This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
CD2AP / CD2-associated protein · IHC design guide
Design Immunohistochemistry for CD2AP
Plan CD2AP chromogenic IHC in paraffin sections using kidney glomerular cells as a positive reference and adipocytes as a low-signal comparison (HPA tissue IHC). The catalog antibody M01756 has an IHC-P range of 0.5–1 μg/mL; evaluate cytoplasmic staining with matched controls (datasheet M01756; HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CD2AP 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 | Cytoplasm in most tissue cells (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in most cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet M01756) | |
| Positive control | Bone marrow+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 consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M01756) | |
| Caveat | Cell type matters: glomerular cells high, adipocytes undetected (HPA tissue IHC) | |
| Regulation | No expression regulator specified (UniProt) | |
| Isoform / epitope | 0 listed isoforms; one 1–639 chain (UniProt) |
Section 1
Recommended CD2AP IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet M01756) is followed by published chromogenic CD2AP protocols for human brain sections (PMC8713526) and a gastric cancer tissue microarray (PMC7078920).
| Sample | Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet M01756) |
| Fixation | Image fixative and duration unreported (datasheet M01756); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet M01756) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M01756) |
| Primary antibody | Mouse monoclonal (clone 5F8) anti-CD2AP, 0.5-1μg/ml (datasheet M01756) |
| Primary incubation | Overnight at 4 °C (datasheet M01756) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet M01756) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CD2AP-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most cells. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 heat retrieval for the catalog antibody (datasheet M01756); the gastric cancer study specifies citrate retrieval without pH or heating details (PMC7078920).
Section 2
What Is the Expected CD2AP Staining Pattern?
CD2AP should produce predominantly cytoplasmic staining in many cell types, with strong signal in kidney glomerular cells and several glandular epithelia (HPA: Enhanced tissue IHC; high in glomerular and glandular cells). Cell junction and actin-associated patterns are biologically plausible (UniProt Q9Y5K6: cytoskeleton and cell junction). CD2AP has no transmembrane segment, so a membrane-associated signal does not imply that it spans the membrane (UniProt Q9Y5K6: topology).
| Clear cytoplasmic staining in glomerular cells or breast, colon, or duodenal glandular cells, with cellular detail preserved. | This fits the reported high IHC staining in those cells (HPA: high in kidney glomeruli and breast, colon, and duodenum glandular cells). Compare the intended cells with neighboring structures; a positive tissue need not stain every cell equally (HPA: cell-specific tissue IHC observations; standard IHC practice). |
| Strong staining is confined to nuclei while cytoplasm in expected-positive cells is blank. | A nuclear-dominant result conflicts with the reported tissue pattern and annotated locations (HPA: cytoplasmic expression in most cells; UniProt Q9Y5K6: cytoplasm, cytoskeleton, and cell junction). Treat it as suspect and check detection and antibody controls before assigning CD2AP positivity (standard IHC practice). |
| Adipocytes or ovarian stromal cells stain strongly while expected-positive cells do not. | Those cell types were reported as not detected, so this distribution warrants investigation (HPA: adipocytes and ovarian stromal cells, not detected). Cross-reactivity or endogenous detection activity is possible; the slide alone cannot distinguish them (standard IHC practice). |
| Chromogen covers tissue and empty spaces uniformly, obscuring cell borders. | This is background rather than an interpretable cell-specific pattern (standard IHC practice). CD2AP tissue IHC is described as cytoplasmic expression in most cells, with differences among cell types; uniform haze cannot establish that distribution (HPA: tissue IHC profile and cell-specific levels). |
| No convincing signal appears in kidney glomerular cells on the test section. | That conflicts with the reported high glomerular staining, but one negative section does not prove absent CD2AP (HPA: kidney glomerular cells, high; standard IHC practice). Check that glomeruli are present and assess a known-positive control processed in the same run (standard IHC practice). |
💡Expected CD2AP appearanceCall a result positive when expected cells show discernible cytoplasmic staining, preferably strong in glomerular or listed glandular cells; isolated nuclear staining or strong adipocyte staining is suspect (HPA: tissue IHC profile and levels; UniProt Q9Y5K6: localization).
| Which compartment should guide IHC scoring? | Use the tissue IHC cytoplasmic profile as the primary guide (HPA: cytoplasmic expression in most cells). Junctional or peripheral accentuation can be compatible with annotated CD2AP locations, but should accompany an interpretable cellular pattern (UniProt Q9Y5K6: cell junction, ruffle, and cytoskeleton; standard IHC practice). |
| How should tissue choice affect expectations? | Kidney glomerular cells and several glandular epithelia offer reported high staining; adipocytes and smooth muscle cells were reported as not detected (HPA: tissue IHC levels). A weak result in a listed low-expression cell type is a less decisive assay check than a result in a reported high-expression cell type (HPA: tissue IHC levels; standard IHC practice). |
| What does antibody validation establish? | HPA reports Enhanced tissue IHC reliability, with high consistency between staining and RNA expression; its listed antibodies also have Enhanced IHC status (HPA: tissue reliability and antibody validation). This supports the reference pattern, while an individual slide still needs appropriate run controls (standard IHC practice). |
| Q: Should IF/ICC look identical to chromogenic tissue IHC? | A: No identical appearance is established: tissue IHC is described as mainly cytoplasmic, whereas ICC-IF reports supported plasma-membrane localization and uncertain centriolar-satellite localization (HPA: tissue IHC and subcellular ICC-IF). Interpret each application against its own reference; the ICC-IF record supplies no IHC protocol setting (HPA: application-specific records). |
| Situation | Likely cause | Next action |
|---|---|---|
| Glomerular cells are unstained. | The section may lack evaluable glomeruli, or the IHC run may have failed; high glomerular staining is the reported reference (HPA: kidney glomerular cells, high; standard IHC practice). | Confirm glomeruli are present, then compare a known-positive section stained in the same run. Review retrieval and detection against the antibody's validated IHC procedure; no CD2AP-specific retrieval setting is supplied here (standard IHC practice). |
| Signal is predominantly nuclear. | The compartment conflicts with reported cytoplasmic tissue staining and annotated CD2AP locations (HPA: tissue IHC profile; UniProt Q9Y5K6: localization). | Check a negative detection control and a known-positive tissue, then reassess antibody specificity if the nuclear pattern persists (standard IHC practice). |
| Adipocytes stain as strongly as expected-positive cells. | This differs from the reported absence of adipocyte staining; cross-reactivity or endogenous detection activity are possible explanations (HPA: adipocytes, not detected; standard IHC practice). | Compare a control without primary antibody and inspect whether signal follows cell boundaries or background. Investigate blocking and detection components using standard IHC controls (standard IHC practice). |
| Diffuse chromogen obscures cellular localization. | Background from the staining workflow may prevent assessment of the reported cytoplasmic pattern (standard IHC practice; HPA: tissue IHC profile). | Inspect the negative detection control; review blocking, washes, detection development, and counterstain so cell-level signal can be scored (standard IHC practice). |
| A low-expression tissue looks negative. | Weak staining may be difficult to distinguish from background in cell types reported at low levels, such as parathyroid glandular cells or cardiomyocytes (HPA: low tissue IHC levels; standard IHC practice). | Check a reported high-expression tissue in the same run before interpreting assay performance; score the intended cell type rather than the entire section (HPA: high and low tissue IHC levels; standard IHC practice). |
Experiment evidence
IHC & IF images showing CD2AP staining
M01756 · IHCIHC analysis of CD2AP using anti-CD2AP antibody (M01756). CD2AP was detected in paraffin-embedded section of human colon cancer. Heat mediat
M01756 · IHCIHC analysis of CD2AP using anti-CD2AP antibody (M01756). CD2AP was detected in paraffin-embedded section of human colon cancer. Heat mediat
M01756 · IFICC analysis of CD2AP using anti-CD2AP antibody (M01756). CD2AP was detected in immunocytochemical section of A431 cell. Enzyme antigen retr
A01756-2 · IHCIHC analysis of CD2AP using anti-CD2AP antibody (A01756-2). CD2AP was detected in a paraffin-embedded section of mouse kidney tissue. Heat m
A01756-2 · IHCIHC analysis of CD2AP using anti-CD2AP antibody (A01756-2). CD2AP was detected in a paraffin-embedded section of rat kidney tissue. Heat med
A01756-2 · IFIF analysis of CD2AP using anti-CD2AP antibody (A01756-2). CD2AP was detected in an immunocytochemical section of Hela cells. Enzyme antigen
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000198087-CD2AP/tissue/cerebral+cortex#imid_1212752
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000198087-CD2AP/tissue/colon#imid_1212670
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000198087-CD2AP/tissue/liver#imid_1212679
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000198087-CD2AP/tissue/kidney#imid_1212672
HPA · ICC-IF RT-4Human Protein Atlas immunofluorescence, RT-4, antibody HPA003267; green = CD2AP, blue = nucleus, red = microtubules.
HPA · ICC-IF RT-4Human Protein Atlas immunofluorescence, RT-4, antibody HPA003267; green = CD2AP, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CD2AP IHC & IF
🧪Run colon first and expect its glandular cells to stain (HPA: High in colon glandular cells). Use adipose tissue adipocytes as the negative comparator (HPA: Not detected in adipocytes); on the colon slide, non-glandular cells should show absent or weaker specific staining, but HPA does not identify a confirmed internal negative cell type there (HPA: colon glandular cells High).
Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CD2AP in RT-4, U-251MG, U2OS, A-431, NIH 3T3, with annotated localisation: Plasma membrane (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a host- and subclass-matched isotype control; a CD2AP knockout sample provides a biological negative when available (standard IHC practice). In colon sections, check the no-primary slide for endogenous peroxidase or biotin signal before interpreting chromogenic staining (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected M01756 paraffin-section caption does not state the fixative (M01756 caption: fixative not stated). The caption uses heat retrieval in citrate buffer at pH 6 for 20 minutes, but does not establish that retrieval is required; the supplied evidence also does not establish whether frozen sections or IF are easier (M01756 caption: retrieval conditions; HPA: ICC-IF images). In colon, inspect gland lumina for trapped chromogen that could be mistaken for cellular staining (standard IHC practice).
HPA tissue IHC evidence for CD2AP
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CD2AP IHC Tips
Use compartment and cell type controls to troubleshoot CD2AP staining in paraffin sections; assess fluorescence separately when planning multiplex imaging.
What retrieval should I try first if CD2AP staining is weak?
Start with heat-mediated retrieval in citrate buffer at pH 6 for 20 minutes (datasheet M01756). Compare the result with a colon section containing glandular cells, which show high CD2AP staining, while keeping section thickness and detection conditions consistent (HPA: High in colon glandular cells; standard IHC practice). If signal remains weak, vary heating time on adjacent sections before considering another retrieval buffer, and watch for tissue damage or rising background (standard IHC practice). Record the retrieval condition alongside the antibody concentration of 1 μg/ml used in the illustrated paraffin-section workflow so comparisons are interpretable (datasheet M01756).
Could fixation explain weak or uneven CD2AP staining?
Target-specific fixation sensitivity is unknown: the M01756 paraffin-section caption does not state a fixative, so its staining result cannot establish an optimal fixation condition (datasheet M01756). Check the specimen’s recorded fixative and processing history, then compare sections processed together before changing retrieval or antibody concentration (standard IHC practice). Use a matched positive-control section in each run; colon glandular cells or kidney glomerular cells are reported with high staining, although either control must share the tested processing conditions (HPA: High in colon glandular cells and kidney glomerular cells; standard IHC practice). Treat a processing-associated difference as a working explanation until it is reproduced in matched material (standard IHC practice).
Which CD2AP staining pattern should I accept in tissue sections?
Expect predominantly cytoplasmic staining across many tissue cells, with particularly high staining reported in kidney glomerular cells and colon glandular cells (HPA: cytoplasmic expression in most cells; High in kidney glomerular cells and colon glandular cells). CD2AP is associated with the cytoskeleton and cell junctions, including the podocyte slit diaphragm, so a junction-adjacent pattern can be biologically plausible where the structure is resolvable (UniProt Q9Y5K6 localisation). Score cytoplasmic and edge-associated signal separately rather than treating every dark cell border as specific (standard IHC practice). Predominantly nuclear staining warrants renewed examination of controls and morphology because the supplied localisation records do not identify the nucleus as a main site (UniProt Q9Y5K6 localisation; HPA tissue IHC).
How should I troubleshoot an apparent epitope-dependent staining difference?
The supplied record lists one CD2AP chain of 1–639 amino acids and no isoforms, so it does not support assigning different tissue patterns to known isoforms (UniProt Q9Y5K6 processing and isoforms). CD2AP has three SH3 domains, including regions 1–59, 108–167 and 269–330, plus reported phosphorylation sites; an antibody’s actual epitope must be established from its own documentation (UniProt Q9Y5K6 domains and modified residues; standard IHC practice). Compare serial sections with matched retrieval and controls when another antibody gives a different pattern (standard IHC practice). Do not attribute a discrepancy to phosphorylation or domain masking without epitope information and direct experimental evidence (UniProt Q9Y5K6 modified residues; standard IHC practice).
How can I check CD2AP localisation by multiplex immunofluorescence?
Pair CD2AP with a marker identifying the expected cell population, such as a validated glomerular-cell marker when examining kidney, and assess overlap at the cell level (HPA: High in kidney glomerular cells; standard IF practice). Select spectrally separated fluorophores after checking the specimen’s autofluorescence in unstained and single-channel controls (standard IF practice). CD2AP has no transmembrane segment and is reported in cytoplasmic, cytoskeletal and junctional locations, so test gentle permeabilisation to expose its intracellular epitope while preserving boundaries (UniProt Q9Y5K6 topology and localisation; standard IF practice). Optimise fixation and permeabilisation for the IF specimen itself; the M01756 paraffin-section caption supplies no IF fixation condition (datasheet M01756; standard IF practice).
What should I check when CD2AP DAB staining looks diffuse?
Run a no-primary control and inspect whether diffuse DAB persists, then review endogenous peroxidase blocking and nonspecific secondary binding (standard chromogenic IHC practice). The illustrated M01756 workflow used 10% goat serum block, 1 μg/ml primary antibody overnight at 4°C, a biotinylated secondary, and DAB development (datasheet M01756). If background rises with primary antibody, compare a lower concentration and shorter incubation on adjacent sections while retaining a positive-control section (standard IHC practice). Judge improvement by preserved cytoplasmic signal in expected cells, rather than by a uniformly pale section (HPA: cytoplasmic expression in most cells; standard IHC practice).
How should I quantify CD2AP staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region before scoring; glandular cells in colon and cells in kidney glomeruli have reported high staining, so whole-section averages can obscure cell composition (HPA: High in colon glandular cells and kidney glomerular cells; standard IHC practice). For a chromogenic readout, report an H-score or percentage of positive cells with the intensity categories and threshold specified in advance (standard IHC practice). If counting discrete positive cells, report density per mm² of evaluable tissue and exclude damaged or necrotic areas by a consistent rule (standard IHC practice). Normalise comparisons to the same cell type, sampled area and staining batch, using matched controls to monitor run variation (standard IHC practice).
How do I distinguish genuine CD2AP staining from artefact?
A convincing result follows the expected cell population and predominantly cytoplasmic distribution: colon glandular cells and kidney glomerular cells are reported high, while adipocytes are reported undetected (HPA: tissue IHC profile and cell-level staining). Interpret a cell-border accent only with intact morphology, given CD2AP’s cytoskeletal and junctional associations (UniProt Q9Y5K6 localisation; standard IHC practice). Be cautious with staining confined to section edges, folds or necrotic regions, and compare those areas with internal tissue and a no-primary control (standard IHC practice). DAB signal that remains without primary antibody calls for review of endogenous enzyme blocking and detection background before assigning CD2AP positivity (standard chromogenic IHC practice).
Boster reagents
Best CD2AP / CD2-associated protein IHC Antibodies
Anti-CD2AP antibodies have paraffin-section IHC images from human colon cancer (M01756 image caption) and mouse and rat kidney (A01756-2 image captions), plus IF data from HeLa cells (A01756-2 image caption).
Real IHC data
Anti-CD2AP Antibody ® (monoclonal, 5F8)
Cat # M01756
Real IHC data
Anti-CD2AP Antibody ®
Cat # A01756-2
M01756 will render with a human colon cancer paraffin-section IHC image; its listed reactivity is human, mouse and rat (catalog: M01756 applications and image caption). A01756-2 will render with a mouse kidney paraffin-section IHC image; rat kidney IHC and HeLa cell IF images are also documented (catalog: A01756-2 image captions).
Which to pick: For human tissue IHC, choose monoclonal M01756, whose own paraffin-section caption documents citrate retrieval at pH 6; the fixative is unreported (catalog: M01756 clone and IHC image caption). For IF/ICC, choose A01756-2 when a fluorescence image is useful: its HeLa cell caption documents fluorescent detection, while M01756 lists IF/ICC but its supplied ICC image uses DAB (catalog: A01756-2 IF image caption; M01756 applications and ICC image caption). For demonstrated kidney IHC across mouse and rat, choose A01756-2; both antibodies list human, mouse and rat reactivity, and A01756-2’s kidney captions document EDTA retrieval at pH 8.0 without reporting the fixative (catalog: reactivity and A01756-2 IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y5K6 (CD2AP_HUMAN, CD2-associated protein).
- Human Protein Atlas. CD2AP tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CD2AP subcellular location (ICC-IF): Mainly localized to the plasma membrane. In addition localized to the centriolar satellites..
- Human Protein Atlas. CD2AP antibody validation summary (3 antibodies).
- Shensu IV maintains the integrity of the glomerular filtration barrier and exerts renal protective effects by regulating endogenous hydrogen sulfide levels. Frontiers in pharmacology 2024 — PMC11667557.
- Association of CD2AP neuronal deposits with Braak neurofibrillary stage in Alzheimer's disease. Brain pathology (Zurich, Switzerland) 2022 — PMC8713526.
- CD2AP inhibits metastasis in gastric cancer by promoting cellular adhesion and cytoskeleton assembly. Molecular carcinogenesis 2020 — PMC7078920.
- Tangzhiqing Granules Alleviate Podocyte Epithelial-Mesenchymal Transition in Kidney of Diabetic Rats. Evidence-based complementary and alternative medicine : eCAM 2017 — PMC5259601.
- PubMed PMID:10339567 — UniProt-cited evidence.
- PubMed PMID:14574404 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CD2AP (CD2-associated protein). Expected localisation Cytoplasm in most tissue cells (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet M01756). Validated IHC antibody: M01756 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet M01756); 2. Fixation: Image fixative and duration unreported (datasheet M01756); 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: Citrate pH 6 , 20 min (datasheet M01756); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M01756); 8. Primary antibody: Mouse monoclonal (clone 5F8) anti-CD2AP, 0.5-1μg/ml (datasheet M01756); 9. Primary incubation: Overnight at 4 °C (datasheet M01756). Watch for: Fixation: Keep fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M01756); Caveat: Cell type matters: glomerular cells high, adipocytes undetected (HPA tissue IHC); Isoform / epitope: 0 listed isoforms; one 1–639 chain (UniProt); Regulation: No expression regulator specified (UniProt). Evidence: UniProt Q9Y5K6, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the plasma membrane. In addition localized to…, Datasheet M01756, PMC8713526, PMC7078920. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CD2AP/immunohistochemistry-ihc-guide.