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CD81 / CD81 antigen · IHC design guide
Design Immunohistochemistry for CD81
Plan chromogenic CD81 IHC on paraffin sections using an IHC-validated antibody (datasheet: A01281-2). Expect cytoplasmic and membranous staining mainly in stroma, with high levels in seminiferous ducts and germinal-center cells (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 | Cytoplasmic and membranous tissue staining (HPA tissue IHC) | |
| Staining pattern | Stroma, seminiferous ducts, germinal centers: cytoplasm/membrane (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 formalin fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Strong stromal and germinal-center staining can confound scoring (HPA tissue IHC) | |
| Regulation | No specific expression regulator reported (UniProt) | |
| Isoform / epitope | No isoforms; extracellular vs cytoplasmic epitope affects access (UniProt) |
Section 1
Recommended CD81 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published CD81 staining methods for breast tissue, lymphoid tissue, and mouse aorta. The aorta method uses frozen sections.
| 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 noteFor paraffin sections, start with citrate pH 6.0 HIER at 95–98 °C for 20 min (page antigen retrieval).
Section 2
What Is the Expected CD81 Staining Pattern?
CD81 is a four-pass membrane protein with extracellular and cytoplasmic loops (UniProt P60033 topology). In paraffin-section IHC, expect membranous and some cytoplasmic staining, chiefly in stromal cells, with strong staining in selected lymphoid and testicular cells (HPA tissue IHC). HPA rates the tissue pattern Supported, with medium consistency between antibody staining and RNA data (HPA tissue IHC).
| Membranous staining, with some cytoplasmic staining, in ovarian stromal cells or decidual cells. | This fits HPA’s High staining in ovarian stroma and placental decidual cells (HPA tissue IHC). Score the named cells and their compartments; a positive field does not mean every neighboring cell should stain. |
| Strong staining in tonsillar germinal center cells or testicular Sertoli cells. | Both are High HPA IHC examples (HPA tissue IHC). Lymph-node germinal center cells are listed at Medium, so compare like cell types rather than expecting identical intensity across organs (HPA tissue IHC). |
| Predominantly nuclear staining, without a convincing membrane or cytoplasmic pattern. | Treat this as discordant: UniProt places CD81 at the cell membrane, including a basolateral membrane complex with CLDN1 (UniProt P60033); HPA reports membranous and cytoplasmic tissue staining (HPA tissue IHC). Check morphology and controls before scoring it as CD81. |
| Strong signal in a cell population HPA lists as not detected, such as adrenal glandular cells. | This is a reason to investigate cross-reactivity or endogenous detection activity, not proof of either (HPA tissue IHC; general IHC practice). HPA’s negative calls apply to the specified cell populations, not necessarily every cell in those tissues. |
| Broad, even chromogen deposition obscures cell borders and tissue structure. | A diffuse field cannot establish CD81 localization (UniProt P60033; HPA tissue IHC). Compare a no-primary control and inspect whether the signal follows cells or background structures; endogenous activity and detection background are general IHC possibilities. |
💡Expected CD81 appearanceCall a section positive when identifiable stromal, germinal center, or Sertoli cells show predominantly membrane-associated staining with some cytoplasmic signal, at the cell-specific Medium or High levels reported by HPA; isolated nuclear signal or diffuse, cell-independent deposit is discordant (HPA tissue IHC; UniProt P60033).
| Cell population and tissue context | HPA reports High staining in ovarian stromal, placental decidual, tonsillar germinal center, and testicular Sertoli cells, but Medium staining in endometrial stromal and lymph-node germinal center cells (HPA tissue IHC). Select and score the named population, not an entire organ. |
| Topology and antibody epitope | CD81 has 4 transmembrane segments, two extracellular loops, and cytoplasmic regions (UniProt P60033 topology). The supplied record does not locate the catalog antibody’s epitope, so topology alone cannot specify an antigen retrieval condition or predict that retrieval will improve staining. |
| Evidence strength | HPA labels the tissue pattern Supported and describes medium staining–RNA consistency; HPA007234 and CAB002507 each have Supported IHC status (HPA tissue IHC; HPA antibodies). These entries support comparison with the observed pattern but do not establish that an unlisted antibody will behave identically. |
| Processing and modification annotations | UniProt lists one CD81 chain, residues 1–236, with no annotated isoforms, glycosylation sites, signal peptide, or propeptide (UniProt P60033). These annotations do not identify the IHC epitope or establish target-specific fixation sensitivity. |
| IF/ICC localization context | For the separate IF/ICC guide, HPA’s approved main location is the plasma membrane, with additional cytosolic localization (HPA subcellular). This supports a localization comparison, but the ICC-IF observation does not prescribe an IHC-P protocol or guarantee the same visual balance in tissue sections. |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in an expected positive section. | The section may lack the relevant cells, or the general IHC workflow may have failed; absence alone does not identify a CD81-specific cause (HPA tissue IHC; general IHC practice). | Verify the named cells are present, then check a known-positive tissue and the detection controls. Tonsillar germinal center cells and testicular Sertoli cells are High HPA examples (HPA tissue IHC). |
| Signal is weak in a chosen comparison tissue. | The sampled population may have a lower reported level: lymph-node germinal center cells and endometrial stromal cells are Medium (HPA tissue IHC). | Score the specified cells against the same tissue context. If controls are also weak, review antibody dilution, detection, and retrieval as general IHC workflow variables; no CD81-specific retrieval effect is supplied. |
| Staining is mostly nuclear. | Nuclear dominance conflicts with the membrane localization in UniProt and the membranous/cytoplasmic HPA tissue pattern (UniProt P60033; HPA tissue IHC). | Inspect a no-primary control and cell morphology, then compare with a known-positive compartment. Do not count nuclear-only signal as the expected CD81 pattern. |
| Chromogen covers cells and spaces indiscriminately. | Diffuse background can arise from general IHC detection or blocking issues; the pattern cannot be assigned to CD81 from appearance alone (general IHC practice). | Compare the no-primary control, review blocking and washes, and judge whether signal tracks the HPA-named cells and expected compartments (HPA tissue IHC). |
| Strong staining appears in an HPA not-detected cell type. | Cross-reactivity or endogenous detection activity is possible; HPA’s call is specific to its listed cell type (HPA tissue IHC; general IHC practice). | Confirm cell identity, inspect a no-primary control, and compare with a named positive population. For example, adrenal glandular cells are listed as Not detected (HPA tissue IHC). |
| IF/ICC Q: What localization should be checked on its separate guide page? | A membrane pattern with additional cytosolic signal is the approved ICC-IF observation (HPA subcellular); tissue IHC and ICC-IF are different preparations. | Use plasma membrane as the main IF/ICC localization check and allow additional cytosolic signal (HPA subcellular). Interpret this as a localization reference, without treating it as an IHC-P protocol. |
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 in adrenal gland glandular cells); on the tonsil slide, treat unstained neighboring cells as internal negatives only after confirming their identity, since the supplied HPA row does not identify a negative tonsil cell type.
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 and an isotype control matched to the primary antibody’s host species and immunoglobulin class; use CD81 knockout material as a biological specificity control where available. For chromogenic tonsil IHC, block endogenous peroxidase and check the controls for background in leukocyte-rich regions (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and retrieval dependency are unreported in the supplied evidence; optimize antigen retrieval for the IHC-validated antibody on the chosen paraffin sections (standard IHC practice). The selected M01281-1 caption shows paraffin-section prostate cancer staining at 1:2000, but its fixative is unreported (selected tissue-IHC caption). The evidence does not establish that frozen sections or IF are easier; tonsil’s dense immune-cell populations can complicate assignment of staining to germinal center cells, so assess morphology alongside the 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 section chromogenic IHC using membrane topology, reported tissue patterns, and appropriate controls (UniProt P60033; HPA tissue IHC).
How should I retrieve CD81 in paraffin sections when membrane staining is weak?
Start with citrate buffer at pH 6.0 for heat induced retrieval at 95–98 °C for 20 min (page retrieval rule). CD81 has 4 transmembrane segments and extracellular regions at residues 34–63 and 113–201, so assess preservation of cell boundaries while optimizing retrieval (UniProt P60033 topology; standard IHC practice). Compare retrieved sections with a no primary control and a consistently positive tissue compartment, keeping section thickness and detection conditions constant (standard IHC practice; HPA tissue IHC). If staining remains weak, test a different retrieval buffer or pH on matched sections and judge signal against background and morphology (standard IHC practice).
Can I attribute weak CD81 staining to formalin fixation?
The selected tissue image reports paraffin embedded human prostate cancer and a 1:2000 antibody dilution, but does not state its fixative (caption: M01281-1). Target specific fixation sensitivity is therefore unknown from the supplied IHC evidence; the HPA tissue pattern does not establish a fixation effect (caption: M01281-1; HPA tissue IHC). For troubleshooting, record fixative, fixation duration, processing history, and section age for each specimen, then compare matched sections through the same retrieval and detection workflow (standard IHC practice). If a known positive compartment stains on the same run while the sample remains weak, investigate specimen handling and cell composition before changing the primary concentration (standard IHC practice).
Should CD81 appear only at cell borders in chromogenic IHC?
CD81 is assigned to the cell membrane, including the basolateral membrane in a complex with CLDN1 (UniProt P60033 subcellular annotation). HPA tissue IHC also describes cytoplasmic and membranous staining, mainly in stroma, so some intracellular signal can be compatible with the reported pattern (HPA tissue IHC). Examine whether staining traces intact cell outlines or reproducible cytoplasmic compartments in relevant cells, using the same counterstain and microscope settings across sections (standard IHC practice). Diffuse staining across damaged tissue, section edges, and acellular material requires comparison with a no primary control before it is scored as CD81 (standard IHC practice).
How does CD81 topology affect epitope troubleshooting in tissue sections?
The supplied record lists 1 chain spanning residues 1–236 and no annotated isoforms, glycosylation sites, or modified residues (UniProt P60033 processing, isoforms and PTMs). Its extracellular loops span residues 34–63 and 113–201, while the short termini and residues 85–89 face the cytoplasm (UniProt P60033 topology). Check the antibody's documented immunogen or epitope before interpreting loss of staining after retrieval changes; the supplied caption does not identify that epitope (caption: M01281-1; standard IHC practice). Use matched sections and a consistent positive compartment to assess epitope accessibility without assuming that an unreported modification explains weak staining (standard IHC practice; HPA tissue IHC).
How can I check a CD81 IHC pattern with multiplex immunofluorescence?
Use IF as a separate check of cell identity and location, multiplexing CD81 with a validated marker for the cell population under examination (standard IF practice). HPA reports high CD81 staining in tonsillar germinal center cells and ovarian stromal cells, which can guide the choice of a relevant expected compartment (HPA tissue IHC). Choose fluorophores after inspecting tissue autofluorescence, and include single color and no primary controls to assess bleed through and background (standard IF practice). For an intracellular epitope, assess permeabilisation; for an extracellular epitope, compare conditions that preserve surface staining, because CD81 contains both cytoplasmic and extracellular regions (UniProt P60033 topology; standard IF practice).
What should I change when CD81 chromogenic staining obscures tissue detail?
First compare the stained section with no primary and detection only controls to locate nonspecific reagent signal or endogenous peroxidase activity (standard IHC practice). Apply an appropriate peroxidase block before chromogenic detection, and check whether longer DAB development increases staining in compartments without clear cell boundaries (standard IHC practice). The selected paraffin section caption documents a 1:2000 primary dilution, but supplies no background measurements or fixative, so treat that dilution as a documented reference rather than an optimum for every specimen (caption: M01281-1). Titrate antibody and detection conditions on matched sections while retaining a positive tissue compartment and consistent counterstaining for comparison (standard IHC practice; HPA tissue IHC).
How should I quantify CD81 when stromal and lymphoid cells both stain? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region before scoring, because HPA reports CD81 staining mainly in tissue stroma and high staining in tonsillar germinal center cells (HPA tissue IHC). For a defined cell population, report the percentage of positive cells and an H score derived from staining intensity; record membranous and cytoplasmic patterns separately (standard IHC practice; HPA tissue IHC). For sparse positive cells, report positive cell density per mm² of viable tissue, with consistent thresholds and excluded artefact regions (standard IHC practice). Normalize cell based results to the counted cells in that population, or area based results to viable tissue area, and apply the same rule across specimens (standard IHC practice).
How do I distinguish credible CD81 staining from artefact?
A credible pattern follows identifiable cells in a relevant compartment, with membranous staining supported by CD81's cell membrane annotation (UniProt P60033 subcellular annotation; standard IHC practice). Cytoplasmic signal alone is not automatically artefactual, since HPA reports cytoplasmic as well as membranous tissue staining, but its reliability assessment notes only medium consistency with RNA expression (HPA tissue IHC). Compare the cell type with reported stromal and lymphoid patterns, then inspect section edges, necrotic areas, and acellular deposits for irregular signal (HPA tissue IHC; standard IHC practice). If staining persists in a no primary control or follows endogenous enzyme activity, exclude it from CD81 scoring and repeat the relevant block or detection check (standard IHC practice).
Boster reagents
Best CD81 / CD81 antigen IHC Antibodies
Both CD81 antibodies have human tissue IHC images (catalog IHC captions); A01281 also has IF images in A549 cells and human brain (A01281 IF captions). Both list human, mouse, and rat reactivity (catalog reactivity).
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 ICC/IF and shows IHC in paraffin-embedded human prostate cancer, testis cancer, Hodgkin’s lymphoma, and tonsil (M01281-1 applications; IHC captions). A01281 lists IHC-P and IF, with IHC images in human lung and brain and IF images in A549 cells and human brain (A01281 applications; image captions).
Which to pick: For paraffin-section tissue IHC, choose M01281-1 if a rabbit monoclonal with human paraffin-section examples suits the experiment; its fixative is unreported (M01281-1 host/clone; IHC captions). For IF, A01281 has its own cell and tissue images; for ICC, M01281-1 lists ICC/IF, although no IF image is supplied for it (A01281 IF captions; M01281-1 applications and image captions). Both list human, mouse, and rat reactivity, while their supplied IHC images show human samples only (catalog reactivity; IHC 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 Cytoplasmic and membranous tissue staining (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 formalin fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Strong stromal and germinal-center staining can confound scoring (HPA tissue IHC); Isoform / epitope: No isoforms; extracellular vs cytoplasmic epitope affects access (UniProt); Regulation: No specific expression regulator reported (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.