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ZAP70 / Tyrosine-protein kinase ZAP-70 · IHC design guide
Design Immunohistochemistry for ZAP70
Plan chromogenic ZAP70 IHC around cytoplasmic staining in immune-cell subsets (HPA tissue IHC). Lymph node non-germinal-center cells provide a high-staining reference (HPA tissue IHC); keep fixation and controls consistent across sections (standard IHC practice).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ZAP70 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 subsets of immune cells (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic signal in non-germinal-center cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB9968) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Presumed off-target staining may confound interpretation (HPA tissue IHC) | |
| Regulation | TCR activation recruits ZAP70 to the membrane (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope differences are unspecified (UniProt) |
Section 1
Recommended ZAP70 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet: PB9968) with 1 published endometrial tissue IHC protocol (PMC10131270 methods).
| Sample | Paraffin-embedded human prostate cancer tissue; fixative not specified (datasheet PB9968) |
| Fixation | Image fixative and duration unreported (datasheet PB9968); 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 PB9968); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9968) |
| Primary antibody | Rabbit anti-ZAP70, 2-5μg/ml (datasheet PB9968) |
| Primary incubation | Overnight at 4 °C (datasheet PB9968) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet PB9968) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ZAP70-positive staining in lymphoid tissue of appendix (HPA tissue IHC: High). HPA tissue profile: Selective cytoplasmic expression in subsets of immune cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet: PB9968); the published protocol does not specify retrieval (PMC10131270 methods).
Section 2
What Is the Expected ZAP70 Staining Pattern?
ZAP70 staining is expected mainly in the cytoplasm of subsets of immune cells in paraffin sections (HPA: selective cytoplasmic expression; IHC reliability Enhanced). T cells and natural killer cells are expected sources, with expression also reported in early thymocytes and pro/pre B cells (UniProt P43403: tissue specificity). Activated T cells may recruit ZAP70 to the plasma membrane (UniProt P43403: subcellular location). ZAP70 has no transmembrane segment (UniProt P43403: topology).
| Distinct cytoplasmic staining in cells of appendix lymphoid tissue and lymph node or tonsil non-germinal-center regions. | This fits the expected immune-cell pattern. HPA reports high staining in these locations and describes selective cytoplasmic expression in immune-cell subsets (HPA: tissue IHC). Score the stained cells and compartment, rather than treating every cell in a positive tissue as positive. |
| Staining is mainly nuclear, or it outlines many cells uniformly at their membranes. | This conflicts with the predominant cytoplasmic tissue pattern (HPA: tissue IHC). Membrane recruitment can occur after T-cell receptor activation (UniProt P43403: subcellular location), so a focal membrane pattern needs context. Widespread nuclear or uniform membrane staining warrants a control review before interpretation. |
| Strong staining appears in adipocytes or respiratory epithelial cells. | HPA reports ZAP70 as not detected in adipocytes of adipose tissue and respiratory epithelial cells of bronchus (HPA: tissue IHC). Consider antibody cross-reactivity or endogenous chromogenic detection activity; compare stained cells with a matched detection-only control and immune-cell positive control (general IHC practice). |
| Weak, diffuse color covers tissue and obscures individual cells. | The result cannot establish the selective cytoplasmic pattern reported by HPA (HPA: tissue IHC). Excess detection background or insufficient blocking or washing can reduce contrast (general IHC practice). Reassess the run with its negative control before scoring weak cells as positive. |
| No staining is visible in an expected positive region. | Absent staining in appendix lymphoid tissue or lymph node non-germinal-center cells conflicts with HPA's high observations there (HPA: tissue IHC). Check tissue preservation and the IHC workflow, including retrieval, antibody incubation and detection (general IHC practice); a blank field alone cannot establish absent ZAP70 expression. |
💡Expected ZAP70 appearanceCall positive a cell-resolved, predominantly cytoplasmic immune-cell signal, with high staining possible in the HPA-listed lymphoid regions (HPA: tissue IHC); diffuse haze or widespread staining of HPA-negative cell types is suspect (HPA: tissue IHC; general IHC practice).
| Activation and compartment | Quiescent T lymphocytes contain cytoplasmic ZAP70; T-cell receptor activation recruits it to the plasma membrane through CD247/CD3Z (UniProt P43403: subcellular location). Assess membrane accentuation in cellular context, without requiring it for a positive IHC result. |
| Tissue and cell selection | HPA reports high staining in appendix lymphoid tissue and lymph node and tonsil non-germinal-center cells, and medium staining in bone marrow hematopoietic cells (HPA: tissue IHC). These are reference patterns, not a promise that every cell will stain. |
| Antibody evidence and discordance | HPA rates tissue IHC reliability Enhanced and lists two antibodies with Enhanced IHC validation, HPA003134 and CAB002625 (HPA: tissue IHC; HPA: antibodies). HPA also records high proximal-tubule microvillus staining while describing selective immune-cell expression; interpret that discordance cautiously (HPA: tissue IHC). |
| Isoforms and modified residues | UniProt lists 3 isoforms and several phosphorylated residues (UniProt P43403: isoforms; modified residues). Without an antibody epitope or phospho-specificity record, these facts cannot predict which forms the IHC stain detects or explain a missing signal. |
| IF/ICC Q&A: Is the same pattern validated? | HPA supplies no ICC-IF images or main subcellular location for ZAP70 (HPA: subcellular record). Cytoplasmic localization and activation-linked membrane recruitment are UniProt expectations (UniProt P43403: subcellular location), not an HPA-validated IF image pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive lymphoid control is blank. | A failed staining step, unsuitable retrieval or weak detection is possible (general IHC practice); HPA reports high staining in appendix lymphoid tissue and lymph node non-germinal-center cells (HPA: tissue IHC). | Review the run controls, antibody preparation, retrieval and detection sequence; repeat alongside a documented positive region before interpreting the test section as negative (general IHC practice). |
| All cells show brown haze. | Excess background can obscure the selective cytoplasmic immune-cell pattern (HPA: tissue IHC; general IHC practice). | Inspect a detection-only control, then review blocking, washing and chromogen development within the established IHC workflow (general IHC practice). Score only cell-resolved signal. |
| Signal is nuclear throughout the section. | A widespread nuclear pattern disagrees with HPA's selective cytoplasmic tissue profile (HPA: tissue IHC). | Check nuclear counterstain and detection-only controls, then compare the compartment in a positive lymphoid region before accepting the signal as ZAP70 (general IHC practice). |
| Epithelial cells or adipocytes stain strongly. | This conflicts with HPA's not detected observations for bronchial respiratory epithelium and adipose-tissue adipocytes (HPA: tissue IHC). Cross-reactivity or endogenous detection activity is possible (general IHC practice). | Compare morphology and a detection-only control; assess the same run's expected immune-cell staining before assigning positivity (general IHC practice; HPA: tissue IHC). |
| Only kidney proximal-tubule microvilli stain. | HPA records high staining there but describes a selective immune-cell profile and notes presumed off-target binding in its reliability assessment (HPA: tissue IHC). The record does not assign that off-target finding to a specific structure. | Do not use the kidney result alone to establish antibody specificity. Compare an HPA-listed lymphoid positive region and the run controls (HPA: tissue IHC; general IHC practice). |
| Membrane accentuation varies among immune cells. | Recruitment of ZAP70 to the plasma membrane depends on T-cell receptor activation (UniProt P43403: subcellular location); activation status cannot be determined from ZAP70 staining alone. | Score the predominant cytoplasmic pattern and document membrane accentuation separately. Compare cell types and controls before treating the variation as a staining failure (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing ZAP70 staining
PB9968 · IHCIHC analysis of ZAP70 using anti-ZAP70 antibody (PB9968). ZAP70 was detected in a paraffin-embedded section of human prostate cancer tissue.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000115085-ZAP70/tissue/cerebral+cortex#imid_1092968
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000115085-ZAP70/tissue/colon#imid_1092894
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000115085-ZAP70/tissue/liver#imid_1092895
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000115085-ZAP70/tissue/kidney#imid_1092896
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ZAP70 IHC & IF
🧪Run lymph node first and look for staining in non-germinal center cells (HPA: High); use adipose tissue adipocytes as the negative tissue (HPA: Not detected). On the positive slide, compare stained cells with morphologically non-lymphoid cells as internal background controls, without assuming every non-germinal center cell must stain (UniProt P43403 tissue specificity; HPA: High in non-germinal center cells).
Positive control tissue: Appendix (Lymphoid tissue, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for ZAP70; derive a cell-line control from the positive tissue's cell type (Lymphoid tissue) and confirm it by RNA or western blot first.
Technical controls: Include no-primary (secondary-only) and concentration-matched rabbit IgG isotype controls (caption: rabbit primary antibody; standard IHC practice), plus ZAP70-knockout material as a biological negative if available (standard IHC practice). Quench endogenous peroxidase and check for DAB signal in non-target cells, including myeloid cells in the lymph node (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected PB9968 paraffin-section caption does not state a fixative (caption: fixative unreported). The caption uses heat retrieval in EDTA at pH 8.0, but it does not establish that retrieval is required (caption: EDTA heat retrieval). There is no supplied evidence that frozen sections or IF are easier; for lymph node DAB staining, endogenous peroxidase is a potential artefact to control (HPA: lymph node non-germinal center cells High; standard chromogenic IHC practice).
HPA tissue IHC evidence for ZAP70
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 |
|---|---|---|---|---|
| Appendix | Lymphoid tissue | High | Protein (IHC) | HPA → |
| Kidney | Proximal tubules (microvilli) | High | Protein (IHC) | HPA → |
| Lymph node | Non-germinal center cells | High | Protein (IHC) | HPA → |
| Tonsil | Non-germinal center cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic 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 → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ZAP70 IHC Tips
Use the catalog antibody’s paraffin-section conditions as the starting point, then judge staining by cell type, compartment and matched controls.
What retrieval should I try first if ZAP70 staining is weak?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet PB9968). The catalog antibody produced a chromogenic tissue image after this retrieval, followed by 2 μg/ml primary antibody overnight at 4°C (datasheet PB9968). If signal is weak, compare retrieval duration and staining intensity across adjacent sections while keeping detection conditions constant; over-retrieval can damage morphology (standard IHC practice). Judge improvement in immune-cell cytoplasm rather than by overall section darkness, because selective cytoplasmic staining in immune-cell subsets is the reported tissue pattern (HPA tissue IHC).
Could fixation explain weak or uneven ZAP70 staining?
Target-specific fixation sensitivity is unknown: the selected tissue caption reports paraffin embedding but does not state a fixative (datasheet PB9968). Record fixation method and duration for each specimen, then compare similarly processed sections before attributing a signal difference to biology (standard IHC practice). Use a positive immune-cell region on the same slide when available, since ZAP70 is expressed in T- and natural killer cells (UniProt P43403 tissue specificity). If staining varies with processing, optimize on matched specimens; neither the reported phosphosites nor the tissue staining profile establishes which fixation conditions preserve this antibody’s epitope (UniProt P43403 modified residues; HPA tissue IHC).
Should ZAP70 stain cytoplasm, membrane, or both?
Expect predominantly cytoplasmic staining in resting immune cells: ZAP70 is cytoplasmic in quiescent T lymphocytes, and tissue IHC reports selective cytoplasmic expression in immune-cell subsets (UniProt P43403 subcellular location; HPA tissue IHC). TCR stimulation can recruit ZAP70 to the plasma membrane through CD247/CD3Z, so a membrane-associated pattern may be biologically plausible in activated cells (UniProt P43403 subcellular location). ZAP70 has no transmembrane segment, so assess membrane staining in cellular context rather than assuming a surface-exposed epitope (UniProt P43403 topology). Compare adjacent immune-cell-rich and nonimmune regions with the same counterstain and exposure conditions to distinguish cellular localisation from diffuse chromogen deposition (standard IHC practice).
Can isoforms or phosphorylation change what this antibody detects in sections?
ZAP70 has 3 listed isoforms, two SH2 domains and a kinase domain, but the supplied product evidence does not map this antibody’s epitope to any of them (UniProt P43403 isoforms and domains; datasheet PB9968). Do not infer isoform specificity from staining intensity or localisation without epitope information and an appropriate validation experiment (standard IHC practice). The record lists phosphorylation sites including Tyr248, Ser289, Tyr292, Tyr315 and Tyr319, but it does not establish that these modifications alter this antibody’s binding (UniProt P43403 modified residues). Keep retrieval and primary-antibody conditions constant across comparison sections so processing differences do not masquerade as biological differences (standard IHC practice).
How should I plan a ZAP70 multiplex IF check?
Pair ZAP70 with a marker identifying the expected immune-cell population, such as a T-cell marker, and assess signal within the same cells (UniProt P43403 tissue specificity; standard IF practice). Choose spectrally separated fluorophores and place the weaker channel away from prominent tissue autofluorescence after checking unstained tissue and single-stain controls (standard IF practice). Permeabilise sufficiently to reach cytoplasmic ZAP70 and the cytoplasmic face of membrane-associated ZAP70, then check whether cell boundaries remain interpretable (UniProt P43403 subcellular location and topology; standard IF practice). Treat IF conditions as needing their own optimization: the supplied PB9968 image establishes a paraffin-section chromogenic workflow, not an IF result (datasheet PB9968).
How can I reduce diffuse brown staining without losing immune-cell signal?
The catalog image used 10% goat serum blocking and 2 μg/ml primary antibody overnight at 4°C (datasheet PB9968). For HRP and DAB detection, include an endogenous-peroxidase block and a no-primary control to identify enzyme activity or detection-reagent background (standard IHC practice). If diffuse signal persists, compare a lower primary concentration and more thorough washes while retaining a matched positive immune-cell region (standard IHC practice; UniProt P43403 tissue specificity). HPA reports presumed off-target binding in its tissue assessment, so a dark region outside the expected cellular pattern warrants independent validation rather than automatic classification as ZAP70 (HPA tissue IHC).
How should I score ZAP70 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region of interest before scoring; ZAP70 tissue staining is selective in immune-cell subsets (HPA tissue IHC). Record the percentage of positive eligible cells and, when intensity matters, an H-score using intensity categories 0–3 multiplied by their respective cell percentages (standard IHC practice). For spatial comparisons, report positive-cell density per mm² of viable tissue or the fraction of positive cells among counted immune cells, with the denominator stated (standard IHC practice). Keep retrieval, staining batch, counterstain and scoring thresholds consistent, and exclude folds, damaged edges and necrotic areas from the measured region (standard IHC practice).
Which staining patterns should make me question a ZAP70-positive call?
A convincing call should identify stained cells and a plausible cytoplasmic or membrane-associated compartment, consistent with ZAP70 localisation and selective immune-cell staining (UniProt P43403 subcellular location; HPA tissue IHC). Diffuse nuclear staining, staining confined to section edges, or signal in necrotic areas calls for a processing and specificity check (standard IHC practice). Compare suspected positive cells with nearby immune-cell populations and a no-primary control; residual endogenous peroxidase can produce brown signal in an HRP and DAB workflow (standard IHC practice; datasheet PB9968). Interpret unexpected cell types cautiously because the HPA tissue assessment explicitly notes presumed off-target binding despite its enhanced reliability rating (HPA tissue IHC).
Boster reagents
Best ZAP70 / Tyrosine-protein kinase ZAP-70 IHC Antibodies
PB9968 has a human paraffin-section IHC image (PB9968 IHC caption). Its catalog lists Human, Mouse and Rat reactivity (PB9968 catalog); M00754-3 lists IF/ICC applications without an image (M00754-3 catalog).
Real IHC data
Anti-ZAP70 Antibody ®
Cat # PB9968
PB9968 is the sole rendered card and has a human prostate cancer paraffin-section IHC image (PB9968 IHC caption). Its listed applications are IHC and WB, and its listed reactivity is Human, Mouse and Rat (PB9968 catalog).
Which to pick: For tissue IHC, pick PB9968: its image shows human paraffin-section staining with EDTA pH 8 retrieval and 2 μg/ml primary antibody; the fixative is unreported (PB9968 IHC caption). For IF/ICC, consider rabbit monoclonal M00754-3, which lists both applications and Human reactivity but has no IF image in the payload (M00754-3 catalog). For cross-species work, PB9968 lists Human, Mouse and Rat reactivity, while its IHC image documents only a human section (PB9968 catalog; PB9968 IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P43403 (ZAP70_HUMAN, Tyrosine-protein kinase ZAP-70).
- Human Protein Atlas. ZAP70 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ZAP70 subcellular location (ICC-IF): Highest expression in JURKAT: 134.1 nTPM.
- Human Protein Atlas. ZAP70 antibody validation summary (2 antibodies).
- Immunohistochemical detection of ZAP70 in chronic lymphocytic leukemia predicts immunoglobulin heavy chain gene mutation status and time to progression. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2010 — PMC2966512.
- Serial minimal residual disease (MRD) monitoring during first-line FCR treatment for CLL may direct individualized therapeutic strategies. Leukemia 2018 — PMC6192870.
- ZAP70 interaction with 13 mRNAs as a potential immunotherapeutic target for endometrial cancer. Oncology letters 2023 — PMC10131270.
- AQP9 and ZAP70 as immune-related prognostic biomarkers suppress proliferation, migration and invasion of laryngeal cancer cells. BMC cancer 2022 — PMC9047300.
- PubMed PMID:1423621 — UniProt-cited evidence.
- PubMed PMID:14985102 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ZAP70 (Tyrosine-protein kinase ZAP-70). Expected localisation Cytoplasmic in subsets of immune cells (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9968). Validated IHC antibody: PB9968 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Lymphoid tissue); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human prostate cancer tissue; fixative not specified (datasheet PB9968); 2. Fixation: Image fixative and duration unreported (datasheet PB9968); 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 PB9968); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9968); 8. Primary antibody: Rabbit anti-ZAP70, 2-5μg/ml (datasheet PB9968); 9. Primary incubation: Overnight at 4 °C (datasheet PB9968). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Presumed off-target staining may confound interpretation (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope differences are unspecified (UniProt); Regulation: TCR activation recruits ZAP70 to the membrane (UniProt). Evidence: UniProt P43403, HPA tissue IHC · Enhanced, HPA cell-line RNA · Highest expression in JURKAT: 134.1 nTPM, Datasheet PB9968, PMC10131270. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ZAP70/immunohistochemistry-ihc-guide.