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ID3 / DNA-binding protein inhibitor ID-3 · IHC design guide
Design Immunohistochemistry for ID3
Plan ID3 paraffin IHC around mainly nuclear staining in most tissues (HPA tissue IHC). Compare high staining in adrenal gland glandular cells with undetected prostate glandular cells (HPA tissue IHC), and optimize the catalog antibody within 1:50–1:200 (datasheet).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ID3 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 | Mainly nuclear staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Most tissue cell types show mainly nuclear staining (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Heart muscle+3 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Staining shows low consistency with RNA levels (HPA tissue IHC) | |
| Regulation | Expression varies by tissue (UniProt) | |
| Isoform / epitope | No annotated isoforms or processing; one 1–119 chain (UniProt) |
Section 1
Recommended ID3 IHC & IF Protocols
The catalog antibody’s IHC protocol is followed by four published ID3 IHC protocols covering lung, vascular, pancreatic and thyroid specimens (PMC3567999; PMC4121297; PMC10361912; PMC8405699).
| Sample | Paraffin-embedded rat brain tissue; fixative not specified (datasheet A01379) |
| Fixation | Image fixative and duration unreported (datasheet A01379); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-ID3, 1:50-1:200 (datasheet A01379) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ID3-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Mainly nuclear expression in most tissues. No signal in the no-primary control. |
💡Decision noteTry Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min first (page retrieval rule: nuclear antigen; UniProt Q02535: nucleus).
Section 2
What Is the Expected ID3 Staining Pattern?
ID3 is a nuclear protein with no transmembrane segment (UniProt Q02535: subcellular location and topology). In paraffin sections, expect mainly nuclear staining in cell types reported by HPA, including adrenal glandular cells, bronchial basal cells and colon endothelial cells (HPA tissue IHC). HPA rates its tissue IHC evidence Approved but reports low consistency between antibody staining and RNA expression; interpret unexpected patterns cautiously (HPA tissue IHC).
| Distinct nuclear staining in adrenal glandular cells, bronchial basal cells or colon endothelial cells. | These cell types have High staining in HPA tissue IHC, which describes expression as mainly nuclear. Assess the labelled cells and compartment together; staining elsewhere in the same tissue does not establish the expected pattern (HPA tissue IHC). |
| Predominantly cytoplasmic or membranous staining, with little nuclear signal. | That distribution conflicts with the reported nuclear location and lack of a transmembrane segment (UniProt Q02535) and HPA's mainly nuclear tissue pattern (HPA tissue IHC). Consider nonspecific staining or a detection artefact; compartment alone cannot identify its cause. |
| Strong staining in a cell type HPA reports as Not detected, such as cardiomyocytes. | This conflicts with the cell-specific HPA observation for heart muscle (HPA: cardiomyocytes Not detected). Check whether the signal follows tissue structures or appears in the negative control; cross-reactivity or endogenous detection activity are possibilities (general IHC practice). |
| Diffuse colour across nuclei, cytoplasm and spaces between cells. | Diffuse staining cannot be scored as the mainly nuclear ID3 pattern (HPA tissue IHC; UniProt Q02535: nucleus). Compare with a section processed without primary antibody; widespread colour may reflect background from the detection system (general IHC practice). |
| No nuclear signal in an adrenal gland section. | HPA reports High staining in adrenal glandular cells (HPA tissue IHC). Confirm those cells are present and the detection run worked before interpreting absence as biological; HPA's Approved rating has a low RNA-to-staining consistency caveat (HPA tissue IHC). |
💡Expected ID3 appearanceCall a result positive when identifiable cells show clear, mainly nuclear staining, especially in HPA High cell types; dominant cytoplasmic staining or uniform colour across unrelated structures is suspect (HPA tissue IHC; UniProt Q02535: nucleus; general IHC practice).
| Cell-specific tissue context | HPA reports High staining in adrenal glandular cells, bronchial basal cells and colon endothelial cells, but Not detected in cardiomyocytes and prostate glandular cells (HPA tissue IHC). Judge the relevant cell population, not a whole-section average. |
| Strength of tissue evidence | The tissue IHC profile is Approved, with low consistency between antibody staining and RNA expression (HPA tissue IHC). Treat an unexpected positive or negative as a finding to check against controls, not a definitive expression call. |
| Antibody-specific validation | HPA lists HPA024677 and CAB021353 as IHC Approved; neither is listed as IHC Enhanced in the supplied antibody record (HPA antibodies). An Approved label does not by itself show that a particular slide's staining is specific. |
| Protein structure | UniProt records one chain spanning residues 1–119, no listed isoforms, no signal peptide and no transmembrane segment (UniProt Q02535). These annotations support assessing nuclear localisation; they do not establish how fixation affects an epitope. |
| What should IF/ICC show? | HPA reports mainly nucleoplasmic localisation in ICC-IF and lists CAB021353 as ICC Supported (HPA subcellular; HPA antibodies). This supports a nuclear localisation check, but the tissue IHC profile remains the basis for paraffin-section expectations (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected nuclear staining is absent in adrenal glandular cells. | A failed staining run or poor visibility of the relevant cells can resemble a negative result (general IHC practice); HPA reports High staining in these cells (HPA tissue IHC). | Confirm cell identity and morphology, review a working positive control from the same run, then check the recorded retrieval, primary-antibody and detection steps against the chosen IHC procedure (general IHC practice). |
| Cytoplasmic colour dominates an otherwise stained section. | The compartment conflicts with nuclear ID3 localisation (UniProt Q02535; HPA tissue IHC). Background or cross-reactivity may contribute (general IHC practice). | Compare the suspect cells with a no-primary control and the nuclear pattern in a known-positive cell population; review blocking and detection conditions if background persists (general IHC practice). |
| Colour appears in cardiomyocytes or prostate glandular cells. | HPA reports ID3 as Not detected in those specific cell types (HPA tissue IHC). Endogenous detection activity or antibody cross-reactivity are possible explanations (general IHC practice). | Verify the cell type, inspect a matched no-primary control and review the detection system's endogenous-activity blocking step; do not assign the colour to ID3 from location alone (general IHC practice). |
| Nearly every structure carries faint colour. | Widespread colour can obscure the mainly nuclear tissue pattern (HPA tissue IHC). Inadequate blocking, washing or detection conditions can increase general background (general IHC practice). | Compare a no-primary section, inspect washes and blocking, and adjust detection or primary-antibody conditions according to the validated IHC procedure before scoring nuclei (general IHC practice). |
| A tissue result disagrees with an RNA-based expectation. | HPA explicitly reports low consistency between antibody staining and RNA expression despite an Approved tissue IHC rating (HPA tissue IHC). RNA and staining should therefore be interpreted as separate observations. | Record the observed cell type, compartment and controls; compare the result with HPA's cell-specific staining entries and avoid treating either assay alone as proof of the other (HPA tissue IHC; general IHC practice). |
| A proposed IF/ICC image is used to judge a paraffin-section result. | HPA's supported nucleoplasmic ICC-IF localisation and Approved tissue IHC profile describe different assay settings (HPA subcellular; HPA tissue IHC). | Use IF/ICC evidence as a compartment check, then judge the paraffin section against its own cell-specific tissue pattern and IHC controls (HPA subcellular; HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing ID3 staining
A01379 · IHCImmunohistochemistry of paraffin-embedded rat brain using ID3 antibody at dilution of 1:200 .Perform microwave antigen retrieval with 10 mM
A01379 · IHCImmunohistochemistry of paraffin-embedded mouse pancreas using ID3 antibody at dilution of 1:200 .Perform microwave antigen retrieval with 1
A01379 · IFImmunofluorescence analysis of HeLa cells using ID3 antibody .
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000117318-ID3/tissue/cerebral+cortex#imid_6318088
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000117318-ID3/tissue/colon#imid_6318014
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000117318-ID3/tissue/liver#imid_6318007
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000117318-ID3/tissue/kidney#imid_6318000
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB021353; green = ID3, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB021353; green = ID3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ID3 IHC & IF
🧪Run bone marrow first: hematopoietic cells should show ID3 staining (HPA: High in hematopoietic cells). Use heart muscle as the negative tissue: cardiomyocytes should lack specific staining (HPA: Not detected in cardiomyocytes). On the bone marrow slide, cells serving as internal negatives should show counterstain without specific nuclear chromogen; confirm their status independently because the supplied HPA row identifies only hematopoietic cells (HPA: bone marrow row; UniProt Q02535: nucleus).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Heart muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ID3 in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control, an isotype control matched to the primary antibody’s host species and clonality, and ID3-knockout tissue as a biological negative (standard IHC practice). In bone marrow, quench endogenous peroxidase before chromogenic detection and check for endogenous biotin if using avidin–biotin detection (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the selected A01379 paraffin-section caption does not report a fixative (A01379 caption). That caption describes microwave retrieval in 10 mM PBS, pH 7.2, but does not establish that retrieval is required; the evidence does not establish whether frozen sections or IF would be easier (A01379 caption). Endogenous peroxidase in marrow cells can create chromogenic background (standard IHC practice).
HPA tissue IHC evidence for ID3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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 → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Basal cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Prostate | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Seminal vesicle | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ID3 IHC Tips
Troubleshoot chromogenic ID3 IHC in paraffin sections by checking retrieval, nuclear localisation, controls and cell-specific scoring (UniProt Q02535; HPA tissue IHC).
What retrieval should I start with when ID3 nuclear staining is weak?
Start with Tris-EDTA at pH 9.0, heated to 95–98 °C for 20 min (page retrieval rule: nuclear antigen). Allow sections to cool in buffer, then compare staining with a matched section processed without retrieval to assess whether heating helps (standard IHC practice). If nuclear staining remains weak, trial microwave retrieval in 10 mM PBS, pH 7.2, the condition reported for the catalog antibody in paraffin-embedded rat brain (A01379 tissue-IHC caption). Keep antibody dilution, detection and development time constant across conditions so differences can be attributed to retrieval (standard IHC practice).
How should I troubleshoot variable ID3 staining after fixation?
Target-specific fixation sensitivity is unknown: the catalog tissue-IHC caption identifies paraffin-embedded rat brain but does not state its fixative (A01379 tissue-IHC caption). Record the fixative, fixation interval and processing history for each specimen before comparing ID3 staining (standard IHC practice). Where fixation histories differ, process control and test sections together, using the same retrieval, 1:200 catalog-antibody dilution and chromogen development time (A01379 tissue-IHC caption; standard IHC practice). If a sample stains poorly, evaluate section integrity and a known positive control before attributing the result to ID3 biology or changing fixation conditions (standard IHC practice).
How can I decide whether the ID3 staining pattern is credible?
Assess ID3 primarily in nuclei: UniProt assigns it to the nucleus, while HPA reports mainly nuclear tissue staining and supported nucleoplasmic localisation (UniProt Q02535; HPA tissue IHC; HPA subcellular). Check whether nuclear signal sits within intact, counterstained cells and is reproducible across comparable regions (standard IHC practice). If staining is predominantly cytoplasmic, diffuse outside cells or concentrated at section edges, review background, retrieval and detection controls before scoring it as ID3 (standard IHC practice). Compare cell populations within the section, since HPA reports high staining in colon endothelial cells and adrenal glandular cells, among others (HPA tissue IHC).
Could an ID3 isoform or epitope explain discordant IHC results?
The supplied UniProt record lists no isoforms and identifies a bHLH domain at residues 28–80 (UniProt Q02535). It reports a 119-aa chain, no transmembrane segment and no annotated glycosylation sites or modified residues (UniProt Q02535). The catalog caption does not identify the antibody epitope, so these annotations cannot establish whether fixation or retrieval masks its binding site (A01379 tissue-IHC caption; standard IHC practice). When results disagree, compare retrieval conditions and antibody controls on adjacent sections, and obtain epitope information before assigning a difference to domain recognition (standard IHC practice).
How should I adapt this IHC assessment to multiplex IF?
For an IF comparison, pair ID3 with a marker identifying the cell population being assessed, such as an endothelial marker when examining the HPA-reported high signal in colon endothelial cells (HPA tissue IHC; standard IF practice). Choose fluorophores after checking the specimen’s autofluorescence, reserving a spectrally distinct channel for the expected nucleoplasmic ID3 signal (HPA subcellular; standard IF practice). Permeabilise sufficiently for antibody access to the nuclear epitope; ID3 has no transmembrane segment, but the catalog epitope is unspecified (UniProt Q02535; A01379 tissue-IHC caption; standard IF practice). Validate antibody performance in IF independently of its paraffin-section IHC image, and include single-colour and secondary-only controls (A01379 tissue-IHC caption; standard IF practice).
What should I check when brown staining obscures ID3 nuclei?
First separate true nuclear staining from diffuse deposits by examining counterstained cell boundaries and the nucleoplasmic pattern expected for ID3 (HPA subcellular; standard IHC practice). Include a primary-antibody omission control to reveal signal from detection reagents, and use a peroxidase block before DAB development in a peroxidase-based workflow (standard IHC practice). If background persists, titrate the catalog antibody around the caption’s 1:200 dilution while holding retrieval and development time fixed (A01379 tissue-IHC caption; standard IHC practice). Inspect tissue edges and damaged or necrotic regions separately; their concentrated staining should not be included in nuclear scoring without control support (standard IHC practice).
How should I quantify ID3 across mixed cell populations? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region before scoring, then count only interpretable nuclei within that population (standard IHC practice; HPA tissue IHC: mainly nuclear expression). Report the percentage of positive nuclei and, when staining intensity is reliable, a nuclear H-score using 0–3 intensity categories (standard IHC practice). For spatial comparisons, report positive nuclear density per mm² of viable tissue and normalise counts to the number of eligible cells or analysed area (standard IHC practice). Keep retrieval, detection, counterstain and scoring thresholds consistent, and report cell-type-specific results because HPA staining varies across tissues and cells (standard IHC practice; HPA tissue IHC).
When should an apparent ID3-positive result be treated cautiously?
A credible result places signal mainly in intact nuclei and matches a relevant cell population, consistent with UniProt’s nuclear assignment and HPA’s mainly nuclear tissue profile (UniProt Q02535; HPA tissue IHC). Treat predominant cytoplasmic or extracellular staining, edge accentuation and staining confined to necrotic areas as potential artefacts pending control review (standard IHC practice). Check the primary-antibody omission and peroxidase-block controls when DAB staining could reflect detection background or endogenous enzyme activity (standard IHC practice). Interpret absence cautiously: HPA rates its tissue antibody staining Approved while reporting low consistency with RNA expression, and the rat-brain catalog image does not state a fixative (HPA tissue IHC; A01379 tissue-IHC caption).
Boster reagents
Best ID3 / DNA-binding protein inhibitor ID-3 IHC Antibodies
A01379 has anti-ID3 IHC images from paraffin-embedded rat brain, mouse pancreas, and human lung cancer (catalog IHC captions), plus IF data from HeLa cells (catalog IF caption).
Real IHC data
Anti-ID3 Antibody
Cat # A01379
A01379 has IHC images of paraffin-embedded rat brain, mouse pancreas, and human lung cancer (catalog IHC captions). It is listed for IHC, IF, and ICC across human, mouse, and rat, with an IF image from HeLa cells (catalog applications; catalog reactivity; catalog IF caption).
Which to pick: For tissue IHC, choose A01379: its paraffin-section captions use 1:200 antibody after microwave retrieval in 10 mM PBS at pH 7.2; the fixative is unreported (catalog IHC captions). For IF/ICC, choose A01379, a rabbit polyclonal listed for both applications with IF data from HeLa cells (catalog host; catalog dilution_raw; catalog applications; catalog IF caption). For work across species, A01379 lists human, mouse, and rat reactivity and has IHC images from each (catalog reactivity; catalog IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q02535 (ID3_HUMAN, DNA-binding protein inhibitor ID-3).
- Human Protein Atlas. ID3 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ID3 subcellular location (ICC-IF): Mainly localized to the nucleoplasm..
- Human Protein Atlas. ID3 antibody validation summary (2 antibodies).
- Id1 and Id3 co-expression correlates with clinical outcome in stage III-N2 non-small cell lung cancer patients treated with definitive chemoradiotherapy. Journal of translational medicine 2013 — PMC3567999.
- PCB153-induced overexpression of ID3 contributes to the development of microvascular lesions. PloS one 2014 — PMC4121297.
- Improved predictability of pancreatic ductal adenocarcinoma diagnosis using a blood immune cell biomarker panel developed from bulk mRNA sequencing and single-cell RNA-sequencing. Cancer immunology, immunotherapy : CII 2023 — PMC10361912.
- Loss of ID3 drives papillary thyroid cancer metastasis by targeting E47-mediated epithelial to mesenchymal transition. Cell death discovery 2021 — PMC8405699.
- PubMed PMID:1628620 — UniProt-cited evidence.
- PubMed PMID:8437843 — UniProt-cited evidence.
- PubMed PMID:7828896 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ID3 (DNA-binding protein inhibitor ID-3). Expected localisation Mainly nuclear staining in most tissues (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A01379 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Heart muscle. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded rat brain tissue; fixative not specified (datasheet A01379); 2. Fixation: Image fixative and duration unreported (datasheet A01379); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-ID3, 1:50-1:200 (datasheet A01379); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Staining shows low consistency with RNA levels (HPA tissue IHC); Isoform / epitope: No annotated isoforms or processing; one 1–119 chain (UniProt); Regulation: Expression varies by tissue (UniProt). Evidence: UniProt Q02535, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm., Datasheet A01379, PMC3567999, PMC4121297. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ID3/immunohistochemistry-ihc-guide.