This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
EHD1 / EH domain-containing protein 1 · IHC design guide
Design Immunohistochemistry for EHD1
Plan EHD1 paraffin IHC around cytoplasmic staining in endothelial, trophoblastic and some germinal center cells (HPA tissue IHC). This guide uses the catalog antibody's 0.5–1 μg/mL range (datasheet A02168-1) and flags the HPA cross-gene specificity caution (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
EHD1 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 | Cytoplasm in tissue (HPA tissue IHC); endosomal membranes (UniProt) | |
| Staining pattern | Cytoplasmic in endothelial, trophoblastic and some germinal center cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A02168-1) | |
| Positive control | Kidney+4 more · see all | |
| Negative control | Adrenal gland+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A02168-1) | |
| Caveat | HPA antibody may detect proteins from more than one gene (HPA tissue IHC) | |
| Regulation | Highly expressed in testis (UniProt) | |
| Isoform / epitope | No annotated isoforms; one 1–534 chain (UniProt) |
Section 1
Recommended EHD1 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A02168-1) is accompanied by four published EHD1 IHC protocols (PMC4565848; PMC5410234; PMC6460645; PMC2856533).
| Sample | Paraffin-embedded human testis cancer tissues; fixative not specified (datasheet A02168-1) |
| Fixation | Image fixative and duration unreported (datasheet A02168-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet A02168-1) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A02168-1) |
| Primary antibody | Rabbit anti-EHD1, 0.5-1μg/ml (datasheet A02168-1) |
| Primary incubation | Overnight at 4 °C (datasheet A02168-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A02168-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | EHD1-positive staining in cells in glomeruli of kidney (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in endothelial, trophoblastic and a subset of germinal center cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate retrieval at pH 6 (datasheet: A02168-1); the three human tissue protocols also specify citrate at pH 6 (PMC4565848; PMC5410234; PMC6460645).
Section 2
What Is the Expected EHD1 Staining Pattern?
EHD1 associates with recycling and early endosome membranes, the cell membrane and ciliary membrane; it has no transmembrane segment (UniProt Q9H4M9). In paraffin-section IHC, expect cytoplasmic staining in endothelial, trophoblastic and some germinal center cells (HPA tissue IHC). Treat the pattern as provisional: HPA rates the tissue staining Approved but cautions that the antibodies target protein from more than one gene and await external verification (HPA tissue IHC).
| Cytoplasmic staining in endothelial or trophoblastic cells, or a subset of germinal center cells (HPA tissue IHC). | This matches the reported tissue pattern (HPA tissue IHC). Assess the named cell population within its tissue; a positive field alone cannot identify which cells carry the signal (standard IHC interpretation). |
| Predominantly nuclear staining, with little signal in the expected cytoplasmic compartment. | This conflicts with the reported tissue pattern and membrane associations (HPA tissue IHC; UniProt Q9H4M9). Review morphology and controls before scoring it as EHD1; HPA's cross-gene antibody caution leaves specificity unresolved (HPA tissue IHC). |
| Strong staining across cell types reported as undetected, such as adrenal glandular cells or bone marrow hematopoietic cells (HPA tissue IHC). | Consider antibody cross-reactivity or endogenous chromogenic activity, then check controls (HPA tissue IHC; standard IHC practice). Bone marrow is RNA-enhanced yet its hematopoietic cells are IHC-undetected; keep those readouts distinct (HPA tissue IHC). |
| Diffuse color obscures cell boundaries or appears in the no-primary control. | The slide cannot support compartment or cell-type scoring; nonspecific reagent binding, endogenous enzyme activity or detection background are possible causes (standard IHC practice). Compare controls before interpreting a weak cytoplasmic signal. |
| No staining in kidney glomerular cells, reported as High by HPA (HPA tissue IHC). | First suspect an assay or tissue-quality problem; check the positive-control section and detection run (standard IHC practice). HPA's Approved rating and cross-gene caution mean its positive pattern alone does not establish EHD1-specific detection (HPA tissue IHC). |
💡Expected EHD1 appearanceCall positive IHC when staining is cytoplasmic in the expected cells, with a High glomerular-cell reference or Medium trophoblastic or germinal-center reference; widespread nuclear or cell-type-independent color is suspect (HPA tissue IHC; standard IHC interpretation).
| Compartment and cell identity | UniProt places EHD1 at endosomal, cell and ciliary membranes; HPA tissue IHC reports cytoplasmic staining in selected cells. Score the visible tissue pattern alongside cell morphology rather than demanding that chromogenic IHC resolve individual endosomes (UniProt Q9H4M9; HPA tissue IHC; standard IHC interpretation). |
| Antibody evidence | HPA049890 and HPA049986 each have Approved IHC status, while the tissue profile warns that the antibodies target protein from more than one gene and await external verification (HPA antibodies; HPA tissue IHC). A plausible pattern therefore remains a provisional identification. |
| Epitope and retrieval choice | UniProt lists a dynamin-type G domain, EH domain and EF-hand, but these records do not map the IHC antibody epitopes or establish EHD1-specific retrieval sensitivity (UniProt Q9H4M9; HPA antibodies). Optimize retrieval against tissue controls as a general IHC step (standard IHC practice). |
| IF/ICC Q&A | What should IF/ICC show? HPA reports mainly plasma membrane staining, with additional primary-cilium and transition-zone localization; this is a separate ICC-IF observation, not an IHC-P protocol choice (HPA subcellular). |
| Situation | Likely cause | Next action |
|---|---|---|
| Kidney glomerular cells lack signal despite their High HPA reference (HPA tissue IHC). | The section, retrieval, primary-antibody step or detection run may have failed (standard IHC practice). | Check tissue integrity and a concurrently processed positive control; review retrieval and detection conditions one at a time (standard IHC practice). Avoid calling the specimen negative until the control stains. |
| Color appears in the no-primary control. | Endogenous detection activity or nonspecific detection-reagent binding can produce chromogenic signal (standard IHC practice). | Address endogenous enzyme activity with the assay's blocking step, check the detection reagents and repeat the no-primary control (standard IHC practice). Do not score matching test-slide color as EHD1. |
| Most cell types stain similarly, including HPA-undetected adrenal glandular cells (HPA tissue IHC). | Excess background or antibody cross-reactivity is plausible; HPA separately cautions that its tissue antibodies target protein from more than one gene (standard IHC practice; HPA tissue IHC). | Compare expected positive and undetected cell populations, review antibody concentration and include appropriate negative controls (HPA tissue IHC; standard IHC practice). Seek independent specificity evidence before assigning widespread signal to EHD1. |
| Nuclear color dominates while expected cytoplasmic staining is absent (HPA tissue IHC). | The compartment disagrees with HPA tissue staining and UniProt membrane localization; background or misidentified cells may explain it (HPA tissue IHC; UniProt Q9H4M9; standard IHC interpretation). | Recheck morphology, counterstain and control slides, then reassess the primary and detection conditions (standard IHC practice). Record the nuclear pattern as discordant unless independent evidence resolves it. |
| Weak cytoplasmic color is hard to distinguish from diffuse slide background. | Background can obscure cell boundaries and compartment assignment (standard IHC interpretation). | Compare a no-primary control and an HPA-reported positive cell population; review blocking, washes and chromogen development (HPA tissue IHC; standard IHC practice). Score only signal whose cells and compartment remain identifiable. |
| Bone marrow is selected as a protein-positive control because its RNA is tissue-enhanced (HPA tissue IHC). | The RNA designation does not match the reported IHC result: hematopoietic cells are Not detected (HPA tissue IHC). | Use HPA-reported High kidney glomerular cells as a tissue-pattern reference and interpret them with HPA's specificity caution (HPA tissue IHC). Keep RNA abundance and observed IHC staining as separate evidence. |
Experiment evidence
IHC & IF images showing EHD1 staining
A02168-1 · IHCIHC analysis of EHD1 using anti-EHD1 antibody (A02168-1). EHD1 was detected in paraffin-embedded section of human testis cancer tissues. Hea
A02168-1 · IHCIHC analysis of EHD1 using anti-EHD1 antibody (A02168-1). EHD1 was detected in paraffin-embedded section of human tonsil tissues. Heat media
A02168-1 · IFIF analysis of EHD1 using anti-EHD1 antibody (A02168-1). EHD1 was detected in immunocytochemical section of A431 cells. Enzyme antigen retri
A02168-2 · IFIF analysis of EHD1 using anti-EHD1 antibody (A02168-2). EHD1 was detected in an immunocytochemical section of SiHa cells. Enzyme antigen re
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000110047-EHD1/tissue/cerebral+cortex#imid_15783191
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000110047-EHD1/tissue/colon#imid_15772741
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000110047-EHD1/tissue/liver#imid_15772750
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000110047-EHD1/tissue/kidney#imid_15772743
![HPA ICC-IF BJ [Human fibroblast] — EHD1 (HPA066751)](https://images.proteinatlas.org/66751/1667_E12_5_blue_red_green.jpg)
HPA · ICC-IF BJ [Human fibroblast]Human Protein Atlas immunofluorescence, BJ [Human fibroblast], antibody HPA066751; green = EHD1, blue = nucleus, red = microtubules.
![HPA ICC-IF BJ [Human fibroblast] — EHD1 (HPA066751)](https://images.proteinatlas.org/66751/1667_E12_6_blue_red_green.jpg)
HPA · ICC-IF BJ [Human fibroblast]Human Protein Atlas immunofluorescence, BJ [Human fibroblast], antibody HPA066751; green = EHD1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for EHD1 IHC & IF
🧪Run kidney first: cells in glomeruli should stain strongly (HPA: High in cells in glomeruli). Use adrenal gland as a negative tissue, where glandular cells are not detected (HPA: Not detected in adrenal gland glandular cells); compare non-glomerular cells on the kidney slide with background, without assuming they are EHD1-negative.
Positive control tissue: Kidney (Cells in glomeruli, HPA High)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show EHD1 in BJ [Human fibroblast], SiHa, U2OS, A-431, A-549, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), Sperm, KOLF2.1J, with annotated localisation: Plasma membrane (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control and a host- and class-matched nonimmune rabbit IgG control (caption: rabbit primary antibody); use a matched EHD1 knockout sample, or peptide blocking if the immunizing peptide is available, to assess target specificity. Check endogenous peroxidase and biotin background in kidney when using the biotin-based DAB detection shown in the caption (caption: biotinylated secondary, streptavidin–biotin complex and DAB).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the selected A02168-1 paraffin-section caption does not state a fixative (caption: fixative unreported). The demonstrated IHC workflow uses heat retrieval in citrate buffer at pH 6 for 20 minutes, followed by 1 μg/mL primary antibody overnight at 4°C (caption: A02168-1 tissue IHC). The evidence does not establish that frozen sections or IF are easier; kidney glomeruli provide a strong IHC starting point (HPA: High in cells in glomeruli), with endogenous biotin a potential concern for the caption’s biotin-based detection.
HPA tissue IHC evidence for EHD1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Caution, targets protein from more than one gene. 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 |
|---|---|---|---|---|
| Kidney | Cells in glomeruli | High | Protein (IHC) | HPA → |
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Appendix | Lymphoid tissue | Medium | Protein (IHC) | HPA → |
| Lung | Alveolar cells | Medium | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced EHD1 IHC Tips
Troubleshoot EHD1 staining in paraffin sections by checking retrieval, cellular distribution, controls, and scoring before interpreting biological differences.
How should I optimize retrieval when EHD1 staining is weak in paraffin sections?
Begin with heat-mediated retrieval in citrate buffer at pH 6 for 20 minutes when staining with A02168-1 (datasheet A02168-1). The selected testis cancer section was blocked with 10% goat serum and incubated with 1 μg/ml primary antibody overnight at 4°C (caption A02168-1). Keep those conditions constant while comparing shorter and longer retrieval on adjacent sections, and check whether tissue morphology survives heating (standard IHC practice). Include a no-primary control and a section with established staining to distinguish inadequate retrieval from a detection problem (standard IHC practice).
Can fixation explain absent EHD1 staining in my paraffin sections?
The selected paraffin-section caption does not state a fixative, and target-specific fixation sensitivity is unknown (caption A02168-1). Record the fixative, fixation duration, tissue thickness, and time before fixation for each specimen before comparing EHD1 intensity (standard IHC practice). If samples differ, stain adjacent sections in the same run with citrate retrieval at pH 6 for 20 minutes and the same detection settings (datasheet A02168-1; standard IHC practice). Interpret a remaining difference only after checking morphology, a no-primary control, and a consistently processed comparison section; those checks can reveal processing variation but cannot establish an EHD1-specific fixation effect (standard IHC practice).
Where should convincing EHD1 staining appear in tissue sections?
Assess staining in the cytoplasm and near cell membranes: EHD1 associates preferentially with tubular recycling endosomes and is also annotated at early endosomes and the cell membrane (UniProt Q9H4M9 localisation). Tissue IHC reports cytoplasmic expression in endothelial, trophoblastic, and some germinal center cells, while its reliability notice flags possible recognition of proteins from more than one gene (HPA tissue IHC). A continuous nuclear-only signal therefore warrants review of the no-primary control, section edges, and antibody specificity before scoring (UniProt Q9H4M9 localisation; standard IHC practice). Resolve fine puncta only where section quality and optical resolution support that distinction; otherwise score the reproducible cellular compartment (standard IHC practice).
Could isoforms or an inaccessible epitope explain discordant EHD1 staining?
The supplied record lists 0 isoforms and one chain spanning residues 1–534, so an isoform-specific tissue pattern is unsupported here (UniProt Q9H4M9 processing and isoforms). EHD1 has no annotated transmembrane segment, while its dynamin-type G domain spans 55–286 and its EH domain spans 444–532 (UniProt Q9H4M9 topology and domains). The antibody epitope is unspecified in the supplied caption, so these domain positions cannot identify what retrieval exposes (caption A02168-1). For discordant sections, compare the established citrate pH 6 retrieval and matched controls before assigning a difference to epitope accessibility (datasheet A02168-1; standard IHC practice).
How can I use IF to investigate an ambiguous chromogenic EHD1 pattern?
Use a separate IF/ICC experiment to compare EHD1 with a marker for the cell population under examination, such as an endothelial marker when assessing endothelial staining (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and favor a far-red channel if tissue autofluorescence obscures shorter-wavelength signals; include single-stain controls (standard IF practice). Because EHD1 has no transmembrane segment and associates with endosomal membranes, test permeabilisation when the antibody must reach intracellular EHD1, while preserving membrane detail (UniProt Q9H4M9 topology and localisation; standard IF practice). Compare the resulting distribution with the supported plasma-membrane and additional ciliary localisations, without treating IF intensity as interchangeable with DAB intensity (HPA subcellular; standard IF and IHC practice).
What should I check when DAB obscures EHD1-positive cells?
First compare a no-primary section with the stained section to identify signal from the detection system or tissue rather than primary-antibody binding (standard IHC practice). The selected workflow used 10% goat serum, 1 μg/ml primary overnight at 4°C, and a biotinylated secondary followed by avidin-biotin detection and DAB (caption A02168-1). Check endogenous peroxidase activity, endogenous biotin, incomplete blocking, and excess DAB development with controls appropriate to that workflow (standard IHC practice). If background persists, adjust blocking, washing, primary concentration, or development time one variable at a time, and retain comparable exposure across specimens (standard IHC practice).
How should I score EHD1 staining across sections with mixed cell populations? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and scoring compartment before analysis, since reported tissue staining includes endothelial, trophoblastic, and a subset of germinal center cells (HPA tissue IHC). For chromogenic sections, report an H-score or the percentage of positive cells within that population, with intensity thresholds set from controls and applied consistently (standard IHC practice). Normalize positive-cell counts to the number of eligible cells, or positive profiles to sampled tissue area in mm² when density is the endpoint (standard IHC practice). Exclude folds, damaged edges, and necrotic regions using the same rules for every section, and report biological replicates separately from sampled fields (standard IHC practice).
How do I distinguish plausible EHD1 signal from staining artefact?
Favor reproducible cytoplasmic or membrane-associated staining in morphologically intact cells, consistent with EHD1's endosomal and cell-membrane annotations (UniProt Q9H4M9 localisation; standard IHC practice). The HPA tissue profile identifies endothelial, trophoblastic, and some germinal center cells, but cautions that its tissue antibody may recognize proteins from more than one gene (HPA tissue IHC). Treat isolated nuclear-only signal, stained tissue edges, necrotic deposits, or signal persisting without primary antibody as reasons for further investigation (UniProt Q9H4M9 localisation; standard IHC practice). Check endogenous peroxidase and, for the selected avidin-biotin DAB workflow, endogenous biotin before calling a cell positive (caption A02168-1; standard IHC practice).
Boster reagents
Best EHD1 / EH domain-containing protein 1 IHC Antibodies
Anti-EHD1 antibodies have IHC images from paraffin sections of human testis cancer, tonsil and placenta, and mouse testis, plus IF/ICC images from A431 and SiHa cells (catalog image captions).
Real IHC data
Anti-EHD1 Antibody ®
Cat # A02168-1
Real IF data
Anti-EHD1 Antibody ®
Cat # A02168-2
A02168-1 has IHC images from human testis cancer, tonsil and placenta, and mouse testis paraffin sections, plus an IF/ICC image from A431 cells (A02168-1 image captions). A02168-2 has an IF/ICC image from SiHa cells and lists no IHC application (A02168-2 IF caption; catalog applications).
Which to pick: Choose A02168-1 for chromogenic IHC in paraffin sections: its IHC captions describe citrate retrieval at pH 6 and DAB detection, but do not report the fixative (A02168-1 IHC captions). For IF/ICC, A02168-2 has a SiHa cell image and A02168-1 has an A431 cell image (catalog IF captions). For cross-species planning, A02168-1 lists human, mouse and rat reactivity, while A02168-2 lists human, mouse and monkey reactivity; neither entry reports a clone, and the images do not demonstrate every listed species (catalog reactivity and clone fields; image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9H4M9 (EHD1_HUMAN, EH domain-containing protein 1).
- Human Protein Atlas. EHD1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. EHD1 subcellular location (ICC-IF): Mainly localized to the plasma membrane. In addition localized to the primary cilium and primary cilium transition zone..
- Human Protein Atlas. EHD1 antibody validation summary (4 antibodies).
- Increased Expression of Eps15 Homology Domain 1 is Associated with Poor Prognosis in Resected Small Cell Lung Cancer. Journal of Cancer 2015 — PMC4565848.
- Mammalian Eps15 homology domain 1 promotes metastasis in non-small cell lung cancer by inducing epithelial-mesenchymal transition. Oncotarget 2017 — PMC5410234.
- The expression of Eps15 homology domain 1 is negatively correlated with disease-free survival and overall survival of osteosarcoma patients. Journal of orthopaedic surgery and research 2019 — PMC6460645.
- The endocytic recycling regulator EHD1 is essential for spermatogenesis and male fertility in mice. BMC developmental biology 2010 — PMC2856533.
- PubMed PMID:9253601 — UniProt-cited evidence.
- PubMed PMID:10395801 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for EHD1 (EH domain-containing protein 1). Expected localisation Cytoplasm in tissue (HPA tissue IHC); endosomal membranes (UniProt). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A02168-1). Validated IHC antibody: A02168-1 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Kidney (Cells in glomeruli); negative Adrenal gland. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human testis cancer tissues; fixative not specified (datasheet A02168-1); 2. Fixation: Image fixative and duration unreported (datasheet A02168-1); 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: Citrate pH 6 , 20 min (datasheet A02168-1); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A02168-1); 8. Primary antibody: Rabbit anti-EHD1, 0.5-1μg/ml (datasheet A02168-1); 9. Primary incubation: Overnight at 4 °C (datasheet A02168-1). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A02168-1); Caveat: HPA antibody may detect proteins from more than one gene (HPA tissue IHC); Isoform / epitope: No annotated isoforms; one 1–534 chain (UniProt); Regulation: Highly expressed in testis (UniProt). Evidence: UniProt Q9H4M9, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the plasma membrane. In addition localized to…, Datasheet A02168-1, PMC4565848, PMC5410234. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/EHD1/immunohistochemistry-ihc-guide.