. EN1 antibody validation summary (2 antibodies).
- Developmental transcription factor EN1--a novel biomarker in human salivary gland adenoid cystic carcinoma. Cancer 2012 — PMC3208084.
- EN1 promotes lung metastasis of salivary adenoid cystic carcinoma by regulating the PI3K-AKT pathway and epithelial-mesenchymal transition. Cancer cell international 2024 — PMC10829235.
- Engrailed 1 overexpression as a potential prognostic marker in quintuple-negative breast cancer. Cancer biology & therapy 2018 — PMC5902237.
- Engrailed 1 mediates correct formation of limb innervation through two distinct mechanisms. PloS one 2015 — PMC4340014.
- PubMed PMID:1363401 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
- PubMed PMID:33568816 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for EN1 (Homeobox protein engrailed-1). Expected localisation Nuclear staining in eccrine sweat glands (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A05395). Validated IHC antibody: A05395 at 1:25 (the product's own IHC validation image is shown). Controls (HPA): positive Skin (Secretory cells); negative Adipose tissue. IHC-P workflow on FFPE sections. Protocol steps: 1. Sample: FFPE human lung carcinoma tissue (datasheet A05395); 2. Fixation: Image formalin-fixed; duration unreported (datasheet A05395); 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: Citrate pH 6 (datasheet A05395); 20 min, 95–100 °C (standard); 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-EN1, 1:25 (datasheet A05395); 9. Primary incubation: 1 hours at 37°C (datasheet A05395). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: Sections lacking eccrine glands may miss the signal (HPA tissue IHC); Isoform / epitope: No isoforms or processing annotated; nuclear target (UniProt); Regulation: Expression regulation unannotated (UniProt). Evidence: UniProt Q05925, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoli rim. In addition localized to the…, Datasheet A05395, PMC3208084, PMC10829235. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/EN1/immunohistochemistry-ihc-guide.