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ECSIT / Evolutionarily conserved signaling intermediate in Toll pathway, mitochondrial · IHC design guide
Design Immunohistochemistry for ECSIT
Plan ECSIT staining in paraffin sections using the observed cytoplasmic tissue pattern (HPA tissue IHC). The catalog antibody was used at 2 μg/mL in human tissue sections (datasheet A07966-3); compare matched sections with appropriate controls (standard IHC practice).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ECSIT 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 | General cytoplasmic staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining; high in colon glandular cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A07966-3) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A07966-3) | |
| Caveat | Low tissue specificity can limit tissue contrast (HPA tissue IHC) | |
| Regulation | Staining-intensity regulation unreported (UniProt) | |
| Isoform / epitope | 4 isoforms; epitope coverage unreported (UniProt) |
Section 1
Recommended ECSIT IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by three published ECSIT IHC methods using paraffin sections or reported IHC antibody conditions.
| Sample | Paraffin-embedded human bladder adenosquamous carcinoma tissue; fixative not specified (datasheet A07966-3) |
| Fixation | Image fixative and duration unreported (datasheet A07966-3); 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 A07966-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A07966-3) |
| Primary antibody | Rabbit anti-ECSIT, 2-5 μg/ml (datasheet A07966-3) |
| Primary incubation | Overnight at 4 °C (datasheet A07966-3) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A07966-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ECSIT-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet: A07966-3); one published paraffin-section method used Tris/borate/EDTA at pH 8.4 (PMC9652319).
Section 2
What Is the Expected ECSIT Staining Pattern?
ECSIT is reported in cytoplasm, nucleus and mitochondria, with no transmembrane segment (UniProt Q9BQ95). In paraffin-section IHC, expect chiefly cytoplasmic staining in the relevant cells; HPA describes general cytoplasmic expression and high staining in several glandular and neuronal cell populations (HPA tissue IHC). HPA rates its tissue IHC profile Approved, with medium consistency between staining and RNA data (HPA tissue IHC).
| Cytoplasmic staining in adrenal glandular cells, bronchial basal cells or cerebellar Purkinje cells. | This matches cell populations scored High by HPA (HPA tissue IHC). Assess staining within the named cells, alongside section morphology and controls; HPA’s overall IHC description is general cytoplasmic expression (HPA tissue IHC). |
| Predominant membrane-edge or extracellular staining, with little intracellular signal. | Treat this as a suspect pattern: ECSIT has no transmembrane segment, and HPA reports general cytoplasmic tissue staining (UniProt Q9BQ95 topology; HPA tissue IHC). Check morphology and detection controls before assigning the deposit to ECSIT. Nuclear signal alone needs separate review because UniProt also lists the nucleus (UniProt Q9BQ95). |
| Strong deposit in a cell population outside the expected stained cells, or in acellular material. | Possible explanations include cross-reactivity or endogenous chromogenic activity; appearance alone cannot distinguish them (general IHC practice). Compare the named HPA-positive cell population on the same or a parallel section and run an appropriate detection control (HPA tissue IHC; general IHC practice). |
| Diffuse colour across tissue, including areas without a clear cellular pattern. | A uniform haze makes cell-level scoring unreliable (general IHC practice). Inspect the detection-only control, washing and reagent concentration before treating the colour as ECSIT signal (general IHC practice). HPA’s reported pattern is cellular and generally cytoplasmic (HPA tissue IHC). |
| No staining in a selected HPA High population. | A blank result in adrenal glandular cells, bronchial basal cells or cerebellar Purkinje cells conflicts with the reported HPA pattern (HPA tissue IHC). First verify tissue identity and the control run, then review retrieval and detection conditions as general IHC troubleshooting steps. |
💡Expected ECSIT appearanceCall a positive IHC result when identifiable HPA High cells show clear, chiefly cytoplasmic staining above local background (HPA tissue IHC); membrane-edge, extracellular or uniform diffuse colour without a cellular pattern warrants a control check (UniProt Q9BQ95 topology; general IHC practice).
| Choice of reference cells | HPA scores adrenal and intestinal glandular cells, bronchial basal cells and selected neurons High, but oral squamous cells and adipocytes Low (HPA tissue IHC). Use a named High population to assess a weak run; Low does not mean a verified negative control. |
| Compartment and protein features | UniProt lists cytoplasm, nucleus and mitochondrion and reports no transmembrane segment or glycosylation sites (UniProt Q9BQ95). HPA’s tissue-level summary is general cytoplasmic expression (HPA tissue IHC). These records do not predict a fixation response. |
| Antibody evidence | HPA lists HPA042979 as IHC Approved and ICC Supported; its overall tissue IHC reliability is Approved with medium staining–RNA consistency (HPA antibodies; HPA tissue IHC). These ratings support comparison with the reported pattern, not automatic assignment of every deposit to ECSIT. |
| IF/ICC Q&A: where is ECSIT seen? | HPA reports mainly nucleoplasmic signal with additional cytosol in ICC-IF, supported for both locations (HPA subcellular). This is an IF/ICC observation; paraffin-section IHC is described separately as generally cytoplasmic (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| HPA High cells are blank. | Tissue selection, retrieval or detection may be at issue; the blank slide does not identify which step failed (HPA tissue IHC; general IHC practice). | Confirm the named cell population, inspect a positive tissue control, and review retrieval and detection settings under the IHC workflow (general IHC practice). |
| Signal is weak but cell-restricted. | The chosen population may be HPA Low, or staining conditions may give limited contrast (HPA tissue IHC; general IHC practice). | Compare with an HPA High population, such as bronchial basal cells, before changing staining conditions; keep comparisons within the same run where possible (HPA tissue IHC; general IHC practice). |
| Colour covers most of the section. | Diffuse background can result from nonspecific reagent binding or excess chromogen development (general IHC practice). | Check the detection-only control, blocking, washes and development time; score ECSIT only where a discernible cellular pattern remains (general IHC practice). |
| Deposit appears along membranes or in acellular spaces. | The location conflicts with the reported intracellular locations and lack of a transmembrane segment (UniProt Q9BQ95; HPA tissue IHC). | Review section morphology and a detection control, then compare intracellular staining in HPA High cells before interpreting the deposit (HPA tissue IHC; general IHC practice). |
| Unexpected cells stain more strongly than the intended reference cells. | Cross-reactivity or endogenous detection activity is possible; HPA’s Low populations are not established negatives (HPA tissue IHC; general IHC practice). | Use the named HPA High cells for pattern comparison and check the detection-only control; assess any endogenous enzyme activity relevant to the chromogen system (HPA tissue IHC; general IHC practice). |
| Nuclear staining dominates an IHC section. | Nuclear localisation is plausible from UniProt and HPA ICC-IF, while HPA summarises tissue IHC as generally cytoplasmic (UniProt Q9BQ95; HPA subcellular; HPA tissue IHC). | Check whether cytoplasmic staining is also present in HPA High cells and whether controls are clean; report the compartment observed without treating ICC-IF as an IHC protocol or identical tissue pattern (HPA tissue IHC; HPA subcellular; general IHC practice). |
Experiment evidence
IHC & IF images showing ECSIT staining
A07966-3 · IHCIHC analysis of ECSIT using anti-ECSIT antibody (A07966-3). ECSIT was detected in a paraffin-embedded section of human bladder adenosquamous
A07966-3 · IHCIHC analysis of ECSIT using anti-ECSIT antibody (A07966-3). ECSIT was detected in a paraffin-embedded section of human colonic adenocarcinom
A07966-3 · IFIF analysis of ECSIT using anti-ECSIT antibody (A07966-3). ECSIT was detected in an immunocytochemical section of PC-3 cells. Enzyme antigen
A07966 · IHCImmunohistochemistry of ECSIT in mouse heart cells with ECSIT antibody at 2 μg/mL.
A07966-1 · IFFluorescent confocal image of Hela cell stained with ECSIT Antibody (C-term). Hela cells were fixed with 4% PFA (20 min), permeabilized with
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000130159-ECSIT/tissue/cerebral+cortex#imid_12164986
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000130159-ECSIT/tissue/colon#imid_12096517
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000130159-ECSIT/tissue/liver#imid_12096526
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000130159-ECSIT/tissue/kidney#imid_12096519
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA042979; green = ECSIT, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA042979; green = ECSIT, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ECSIT IHC & IF
🧪Run bronchus first and score basal cells, which show high ECSIT staining (HPA: High in bronchus basal cells). HPA lists no negative tissue because ECSIT is detected in all 45 scored tissues; use no-primary and isotype controls, and regard cells showing only counterstain as within-slide background comparators, not verified ECSIT-negative cells (HPA: no negative rows; standard IHC control practice).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: ECSIT is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ECSIT in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, a matched rabbit IgG isotype control, and an ECSIT-knockout specimen or validated peptide-block control (selected-SKU caption: rabbit anti-ECSIT; standard IHC control practice). For bronchus, quench endogenous peroxidase and check endogenous biotin before the biotin-based DAB detection used in the caption (selected-SKU caption: biotinylated secondary, SABC and DAB; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A07966-3 paraffin-section caption does not state the fixative; fixation cannot be inferred (selected-SKU IHC caption). The reported staining used heat-mediated retrieval in EDTA at pH 8.0, but retrieval dependence was not tested (selected-SKU IHC caption). Frozen-section or tissue-IF ease cannot be established from the supplied evidence; in bronchus, endogenous peroxidase can produce misleading DAB signal (HPA: ICC-IF images; standard IHC practice). The selected A07966-3 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image A07966-3).
HPA tissue IHC evidence for ECSIT
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Basal cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: ECSIT is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced ECSIT IHC Tips
Use the catalog antibody’s paraffin-section IHC conditions as the starting point, then assess staining by cell type and compartment.
What retrieval should I use when ECSIT staining is weak?
Use heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A07966-3). The selected tissue image used this retrieval before incubation with 2 μg/ml antibody overnight at 4°C (datasheet A07966-3). If staining is weak, check section adhesion, heating consistency and complete cooling before changing the retrieval condition (standard IHC practice). Run any alternative buffer or pH as a separately labelled fallback beside the EDTA control, because its performance with this antibody is unreported (datasheet A07966-3). Compare staining within the same cell population and inspect tissue edges for retrieval-related artefact (standard IHC practice).
Could fixation explain inconsistent ECSIT staining between paraffin blocks?
The selected image identifies a paraffin section but does not state its fixative, so ECSIT-specific fixation sensitivity is unknown (datasheet A07966-3). Record each block’s fixative, fixation duration and processing history before attributing a difference to ECSIT biology (standard IHC practice). Compare matched sections under the same EDTA pH 8.0 retrieval and 2 μg/ml primary conditions used for the selected image (datasheet A07966-3). If archival blocks vary, include a consistently processed reference section in each run and assess morphology alongside staining (standard IHC practice). Neither the reported tissue staining nor ECSIT’s lack of a transmembrane segment establishes a fixation effect (HPA tissue IHC; UniProt Q9BQ95 topology).
How should I assess nuclear versus cytoplasmic ECSIT staining?
Score nuclear and cytoplasmic DAB separately: ECSIT is annotated in the cytoplasm, nucleus and mitochondrion (UniProt Q9BQ95 subcellular). Tissue IHC shows general cytoplasmic expression, while cell imaging reports mainly nucleoplasmic and additional cytosolic localisation (HPA tissue IHC; HPA subcellular). A fine cytoplasmic pattern may be compatible with mitochondrial ECSIT, but DAB alone cannot establish organelle identity (UniProt Q9BQ95 subcellular; standard IHC interpretation). Compare intact cells with the same morphology across sections, and record the fraction showing each compartment pattern (standard IHC practice). Treat an abrupt compartment shift confined to damaged tissue as a staining concern before assigning a biological explanation (standard IHC practice).
Can this stain distinguish ECSIT isoforms or a processed form?
ECSIT has 4 annotated isoforms, and the processed chain is annotated as residues 49–431 (UniProt Q9BQ95 isoforms and processing). The supplied tissue image does not identify the antibody epitope or demonstrate isoform-specific staining (datasheet A07966-3). Do not assign a DAB-positive cell to a particular isoform, or infer cleavage from staining intensity alone (standard IHC interpretation). Ask for documented epitope mapping and compare it with isoform sequences before designing a discriminating antibody panel (UniProt Q9BQ95 isoforms; standard IHC practice). Because ECSIT has no annotated transmembrane segment or glycosylation sites, neither feature identifies this antibody’s accessible epitope (UniProt Q9BQ95 topology and glycosylation).
How could IF help investigate the ECSIT pattern seen by IHC?
Use IF as a separate localisation check for an ambiguous IHC pattern, since cell imaging supports nucleoplasmic and cytosolic ECSIT (HPA subcellular). Multiplex ECSIT with a validated marker for the cell type being evaluated; for example, HPA reports high ECSIT staining in colon glandular cells (HPA tissue IHC). Select fluorophores after checking tissue autofluorescence, with a spectrally separate channel for the cell marker and single-stain controls (standard IF practice). Permeabilisation must provide access to the epitope’s compartment, but its location and membrane-facing side are unreported for this antibody (datasheet A07966-3). Titrate permeabilisation while checking morphology; do not treat IF agreement as proof of mitochondrial localisation (standard IF practice).
How can I reduce diffuse or patchy DAB background?
First compare a no-primary control with the stained section to identify signal from detection reagents or endogenous enzyme activity (standard chromogenic IHC practice). The selected image used 10% goat serum block, a biotinylated secondary, streptavidin–biotin detection and DAB (datasheet A07966-3). Include a peroxidase block and assess whether endogenous biotin contributes to staining with this detection scheme (standard chromogenic IHC practice). If background persists, check washes and titrate the primary around the reported 2 μg/ml condition while keeping retrieval constant (datasheet A07966-3; standard IHC practice). Note whether diffuse colour follows tissue edges, folds or damaged areas before scoring cells (standard IHC practice).
How should I quantify ECSIT staining across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because tissue IHC describes general cytoplasmic expression while cell imaging also supports nucleoplasmic ECSIT (HPA tissue IHC; HPA subcellular). For each compartment, record the percentage of positive cells and intensity grades, then calculate an H-score if intensity is reproducible (standard IHC practice). Alternatively, report positive cells per mm² when cell density changes across regions, with the assessed area documented (standard IHC practice). Normalise counts to viable cells of the chosen type or to their measured tissue area, and apply one threshold across the comparison set (standard IHC practice). Keep retrieval and chromogen development consistent between slides (standard IHC practice).
When is ECSIT staining credible rather than artefactual?
Credible staining should recur in intact cells of a defined type and a plausible compartment: ECSIT is annotated in cytoplasm, nucleus and mitochondrion (UniProt Q9BQ95 subcellular). HPA reports high staining in colon glandular cells and low staining in oral mucosal squamous epithelial cells, which can inform within-study comparisons without serving as absolute controls (HPA tissue IHC). Reconsider colour limited to section edges, necrotic regions or unexpected cells, especially if the no-primary control also stains (standard IHC practice). Check endogenous peroxidase and biotin contributions when using the reported streptavidin–biotin DAB scheme (datasheet A07966-3; standard chromogenic IHC practice). A compartment shift requires orthogonal support before assigning signaling activity (UniProt Q9BQ95 function; standard IHC interpretation).
Boster reagents
Best ECSIT / Evolutionarily conserved signaling intermediate in Toll pathway, mitochondrial IHC Antibodies
ECSIT antibodies have IHC images from human paraffin sections and mouse heart cells (catalog image captions), plus IF images from human cells (catalog IF image captions).
Real IHC data
Anti-ECSIT Antibody ®
Cat # A07966-3
Real IHC data
Anti-ECSIT Antibody
Cat # A07966
Real IF data
Anti-ECSIT Antibody (C-term)
Cat # A07966-1
A07966-3 has human paraffin-section IHC images and a PC-3 IF image (A07966-3 image captions); A07966 has a mouse heart IHC image (A07966 image caption). A07966-1 has a HeLa IF image and lists IF as a tested application (A07966-1 image caption and catalog applications).
Which to pick: For human tissue IHC, choose A07966-3: its own caption documents paraffin sections, EDTA retrieval at pH 8.0, and 2 μg/ml primary antibody; the fixative is unreported (A07966-3 IHC caption). For IF/ICC, A07966-3 lists both applications and has a PC-3 IF image, while A07966-1 lists IF and has a HeLa IF image (catalog applications and IF captions). For work involving human and mouse samples, consider A07966, which lists both species, although its IHC image shows mouse heart cells and reports no fixative (A07966 catalog reactivity and IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9BQ95 (ECSIT_HUMAN, Evolutionarily conserved signaling intermediate in Toll pathway, mitochondrial).
- Human Protein Atlas. ECSIT tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ECSIT subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol..
- Human Protein Atlas. ECSIT antibody validation summary (1 antibodies).
- Impaired mitochondrial oxidative metabolism in skeletal progenitor cells leads to musculoskeletal disintegration. Nature communications 2022 — PMC9652319.
- ECSIT Is a Critical Factor for Controlling Intestinal Homeostasis and Tumorigenesis through Regulating the Translation of YAP Protein. Advanced science (Weinheim, Baden-Wurttemberg, Germany) 2023 — PMC10477885.
- ECSIT inhibits cell death to increase tumor progression and metastasis via p53 in human breast cancer. Translational cancer research 2022 — PMC9091042.
- Ligand-independent EPHA2 signaling sustains neural progenitors via ECSIT and NAD(). EMBO reports 2026 — PMC13503776.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15057824 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ECSIT (Evolutionarily conserved signaling intermediate in Toll pathway, mitochondrial). Expected localisation General cytoplasmic staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A07966-3). Validated IHC antibody: A07966-3 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human bladder adenosquamous carcinoma tissue; fixative not specified (datasheet A07966-3); 2. Fixation: Image fixative and duration unreported (datasheet A07966-3); 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 A07966-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A07966-3); 8. Primary antibody: Rabbit anti-ECSIT, 2-5 μg/ml (datasheet A07966-3); 9. Primary incubation: Overnight at 4 °C (datasheet A07966-3). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A07966-3); Caveat: Low tissue specificity can limit tissue contrast (HPA tissue IHC); Isoform / epitope: 4 isoforms; epitope coverage unreported (UniProt); Regulation: Staining-intensity regulation unreported (UniProt). Evidence: UniProt Q9BQ95, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A07966-3, PMC9652319, PMC10477885. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ECSIT/immunohistochemistry-ihc-guide.