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CD3D / T-cell surface glycoprotein CD3 delta chain · IHC design guide
Design Immunohistochemistry for CD3D
Plan chromogenic CD3D IHC in paraffin sections using the cytoplasmic immune-cell pattern and tonsil staining as references (HPA tissue IHC). Compare observed staining with CD3D’s annotated membrane location (UniProt), and assess unexpected tissue staining cautiously because presumed off-target binding was reported (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 a subset of immune cells (HPA tissue IHC) | |
| Staining pattern | A subset of immune cells shows cytoplasmic staining (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 tissue staining can mislead scoring (HPA tissue IHC) | |
| Regulation | TCR-CD3 assembly supports surface expression (UniProt) | |
| Isoform / epitope | 2 isoforms; check extracellular vs cytoplasmic epitope (UniProt) |
Section 1
Recommended CD3D IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published CD3D staining workflows 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 retrieval at 95–98 °C for 20 min (page antigen-retrieval setting); adjust for tissue and antibody performance.
Section 2
What Is the Expected CD3D Staining Pattern?
CD3D is a cell membrane protein with an extracellular region at 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 describes cytoplasmic staining in a subset of immune cells; its tissue IHC reliability is Enhanced, with medium consistency between staining and RNA data and presumed off target binding disregarded (HPA tissue IHC).
| Discrete staining in a subset of immune cells in tonsil, with signal outside germinal centers (HPA tissue IHC: medium in non-germinal center cells). | This fits a useful positive pattern: CD3D is mostly present on T lymphocytes (UniProt P04234 tissue specificity). Judge the stained cells and their distribution together; HPA describes cytoplasmic staining in a subset of immune cells, while UniProt places the mature protein at the cell membrane (HPA tissue IHC; UniProt P04234 subcellular location). |
| Strong nuclear staining dominates the section, with little staining in the expected immune-cell population. | Nuclear dominance is unsupported by the reported cell membrane location or HPA tissue IHC profile (UniProt P04234 subcellular location; HPA tissue IHC). Treat it as a suspect pattern and check controls and morphology before interpreting it as CD3D; cytoplasmic immune-cell staining alone is reported by HPA (HPA tissue IHC). |
| Prominent staining appears in nonimmune structures, including distal tubules in kidney (HPA tissue IHC: medium in distal tubules). | HPA reports this kidney observation but also notes presumed off target binding in its tissue IHC assessment (HPA tissue IHC). Do not equate every stained tubule with CD3D expression. Review cell identity and antibody controls; the expected biological population remains T lymphocytes (UniProt P04234 tissue specificity). |
| Pale, widespread color obscures the distinction between immune cells and surrounding tissue. | The distribution no longer matches HPA's subset-of-immune-cells profile (HPA tissue IHC). As a general IHC troubleshooting step, compare the negative control and inspect blocking, washing, and detection conditions. Background alone cannot establish whether the target is present in individual cells. |
| No convincing immune-cell signal appears in tonsil or thymus. | HPA reports medium staining in tonsil non-germinal center cells and thymus cortical cells (HPA tissue IHC). First verify that the selected region contains the expected cells; then review antibody validation, retrieval, and detection as general IHC checks. A blank section alone does not show that CD3D is absent. |
💡Expected CD3D appearanceCall a section positive when a subset of immune cells stains distinctly, as reported by HPA, in a distribution consistent with T lymphocytes; HPA records medium staining in tonsil non-germinal center cells and thymus cortical cells, whereas dominant nuclear or diffuse nonimmune staining is suspect (HPA tissue IHC; UniProt P04234 tissue specificity and topology).
| Cell type and tissue region | CD3D is mostly present on T lymphocytes (UniProt P04234 tissue specificity). HPA records medium staining in tonsil non-germinal center cells and thymus cortical cells, so evaluate those compartments and the stained cells' morphology rather than treating all tissue cells as expected positives (HPA tissue IHC). |
| Compartment and epitope location | The protein spans the membrane, with extracellular residues 22–105 and cytoplasmic residues 127–171 (UniProt P04234 topology). The antibody epitope is unspecified here, so topology cannot predict its staining outline or a retrieval requirement. HPA reports cytoplasmic staining in a subset of immune cells in tissue IHC (HPA tissue IHC). |
| Strength of the tissue evidence | HPA rates tissue IHC reliability Enhanced but reports only medium consistency with RNA and says presumed off target binding was disregarded (HPA tissue IHC). Its listed medium kidney distal-tubule staining therefore warrants careful cell-level interpretation; the rating does not validate every stained cell type (HPA tissue IHC). |
| Isoforms and protein processing | UniProt lists two isoforms and a cleaved signal peptide at residues 1–21 (UniProt P04234 isoforms and processing). The supplied evidence does not identify the antibody epitope or show isoform-specific staining. Avoid assigning a particular tissue pattern to an isoform or processing state. |
| IF/ICC Q&A: should an intracellular pattern be expected? | HPA lists endoplasmic reticulum and vesicles as uncertain ICC-IF locations, and rates one listed antibody's ICC result Uncertain (HPA subcellular; HPA antibodies). Those observations do not establish the expected pattern for paraffin IHC; use the tissue IHC profile for this section (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive tonsil has no discrete signal in the expected region. | The sampled area may lack the relevant non-germinal center cells, or the IHC workflow may have failed; HPA reports medium staining in those cells (HPA tissue IHC). | Check region and cell morphology, then confirm antibody validation and positive-control performance. Review retrieval and detection using the antibody's IHC instructions as general workflow checks; no CD3D-specific retrieval condition is supplied. |
| The entire section looks lightly stained, including areas without immune-cell clusters. | Diffuse color is inconsistent with HPA's subset-of-immune-cells tissue profile (HPA tissue IHC). General IHC possibilities include background from detection reagents or incomplete washing. | Compare a negative control and inspect blocking, wash, and detection steps. Score cells only after their boundaries and tissue context can be distinguished. |
| Strong nuclear signal exceeds staining in immune cells. | A nuclear-dominant pattern is unsupported by the UniProt cell membrane location and HPA tissue IHC description (UniProt P04234 subcellular location; HPA tissue IHC). | Check the negative control and confirm that chromogen or counterstain is being interpreted correctly. Reassess with the expected immune-cell distribution before assigning nuclear staining to CD3D. |
| Kidney tubules stain as strongly as, or more strongly than, nearby immune cells. | HPA lists medium staining in distal tubules while also flagging presumed off target binding in its tissue assessment (HPA tissue IHC); that record does not resolve the identity of a particular tubular signal. | Record tubular and immune-cell staining separately. Use morphology and appropriate controls to assess the pattern; avoid scoring tubular staining alone as evidence of T-cell CD3D. |
| A section contains few or no apparently positive cells outside lymphoid regions. | CD3D is mostly present on T lymphocytes, so the relevant cells may be sparse in the sampled field (UniProt P04234 tissue specificity). HPA describes staining in a subset of immune cells (HPA tissue IHC). | Inspect additional fields for immune cells and compare an HPA-supported positive tissue such as tonsil. Interpret a blank field in light of cell content and positive-control performance. |
| IF/ICC shows puncta or reticular staining that differs from the IHC slide. | HPA's endoplasmic reticulum and vesicle ICC-IF assignments are uncertain, while its tissue IHC profile reports cytoplasmic staining in a subset of immune cells (HPA subcellular; HPA tissue IHC). | Assess each application against its own validation evidence. Do not use the uncertain ICC-IF localization to redefine a positive paraffin IHC result (HPA subcellular; HPA tissue IHC). |
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: CD3D staining should appear in T lymphocytes among the non-germinal center cells scored Medium (HPA: Tonsil non-germinal center cells, Medium; UniProt P04234 tissue specificity). Use adipose tissue as a negative comparator: adipocytes are Not detected by HPA, while tonsillar epithelial cells should remain unstained as an internal negative population (HPA: Adipose tissue adipocytes, Not detected; 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 a no-primary, secondary-only section and an isotype-matched control using the primary antibody’s host species and clonality; use a CD3D knockout specimen, if available, as a biological negative (standard IHC practice). For chromogenic tonsil IHC, block endogenous peroxidase and check whether background persists in the control sections (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence; the selected M04405 paraffin-section human spleen caption does not state a fixative (M04405 caption). Antigen retrieval dependence and whether frozen sections or IF are easier are unreported, so assess retrieval with matched positive and negative sections under the catalog antibody’s IHC-P protocol (supplied application evidence: IHC-P protocol). Dense lymphoid areas in tonsil can make background hard to distinguish from specific staining; assess the expected T-cell surface pattern against the controls (HPA: Tonsil non-germinal center cells, Medium; UniProt P04234 subcellular location).
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 staining in paraffin sections by checking retrieval, cellular pattern, controls and scoring before interpreting chromogenic signal.
How should I adjust retrieval when CD3D staining is weak or uneven?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval rule). If staining remains weak, compare a small retrieval series on matched sections while keeping antibody concentration, detection and imaging conditions constant (standard IHC practice). Use the same tissue area and assess whether improved signal appears in immune cells without a parallel rise in epithelial or diffuse background staining (UniProt P04234 tissue specificity; HPA tissue IHC). Document the buffer, temperature, heating interval and cooling conditions for every run so an apparent gain can be distinguished from inconsistent processing (standard IHC practice).
Could fixation explain weak CD3D staining in paraffin sections?
Target-specific sensitivity of CD3D to fixation is unknown from the supplied evidence; the M04405 caption identifies paraffin-embedded human spleen but does not report its fixative (M04405 tissue-IHC caption). Record the fixative and fixation interval for each specimen, then compare matched tissue regions processed under known conditions (standard IHC practice). If archival material stains weakly, check section integrity and run a positive tissue control before changing the antibody concentration or retrieval conditions (standard IHC practice). Do not assign weak signal to a particular fixation mechanism on the basis of CD3D topology, phosphorylation sites or HPA staining patterns alone (UniProt P04234 topology and modified residues; HPA tissue IHC).
What CD3D staining pattern is credible in tissue sections?
Look first for signal associated with lymphocyte-sized immune cells, with membranous staining consistent with CD3D in the surface TCR–CD3 complex (UniProt P04234 subcellular location, function and tissue specificity). A section can also show cytoplasmic immune-cell staining, as reported in the HPA tissue profile, so compartment alone should not determine whether a cell is positive (HPA tissue IHC). Interpret intracellular signal cautiously because the HPA subcellular record lists endoplasmic reticulum and vesicles as uncertain locations (HPA subcellular). Compare the staining pattern with morphology, a matched negative control and the expected distribution of immune cells before calling diffuse staining in another cell population CD3D (standard IHC practice; UniProt P04234 tissue specificity).
How can epitope position change the CD3D IHC pattern?
Confirm the antibody’s documented immunogen or epitope before interpreting a missing or predominantly intracellular signal; the supplied M04405 caption does not identify its epitope (M04405 tissue-IHC caption). CD3D has an extracellular region at residues 22–105, a membrane-spanning segment at 106–126 and a cytoplasmic region at 127–171 (UniProt P04234 topology). The record lists 2 isoforms, glycosylation sites at residues 38 and 74, and cytoplasmic phosphotyrosines at 149 and 160; it does not establish which features this antibody recognizes (UniProt P04234 isoforms, glycosylation and modified residues). Compare antibodies or controls only after checking their stated epitope coverage and staining conditions (standard IHC practice).
How should I assess CD3D in a multiplex IF follow-up?
Treat IF/ICC as a separate assay from the paraffin-section chromogenic result: the M04405 image caption documents tissue IHC and supplies no IF/ICC fixation or permeabilisation conditions (M04405 tissue-IHC caption). Pair CD3D with an independently validated T-cell marker and inspect single channels as well as the merged image to assess cellular co-localisation (UniProt P04234 tissue specificity; standard IF practice). Choose fluorophores in channels with low tissue autofluorescence, and include unstained and single-label controls before assigning dim puncta to CD3D (standard IF practice). Set permeabilisation according to the antibody’s documented epitope: access to a cytoplasmic epitope generally requires it, whereas an accessible extracellular epitope may be assessed without it (UniProt P04234 topology; standard IF practice).
How do I reduce diffuse or misleading chromogenic background?
First compare the stained section with a no-primary control and inspect whether colour follows tissue edges, damaged regions or broad non-immune cell populations (standard IHC practice; UniProt P04234 tissue specificity). For peroxidase-based detection, apply an endogenous peroxidase block and review secondary reagent specificity and wash adequacy before increasing antibody dilution (standard IHC practice). The HPA tissue record reports presumed off-target binding that was disregarded, so isolated staining outside the expected immune-cell pattern needs independent support (HPA tissue IHC). Keep retrieval and detection settings consistent while changing one variable at a time, and judge any improvement against both positive immune cells and negative tissue compartments (standard IHC practice).
What should I count when comparing CD3D staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the region of interest and cell-identification rules before scoring, then report either the percentage of CD3D-positive immune cells, positive-cell density per mm², or an H-score when intensity is reproducible (standard IHC practice). Normalise cell counts to viable tissue area or to the number of evaluable immune cells, and state which denominator was used (standard IHC practice). Exclude folds, section edges and necrotic areas using the same rule in every specimen (standard IHC practice). Because HPA reports cytoplasmic staining in a subset of immune cells and medium concordance with RNA, record the accepted compartment and threshold explicitly rather than treating all brown pixels as CD3D-positive cells (HPA tissue IHC; standard IHC practice).
When is a CD3D-positive result likely to be artefactual?
A credible result tracks immune-cell morphology and distribution, consistent with CD3D being mostly present on T lymphocytes with TCR–CD3 partners (UniProt P04234 tissue specificity and subunit). Diffuse staining of unrelated cell populations warrants scrutiny, particularly because HPA reports presumed off-target binding and only medium consistency between antibody staining and RNA expression (HPA tissue IHC). Check whether signal concentrates at section edges or necrotic areas, persists in a no-primary control, or follows endogenous enzyme activity rather than individual cells (standard IHC practice). Reconcile any predominantly intracellular pattern with HPA’s cytoplasmic tissue staining and uncertain endoplasmic-reticulum or vesicle localisation before making a cell-type claim (HPA tissue IHC; HPA subcellular).
Boster reagents
Best CD3D / T-cell surface glycoprotein CD3 delta chain IHC Antibodies
Catalog anti-CD3D antibodies have IHC data from human paraffin-embedded spleen (M04405 IHC image caption) and fluorescence data from HeLa ICC and Jurkat IF (A04405-1 and M04405 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 IHC shown in paraffin-embedded spleen and IF shown in Jurkat cells (M04405 catalog and image captions). A04405-1 is listed for human IHC and ICC, with fluorescence shown in paraformaldehyde-fixed, permeabilised HeLa cells (A04405-1 catalog and image caption).
Which to pick: Choose M04405 for tissue IHC: its own image shows paraffin-embedded human spleen (M04405 IHC image caption), and it is a rabbit monoclonal antibody, clone IDE-3 (M04405 catalog); the spleen fixative is unreported (M04405 IHC image caption). For cell fluorescence, choose M04405 for IF in Jurkat cells (M04405 catalog and IF image caption) or A04405-1 for ICC in HeLa cells (A04405-1 catalog and image caption). Neither SKU has stated support for cross-species samples; both list human reactivity only (catalog: reactivity fields).
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 a subset of immune cells (HPA tissue IHC). 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 tissue staining can mislead scoring (HPA tissue IHC); Isoform / epitope: 2 isoforms; check extracellular vs cytoplasmic epitope (UniProt); Regulation: TCR-CD3 assembly supports surface expression (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.