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CD3D / T-cell surface glycoprotein CD3 delta chain · IHC design guide
Design Immunohistochemistry for CD3D
Plan CD3D paraffin-section IHC using tonsil non-germinal center cells as a positive control (HPA tissue IHC) and an IHC-validated antibody at 1:50 (datasheet: M04405). Interpret cytoplasmic staining in a subset of immune cells (HPA tissue IHC) alongside the expected membrane location (UniProt) and presumed off-target staining (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CD3D 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 | Cytoplasmic in immune cells (HPA tissue IHC); membrane expected (UniProt) | |
| Staining pattern | Cytoplasmic staining in a subset of immune cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Kidney+2 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep formalin fixation consistent across paraffin sections (standard IHC practice; not target-specific) | |
| Caveat | Presumed off-target staining can complicate interpretation (HPA tissue IHC) | |
| Regulation | Staining may vary with T-cell abundance (UniProt) | |
| Isoform / epitope | 2 isoforms; extracellular vs cytoplasmic epitope coverage unknown (UniProt) |
Section 1
Recommended CD3D IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with published CD3D protocols for gastric, liver, and cervical cancer tissues (PMC9198637; PMC8192185; PMC11375541).
| Sample | Paraffin-embedded human spleen tissue; fixative not specified (datasheet M04405) |
| Fixation | Image fixative and duration unreported (datasheet M04405); 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 citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane 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 monoclonal (clone IDE-3) anti-CD3D, 1:50 (datasheet M04405) |
| 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 | CD3D-positive staining in distal tubules of kidney (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in a subset of immune cells. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval rule); the liver protocol uses 115 °C for 3 min (PMC8192185).
Section 2
What Is the Expected CD3D Staining Pattern?
CD3D is a cell-membrane component of the TCR–CD3 complex, with extracellular residues 22–105, a transmembrane segment at 106–126, and a cytoplasmic tail at 127–171 (UniProt P04234 topology). Expect staining chiefly in T lymphocytes (UniProt P04234 tissue specificity). HPA reports cytoplasmic staining in a subset of immune cells; its tissue IHC reliability is Enhanced, with presumed off-target binding observed and disregarded (HPA tissue IHC).
| Discrete staining in immune cells of tonsil or thymus, with a membranous outline or cytoplasmic signal. | This fits CD3D’s T-cell membrane location (UniProt P04234 subcellular location) and HPA’s observed cytoplasmic staining in a subset of immune cells (HPA tissue IHC). HPA reports Medium staining in tonsil non-germinal center cells and thymus cortical cells (HPA tissue IHC). Judge the stained cells and their location together, rather than requiring a perfect membrane ring in every section. |
| Strong nuclear staining dominates cells that otherwise lack a plausible immune-cell pattern. | Nuclear localization is unsupported by the supplied CD3D record: UniProt places CD3D at the cell membrane, while HPA describes cytoplasmic immune-cell staining in tissue IHC (UniProt P04234 subcellular location; HPA tissue IHC). Treat the nuclear pattern as suspect and review controls and detection conditions before scoring it as CD3D (general IHC practice). |
| Staining is concentrated in glandular or tubular cells instead of recognizable immune cells. | This does not, by itself, establish a T-cell result (UniProt P04234 tissue specificity). HPA reports Medium staining in kidney distal tubules and Low staining in stomach and small-intestine glandular cells, despite noting presumed off-target binding (HPA tissue IHC). Record the cell type and assess possible cross-reactivity or endogenous detection activity with appropriate controls (general IHC practice). |
| Broad, diffuse color covers tissue structures and obscures individual positive cells. | A field-wide haze cannot be reconciled confidently with HPA’s subset-of-immune-cells profile (HPA tissue IHC). Background from detection reagents, incomplete blocking, or overly strong assay conditions is possible (general IHC practice). Compare a negative reagent control and reduce background before interpreting faint cells; diffuse staining alone is not evidence of CD3D. |
| No convincing signal appears in tonsil or thymus where positive cells were expected. | HPA reports Medium staining in tonsil non-germinal center cells and thymus cortical cells (HPA tissue IHC). A blank result in suitable tissue calls for an assay check, including antibody dilution, retrieval, detection reagents, and section quality (general IHC practice). First verify that the expected cellular area is present; a single blank section cannot establish absent CD3D expression. |
💡Expected CD3D appearanceCall a result positive when discrete immune cells show a membranous or cytoplasmic pattern, typically at the Medium level reported for selected tonsil and thymus cells; isolated nuclear or widespread epithelial staining warrants control-based review (UniProt P04234 topology and tissue specificity; HPA tissue IHC; general IHC practice).
| Cell compartment and epitope position | CD3D spans the membrane, with extracellular residues 22–105 and cytoplasmic residues 127–171 (UniProt P04234 topology). The supplied record does not map the antibody epitope, so an exclusively extracellular or intracellular antibody-binding claim is unsupported. |
| Tissue pattern and validation | HPA rates tissue IHC reliability Enhanced and reports cytoplasmic staining in a subset of immune cells, while noting presumed off-target binding (HPA tissue IHC). HPA071778 and CAB013055 both have Enhanced IHC validation; this supports using the cellular pattern, while leaving anomalous staining open to review (HPA antibodies; HPA tissue IHC). |
| Protein processing and variants | The UniProt precursor has a signal peptide at residues 1–21 and a mature chain spanning 22–171; two isoforms are listed (UniProt P04234 processing; isoforms). No epitope sequence or isoform-specific IHC performance is supplied, so these features do not predict a distinct staining pattern for this antibody. |
| Antigen retrieval | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. |
| IF/ICC Q&A: what localization is reported? | HPA reports uncertain endoplasmic-reticulum and vesicle localization in ICC-IF, and rates HPA071778 ICC validation Uncertain (HPA subcellular ICC-IF; HPA antibodies). Interpret that observation within an IF/ICC experiment; it does not replace the tissue-IHC immune-cell pattern (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Tonsil or thymus has no discrete positive cells. | The sampled area may lack the HPA-listed cell population, or the IHC assay may have failed (HPA tissue IHC; general IHC practice). | Confirm the relevant region on the section, then check the positive control, antibody dilution, retrieval, and detection steps (general IHC practice). HPA lists Medium staining in tonsil non-germinal center and thymus cortical cells (HPA tissue IHC). |
| A nuclear pattern is stronger than immune-cell staining. | This location conflicts with UniProt’s cell-membrane assignment and HPA’s cytoplasmic immune-cell profile (UniProt P04234 subcellular location; HPA tissue IHC). | Compare reagent controls and a known-positive immune-cell area; review primary-antibody and detection conditions before accepting the signal (general IHC practice). |
| Kidney tubules or glandular cells appear positive. | HPA records Medium distal-tubule staining and Low stomach or small-intestine glandular staining, while noting presumed off-target binding (HPA tissue IHC). | Document the exact cell type and compare appropriate controls and immune-cell staining. Do not score epithelial color as T-cell localization without cellular evidence (UniProt P04234 tissue specificity; general IHC practice). |
| Diffuse chromogen masks individual cells. | The result cannot be matched reliably to HPA’s subset-of-immune-cells profile; assay background is possible (HPA tissue IHC; general IHC practice). | Inspect negative reagent controls and review blocking, antibody dilution, washes, and chromogen development (general IHC practice). Reassess whether discrete immune cells remain visible. |
| A faint cytoplasmic signal appears without a crisp membrane rim. | HPA describes cytoplasmic staining in a subset of immune cells, although UniProt assigns CD3D to the cell membrane (HPA tissue IHC; UniProt P04234 subcellular location). | Use cell identity, distribution, and controls to judge the signal; do not reject it solely for lacking a continuous rim (HPA tissue IHC; general IHC practice). |
| ICC-IF shows punctate intracellular staining that differs from the tissue slide. | HPA’s ICC-IF summary lists uncertain endoplasmic-reticulum and vesicle localization, whereas tissue IHC reports cytoplasmic immune-cell staining (HPA subcellular ICC-IF; HPA tissue IHC). | Assess the ICC-IF result within its own controls and validation context. HPA071778 is ICC Uncertain but IHC Enhanced (HPA antibodies); use the tissue-IHC pattern to interpret the paraffin section. |
Experiment evidence
IHC & IF images showing CD3D staining
M04405 · IHCImmunohistochemical analysis of paraffin-embedded human spleen, using CD3D Antibody.
M04405 · IFImmunofluorescent analysis of Jurkat cells, using CD3D Antibody.
A04405-1 · IFICC staining CD3D in Hela cells (green). The nuclear counter stain is DAPI (blue). Cells were fixed in paraformaldehyde, permeabilised with
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000167286-CD3D/tissue/cerebral+cortex#imid_21131331
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000167286-CD3D/tissue/colon#imid_21131257
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000167286-CD3D/tissue/liver#imid_21131266
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000167286-CD3D/tissue/kidney#imid_21131259
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA071778; green = CD3D, blue = nucleus, red = microtubules.
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA071778; green = CD3D, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CD3D IHC & IF
🧪Run tonsil first: non-germinal center cells should stain (HPA: Medium in tonsil non-germinal center cells). Run adipose tissue as a negative: adipocytes should remain unstained (HPA: Not detected in adipocytes); on the tonsil slide, epithelial cells provide an internal negative because CD3D is mostly present on T lymphocytes (UniProt P04234 tissue specificity).
Positive control tissue: Kidney (Distal tubules, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CD3D in Hep-G2, JURKAT, U2OS, with annotated localisation: Endoplasmic reticulum (uncertain), Vesicles (uncertain) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host-species- and clonality-matched isotype controls, plus CD3D-knockout material if available as a biological negative (standard IHC practice). For chromogenic tonsil IHC, block endogenous peroxidase; block endogenous biotin if using biotin-based detection (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and retrieval dependency are unreported in the supplied evidence; the selected paraffin-section spleen caption also leaves the fixative unreported (SKU M04405 caption). Optimize antigen retrieval empirically for paraffin IHC; the supplied evidence does not establish that frozen sections or IF are easier (standard IHC practice; SKU M04405 caption). In tonsil, score the specified non-germinal center cells rather than treating all cells as expected positives (HPA: Medium in tonsil non-germinal center cells; UniProt P04234 tissue specificity).
HPA tissue IHC evidence for CD3D
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded.). 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 | Distal tubules | Medium | Protein (IHC) | HPA → |
| Thymus | Cortical cells | Medium | Protein (IHC) | HPA → |
| Tonsil | Non-germinal center 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 | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CD3D IHC Tips
Troubleshoot CD3D chromogenic staining in paraffin sections using the stated retrieval method, membrane topology, tissue evidence, and appropriate controls.
How should I retrieve CD3D in paraffin sections when staining is weak?
Start with citrate at pH 6.0, heated to 95–98 °C for 20 min (page retrieval rule). Compare a stained section with a no-primary control and inspect immune-cell staining after retrieval, keeping section thickness and detection conditions consistent across the comparison (standard IHC practice; UniProt P04234 tissue specificity). If staining remains weak, test another retrieval condition on matched serial sections and assess both signal and tissue damage before adopting it (standard IHC practice). Record the retrieval method with each result, because the catalog antibody’s paraffin-section caption does not report retrieval conditions (catalog antibody M04405 caption).
Could fixation explain inconsistent CD3D staining between paraffin specimens?
Target-specific sensitivity of CD3D staining to fixation is unknown from the supplied evidence; the catalog image identifies paraffin-embedded spleen but does not state its fixative (catalog antibody M04405 caption). Document the fixative and processing history for each specimen, then compare sections processed as similarly as possible while holding citrate pH 6.0 retrieval constant (standard IHC practice; page retrieval rule). Include an expected positive immune-cell population and a no-primary control in each run to distinguish signal loss from background (UniProt P04234 tissue specificity; standard IHC practice). Do not attribute a specimen difference to fixation without a controlled processing comparison (standard IHC practice).
Should CD3D staining appear at the membrane or in the cytoplasm?
Expect staining associated with T-lymphocytes and assess membrane-associated signal first, because CD3D is a TCR-CD3 cell-membrane component (UniProt P04234 subcellular location and tissue specificity). Its extracellular region spans residues 22–105, its membrane segment 106–126, and its cytoplasmic tail 127–171 (UniProt P04234 topology). Cytoplasmic staining in a subset of immune cells is also reported, while endoplasmic-reticulum and vesicle assignments from cell imaging are marked uncertain (HPA tissue IHC; HPA subcellular). Resolve apparently diffuse staining by checking cell morphology and no-primary background on adjacent sections, rather than treating every cytoplasmic deposit as surface CD3D (standard IHC practice; UniProt P04234 subcellular location).
How can epitope position affect CD3D staining after retrieval?
Check whether the catalog antibody’s stated epitope lies in the extracellular residues 22–105 or cytoplasmic residues 127–171, if epitope information is available (UniProt P04234 topology). CD3D has 2 listed isoforms, so an antibody’s isoform coverage cannot be assumed without an epitope map or direct validation (UniProt P04234 isoforms; standard IHC practice). Extracellular glycosylation sites at residues 38 and 74, and cytoplasmic phosphotyrosines at 149 and 160, are relevant when evaluating an epitope claim, but their effects on this antibody are unreported (UniProt P04234 glycosylation and modified residues; catalog antibody M04405 caption). Compare retrieval conditions on serial sections before attributing a staining change to an isoform or modification (standard IHC practice).
How should I investigate CD3D staining in a companion immunofluorescence assay?
For a separate IF assay, pair CD3D with a T-cell marker in different fluorescence channels and verify concordance at the single-cell level (UniProt P04234 tissue specificity; standard IF practice). Select fluorophores after inspecting unstained tissue autofluorescence, and use single-stain controls to assess channel spillover (standard IF practice). Match permeabilisation to the antibody’s known epitope: an extracellular epitope may be assessed without permeabilisation, whereas a cytoplasmic-tail epitope requires membrane access (UniProt P04234 topology; standard IF practice). The paraffin-section catalog image supplies no IF fixation or permeabilisation conditions, so establish those conditions with IF controls rather than transferring the IHC workflow (catalog antibody M04405 caption; standard IF practice).
What should I check when CD3D chromogenic staining is widespread?
First inspect no-primary and detection-only controls for chromogen deposition, then check whether staining follows tissue edges, damaged areas, or endogenous enzyme activity (standard IHC practice). Apply an appropriate peroxidase block before peroxidase-based DAB detection and optimise nonspecific blocking and washes using matched sections (standard IHC practice). The catalog image documents staining of paraffin-embedded human spleen but gives no antibody dilution or detection settings, so it cannot set those variables for a new specimen (catalog antibody M04405 caption). Treat staining outside plausible immune-cell populations cautiously, especially because presumed off-target binding was observed in the tissue survey (UniProt P04234 tissue specificity; HPA tissue IHC reliability description).
How should I quantify CD3D-positive cells in chromogenic sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue region and counting rules before scoring, then report CD3D-positive immune cells as a percentage of eligible nucleated cells or as density per mm² of analysed tissue (standard IHC practice; UniProt P04234 tissue specificity). If intensity matters, use an H-score based on the percentages at intensity grades 0–3, giving a possible range of 0–300 (standard IHC practice). Keep retrieval, chromogen development, image settings, and positivity thresholds consistent across specimens, and exclude folds, edges, and necrotic regions by a predefined rule (standard IHC practice). State the denominator and tissue area so variation in immune-cell content is distinguishable from variation in staining per cell (standard IHC practice).
When is an apparent CD3D-positive result more likely to be artefact?
A credible result should localise to plausible T-lymphocytes and be consistent with CD3D’s role in the cell-surface TCR-CD3 complex (UniProt P04234 tissue specificity, function, and subcellular location). Diffuse staining in unrelated cells warrants scrutiny, since the tissue survey reports cytoplasmic immune-cell signal alongside presumed off-target binding (HPA tissue IHC profile and reliability description). Recheck deposits concentrated at section edges or necrotic areas, and compare them with no-primary and peroxidase-block controls before scoring (standard IHC practice). Cytoplasmic signal alone does not prove artefact, but an unexpected compartment or cell type needs corroboration with morphology, controls, and an independent T-cell marker (HPA tissue IHC profile; UniProt P04234 tissue specificity; standard IHC practice).
Boster reagents
Best CD3D / T-cell surface glycoprotein CD3 delta chain IHC Antibodies
Human-reactive anti-CD3D antibodies have IHC images from paraffin-embedded spleen and fluorescence images from HeLa and Jurkat cells (catalog reactivity; image captions).
Real IHC data
Anti-CD3D Rabbit Monoclonal Antibody
Cat # M04405
Real IF data
Anti-CD3D Antibody
Cat # A04405-1
M04405 is listed for human IHC and IF, with paraffin-section spleen IHC and Jurkat IF images (M04405 applications, reactivity, image captions). A04405-1 is listed for human IHC and ICC, with a paraformaldehyde-fixed HeLa ICC image (A04405-1 applications, reactivity, image caption).
Which to pick: Choose M04405 for tissue IHC: this rabbit monoclonal has a human spleen paraffin-section IHC image; its caption does not report the fixative (M04405 catalog clone, reactivity, IHC caption). For cellular fluorescence, choose A04405-1 for ICC in paraformaldehyde-fixed, Triton-permeabilised HeLa cells, or M04405 for IF in Jurkat cells (A04405-1 ICC caption; M04405 applications, IF caption). Neither SKU has listed nonhuman reactivity to support cross-species use (catalog reactivity: Human for both).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P04234 (CD3D_HUMAN, T-cell surface glycoprotein CD3 delta chain).
- Human Protein Atlas. CD3D tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CD3D subcellular location (ICC-IF): Localized to the endoplasmic reticulum and vesicles..
- Human Protein Atlas. CD3D antibody validation summary (2 antibodies).
- CD3D Is an Independent Prognostic Factor and Correlates With Immune Infiltration in Gastric Cancer. Frontiers in oncology 2022 — PMC9198637.
- Identification of CD8+ T Cell-Related Genes: Correlations with Immune Phenotypes and Outcomes of Liver Cancer. Journal of immunology research 2021 — PMC8192185.
- Dual Role of Necroptosis in Cervical Cancer: Promoting Tumor Aggression and Modulating the Immune Microenvironment via the JAK2-STAT3 Pathway. Journal of Cancer 2024 — PMC11375541.
- A Standardized Approach to Orthotopic (Life-supporting) Porcine Cardiac Xenotransplantation in a Nonhuman Primate Model. Transplantation 2023 — PMC12805938.
- PubMed PMID:2939461 — UniProt-cited evidence.
- PubMed PMID:6095101 — UniProt-cited evidence.
- PubMed PMID:3488209 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CD3D (T-cell surface glycoprotein CD3 delta chain). Expected localisation Cytoplasmic in immune cells (HPA tissue IHC); membrane expected (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: M04405 at 1:50 (the product's own IHC validation image is shown). Controls (HPA): positive Kidney (Distal tubules); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human spleen tissue; fixative not specified (datasheet M04405); 2. Fixation: Image fixative and duration unreported (datasheet M04405); 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 citrate buffer, pH 6.0 , 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 monoclonal (clone IDE-3) anti-CD3D, 1:50 (datasheet M04405); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep formalin fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: Presumed off-target staining can complicate interpretation (HPA tissue IHC); Isoform / epitope: 2 isoforms; extracellular vs cytoplasmic epitope coverage unknown (UniProt); Regulation: Staining may vary with T-cell abundance (UniProt). Evidence: UniProt P04234, HPA tissue IHC · Enhanced, HPA ICC-IF · endoplasmic reticulum and vesicles., Datasheet M04405, PMC9198637, PMC8192185. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CD3D/immunohistochemistry-ihc-guide.