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CD81 / CD81 antigen · IHC design guide
Design Immunohistochemistry for CD81
Plan chromogenic CD81 IHC in paraffin sections using tonsil germinal centers as a high-staining reference (HPA tissue IHC). Start the IHC-P catalog antibody A01281 at 2 μg/mL and assess membrane and cytoplasmic staining by cell population (datasheet: A01281; HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CD81 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 | Stromal cytoplasm and membranes (HPA tissue IHC) | |
| Staining pattern | Mainly stromal staining; high in germinal center cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Endometrium+4 more · see all | |
| Negative control | Adrenal gland+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Stromal staining may complicate germinal center scoring (HPA tissue IHC) | |
| Regulation | No expression regulator specified (UniProt) | |
| Isoform / epitope | No isoforms annotated; extracellular versus cytoplasmic epitopes matter (UniProt) |
Section 1
Recommended CD81 IHC & IF Protocols
The catalog antibody protocol is followed by published CD81 staining methods for human breast tissue, human tonsil and lymphoma tissue, and mouse aorta (PMC6109364; PMC2813949; PMC4523685).
| Sample | Paraffin-embedded Human prostate cancer tissue; fixative not specified (datasheet M01281-1) |
| Fixation | Image fixative and duration unreported (datasheet M01281-1); 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 GAA-3) anti-CD81, 1:50-1:200 recommended; image 1:2000 (datasheet M01281-1) |
| 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 | CD81-positive staining in cells in endometrial stroma of endometrium (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic and membranous expression in mainly in tissue stroma with additional high levels in seminiferous ducts and lymphoid germinal center cells. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval rule); clone 1D6 was published with 10 min microwave retrieval (PMC2813949 methods).
Section 2
What Is the Expected CD81 Staining Pattern?
CD81 is a four-pass membrane protein (UniProt P60033 topology); stroma and germinal centers stain (HPA IHC: Supported).
| Ovarian stromal cells stain strongly (HPA: High in ovarian stroma). | Membranous and cytoplasmic staining fits the observed pattern (HPA tissue IHC). |
| Nuclear-only staining (HPA tissue IHC: membrane and cytoplasm). | Suspect artefact; nuclear localisation is outside the reported pattern (HPA tissue IHC). |
| Strong bronchial epithelial staining (HPA: Not detected). | Check cross-reactivity or endogenous detection activity (standard IHC practice). |
| Diffuse staining across the section (standard IHC practice). | Suspect background from blocking, washing or detection (standard IHC practice). |
| No germinal center signal in tonsil (HPA: High). | The expected positive pattern is missing; assess the IHC run (HPA tissue IHC; standard IHC practice). |
💡Expected CD81 appearanceHigh stromal or germinal center membrane ± cytoplasm (HPA IHC); diffuse haze is suspect (standard IHC practice).
| Protein topology (UniProt P60033). | Four transmembrane spans and two extracellular loops (UniProt P60033 topology). |
| IHC antibody validation (HPA antibodies). | HPA007234 and CAB002507 have Supported IHC status (HPA antibodies). |
| IF/ICC: where is CD81 seen? (HPA ICC-IF) | Mainly plasma membrane, with some cytosol (HPA ICC-IF: approved). |
| Situation | Likely cause | Next action |
|---|---|---|
| No tonsil germinal center signal (HPA: High). | An expected positive control failed (HPA tissue IHC). | Review the run and IHC-validated antibody (standard IHC practice). |
| Nuclear-only staining (HPA tissue IHC: membrane and cytoplasm). | Possible nonspecific staining (standard IHC practice). | Compare with a positive tissue and detection control (standard IHC practice). |
| Strong bronchial epithelial stain (HPA: Not detected). | Possible cross-reactivity or endogenous activity (standard IHC practice). | Check a detection-only control (standard IHC practice). |
| Diffuse section-wide haze (standard IHC practice). | Possible background from blocking or washing (standard IHC practice). | Review blocking and washes (standard IHC practice). |
| Signal only in cytoplasm (HPA tissue IHC: membrane and cytoplasm). | The expected membrane component is absent (HPA tissue IHC). | Compare with the membrane pattern in a positive control (standard IHC practice). |
Experiment evidence
IHC & IF images showing CD81 staining
M01281-1 · IHCImmunohistochemical analysis of paraffin-embedded Human prostate cancer, using the Antibody at 1:2000 dilution.
M01281-1 · IHCImmunohistochemical analysis of paraffin-embedded Human testis cancer, using the Antibody at 1:400 dilution.
A01281 · IHCImmunohistochemistry of CD81 in human lung tissue with CD81 antibody at 5 μg/mL.
A01281 · IHCImmunohistochemistry of CD81 in human brain tissue with CD81 antibody at 2 μg/ml.
A01281 · IFImmunofluorescence of CD81 in A549 cells with CD81 antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000110651-CD81/tissue/cerebral+cortex#imid_805664
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000110651-CD81/tissue/colon#imid_805598
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000110651-CD81/tissue/liver#imid_805599
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000110651-CD81/tissue/kidney#imid_805584
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB002507; green = CD81, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB002507; green = CD81, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CD81 IHC & IF
🧪Run tonsil first: germinal center cells should stain (HPA: High in tonsil germinal center cells); use adrenal gland glandular cells as the negative tissue (HPA: Not detected). On the tonsil slide, cells without specific CD81 signal should show counterstain without a membrane rim; do not designate a particular neighboring cell type as negative from the supplied HPA rows (HPA: tonsil row identifies germinal center cells only).
Positive control tissue: Endometrium (Cells in endometrial stroma, HPA Medium)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CD81 in A-431, U-251MG, U2OS, with annotated localisation: Plasma membrane (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; an isotype control matched to the primary antibody’s host species and clonality; and CD81 knockout tissue as a biological negative where available (standard IHC practice). Block endogenous peroxidase in tonsil before chromogenic detection to assess background from blood and immune cells (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the selected paraffin-section caption does not state a fixative (M01281-1 caption: fixative not stated). Antigen retrieval may require optimization for paraffin IHC; the supplied evidence does not establish whether frozen sections or IF are easier (standard IHC practice; M01281-1 caption: paraffin-embedded section). In tonsil, dense germinal center cells and endogenous peroxidase can complicate assessment of membrane staining, so interpret the signal against morphology and the negative controls (HPA: High in tonsil germinal center cells; standard IHC practice).
HPA tissue IHC evidence for CD81
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Medium consistency between antibody staining and RNA expression data. Antibody staining in cells/structures not annotated, view images.). 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 |
|---|---|---|---|---|
| Endometrium | Cells in endometrial stroma | Medium | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | High | Protein (IHC) | HPA → |
| Placenta | Decidual cells | High | Protein (IHC) | HPA → |
| Testis | Sertoli cells | High | Protein (IHC) | HPA → |
| Tonsil | Germinal center cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| 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 → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CD81 IHC Tips
Troubleshoot CD81 staining in paraffin sections by checking retrieval, cell identity, membrane localisation and assay controls before interpreting chromogenic signal.
How should I retrieve CD81 in paraffin sections with weak staining?
Start with citrate pH 6.0 heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval specification). Run a known positive section alongside the test section, then compare membrane staining and tissue preservation under the same detection conditions (standard IHC practice). CD81 has four transmembrane segments and extracellular loops at residues 34–63 and 113–201, but the catalog antibody’s epitope is unspecified (UniProt P60033 topology; supplied product evidence). If staining remains weak, adjust retrieval intensity in a controlled comparison and check whether the positive control improves without excess background or tissue damage (standard IHC practice).
Could fixation explain weak or uneven CD81 staining?
Target-specific fixation sensitivity is unknown from the supplied evidence; the selected caption reports paraffin embedding but does not state a fixative (selected M01281-1 IHC caption). Record fixation conditions and compare sections processed under consistent conditions before attributing intensity differences to biology (standard IHC practice). Uneven staining may reflect processing variation, so inspect morphology and compare an internal positive cell population across the section (standard IHC practice). If a fixation change is suspected, test matched sections with the same retrieval, antibody dilution and chromogenic detection settings; this establishes whether the change affects this assay without assuming a CD81-specific fixation effect (standard IHC practice).
Where should convincing CD81 chromogenic staining appear?
Look first for staining at cell boundaries: CD81 is annotated at the cell membrane, including the basolateral membrane when associated with CLDN1 (UniProt P60033 localisation). Cytoplasmic signal can also occur in tissue IHC, and an additional cytosolic location is reported, so judge it alongside morphology and membrane staining (HPA tissue IHC profile; HPA subcellular profile). Stroma and lymphoid germinal center cells can show CD81 staining, while signal intensity varies by cell population (HPA tissue IHC profile). Review high magnification fields to distinguish a crisp cellular pattern from diffuse pigment, and compare the pattern with a control section processed in the same run (standard IHC practice).
How does CD81 topology affect epitope troubleshooting?
CD81 is a 236 amino acid, four-pass membrane protein with extracellular regions at residues 34–63 and 113–201 (UniProt P60033 topology). The supplied record annotates no isoforms or glycosylation sites, so those annotations do not identify the catalog antibody’s binding site (UniProt P60033 isoform and glycosylation annotations; supplied product evidence). Ask which region the antibody recognizes before interpreting a change in retrieval response as an extracellular or cytoplasmic epitope effect (standard IHC practice). If epitope information is unavailable, compare retrieval conditions on matched sections and require preserved morphology plus expected cellular localisation before selecting a condition (standard IHC practice).
How should I assess CD81 in a multiplex IF experiment?
For multiplex IF, pair CD81 with a marker that identifies the expected cell population, such as a B-cell marker when evaluating germinal center cells (HPA tissue IHC profile; UniProt P60033 tissue specificity). Choose fluorophores after checking the specimen’s autofluorescence in unstained controls, and place the CD81 signal in a channel with adequate separation from that background (standard IF practice). CD81 has extracellular and cytoplasmic regions, but the supplied evidence does not identify the catalog antibody’s epitope (UniProt P60033 topology; supplied product evidence). Set permeabilisation according to the verified epitope side and antibody performance, then compare membrane localisation and single-stain controls before interpreting overlap (standard IF practice).
What should I check when CD81 DAB staining is diffuse?
First compare the stained section with a no-primary control to assess secondary reagent, endogenous peroxidase and chromogen background (standard chromogenic IHC practice). Apply a peroxidase block and appropriate protein blocking as general workflow steps, then reassess whether cellular staining remains distinct from diffuse deposit (standard chromogenic IHC practice). CD81 is primarily a membrane protein, although cytoplasmic tissue staining is reported; neither observation alone makes every brown area specific (UniProt P60033 localisation; HPA tissue IHC profile). Check edges, folds and damaged regions separately, and titrate the primary antibody if background obscures the expected cell-boundary pattern (standard IHC practice).
How can I quantify CD81 staining across heterogeneous tissue? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue compartment before scoring, because CD81 tissue staining includes stroma and lymphoid germinal center cells (HPA tissue IHC profile). For chromogenic IHC, report the percentage of positive cells and staining intensity, or calculate an H-score from intensity categories using fixed thresholds (standard IHC quantification practice). Normalise each score to the number of evaluable cells in that population; for area-based counts, report positive cells per mm² of evaluable tissue (standard IHC quantification practice). Keep section thickness, imaging and threshold rules consistent, and exclude folds, necrosis and section edges by predefined criteria (standard IHC quantification practice).
How can I distinguish genuine CD81 staining from artefact?
A convincing result follows identifiable cell boundaries or a reproducible cellular pattern in an expected population, supported by an appropriate control section (UniProt P60033 localisation; HPA tissue IHC profile; standard IHC practice). Germinal center cells and several stromal populations provide documented contexts for comparison, but intensity depends on the cell type (HPA tissue IHC profile). Treat isolated nuclear staining, deposits at section edges or necrotic areas, and widespread signal in a no-primary control as reasons to investigate artefact (UniProt P60033 localisation; standard IHC practice). Check the peroxidase block when pigment persists without primary antibody, and interpret a positive field only after morphology and controls agree (standard chromogenic IHC practice).
Boster reagents
Best CD81 / CD81 antigen IHC Antibodies
The catalog antibodies have IHC images from human tissues and IF images from A549 cells and human brain (catalog image captions). Both list human, mouse, and rat reactivity (catalog: A01281, M01281-1).
Real IHC data
Anti-CD81 Rabbit Monoclonal Antibody
Cat # M01281-1
Real IHC data
Anti-CD81 antigen CD81 Antibody
Cat # A01281
M01281-1 lists IHC and IF/ICC applications and has paraffin-section IHC images from human prostate cancer, testis cancer, Hodgkin’s lymphoma, and tonsil (catalog applications; M01281-1 image captions). A01281 lists IHC-P and IF applications, with IHC images from human lung and brain and IF images from A549 cells and human brain (catalog applications; A01281 image captions).
Which to pick: For paraffin-section tissue IHC, choose M01281-1 when its rabbit monoclonal format suits the experiment: its own caption documents a human prostate cancer paraffin section, but does not report the fixative (catalog: clone GAA-3; M01281-1 image caption). For IF, choose A01281 because its own captions show A549 cells and human brain; M01281-1 lists ICC/IF, but its supplied images document IHC only (A01281 image captions; catalog applications; M01281-1 image captions). For mouse or rat samples, either SKU lists reactivity, while the supplied IHC and IF images document human samples or A549 cells; confirm performance in the chosen species (catalog reactivity; catalog image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P60033 (CD81_HUMAN, CD81 antigen).
- Human Protein Atlas. CD81 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. CD81 subcellular location (ICC-IF): Mainly localized to the plasma membrane. In addition localized to the cytosol..
- Human Protein Atlas. CD81 antibody validation summary (2 antibodies).
- Gemcitabine-cisplatin chemotherapy plus anti-PD-L1 therapy reinvigorates antitumor immune response by reprogramming the intrahepatic cholangiocarcinoma microenvironment. Frontiers in immunology 2025 — PMC12706460.
- Increased Expression of CD81 in Breast Cancer Tissue is Associated with Reduced Patient Prognosis and Increased Cell Migration and Proliferation in MDA-MB-231 and MDA-MB-435S Human Breast Cancer Cell Lines In Vitro. Medical science monitor : international medical journal of experimental and clinical research 2018 — PMC6109364.
- CD81 protein is expressed at high levels in normal germinal center B cells and in subtypes of human lymphomas. Human pathology 2010 — PMC2813949.
- Magnetic Resonance Imaging of Atherosclerosis Using CD81-Targeted Microparticles of Iron Oxide in Mice. BioMed research international 2015 — PMC4523685.
- PubMed PMID:1695320 — UniProt-cited evidence.
- PubMed PMID:16554811 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CD81 (CD81 antigen). Expected localisation Stromal cytoplasm and membranes (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: M01281-1 at 1:50-1:200 recommended; image 1:2000 (the product's own IHC validation image is shown). Controls (HPA): positive Endometrium (Cells in endometrial stroma); negative Adrenal gland. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded Human prostate cancer tissue; fixative not specified (datasheet M01281-1); 2. Fixation: Image fixative and duration unreported (datasheet M01281-1); 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 GAA-3) anti-CD81, 1:50-1:200 recommended; image 1:2000 (datasheet M01281-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Stromal staining may complicate germinal center scoring (HPA tissue IHC); Isoform / epitope: No isoforms annotated; extracellular versus cytoplasmic epitopes matter (UniProt); Regulation: No expression regulator specified (UniProt). Evidence: UniProt P60033, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the plasma membrane. In addition localized to…, Datasheet M01281-1, PMC6109364, PMC2813949. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CD81/immunohistochemistry-ihc-guide.