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CD28 / T-cell-specific surface glycoprotein CD28 · IHC design guide
Design Immunohistochemistry for CD28
Plan CD28 paraffin-section IHC using lymph node or tonsil, where immune-cell subsets show cytoplasmic staining (HPA tissue IHC). Compare that pattern with CD28’s expected cell-surface location (UniProt), and start the catalog antibody at 2 μg/mL (datasheet: 2 μg/mL).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CD28 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 | Observed immune-cell cytoplasm (HPA tissue IHC); expected cell surface (UniProt) | |
| Staining pattern | Selective cytoplasmic staining in immune-cell subsets (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Lymph node+1 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 across sections. (standard IHC practice; not target-specific) | |
| Caveat | Tissue IHC shows cytoplasm despite surface topology (HPA tissue IHC; UniProt) | |
| Regulation | Regulation of staining intensity unreported (UniProt) | |
| Isoform / epitope | 7 isoforms; check extracellular versus cytoplasmic epitope (UniProt) |
Section 1
Recommended CD28 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by a published CD28 protocol for FFPE tissue from patients with adult T-cell leukemia/lymphoma (PMC8748252).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A00065); 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 anti-CD28, 2 μg/mL (datasheet A00065) |
| 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 | CD28-positive staining in non-germinal center cells of lymph node (HPA tissue IHC: Medium). HPA tissue profile: Selective cytoplasmic expression in subsets of immune cells. No signal in the no-primary control. |
💡Decision noteTry citrate pH 6.0 HIER at 95–98 °C for 20 min first for membrane CD28 (page retrieval rule; UniProt P10747 topology).
Section 2
What Is the Expected CD28 Staining Pattern?
CD28 is a cell-surface receptor with an extracellular region at residues 19–152 and a membrane-spanning segment at 153–179 (UniProt P10747 topology). In tissue IHC, expect selective staining of immune-cell subsets; HPA reports medium cytoplasmic staining in lymph-node and tonsil non-germinal-center cells (HPA tissue IHC). HPA rates the tissue pattern Enhanced for consistency with RNA expression, pending external verification (HPA tissue IHC).
| Medium staining in subsets of lymph-node or tonsil non-germinal-center cells (HPA tissue IHC). | This matches the reported tissue IHC distribution and intensity (HPA tissue IHC). Score the stained immune-cell subsets separately from surrounding unstained cells; a uniformly positive field would exceed the selective pattern reported by HPA (HPA tissue IHC). |
| Predominantly nuclear staining, without the reported immune-cell pattern (HPA tissue IHC; UniProt P10747 location). | Treat a nuclear-only result as suspect: CD28 is assigned to the cell surface by UniProt, while HPA describes selective cytoplasmic tissue IHC staining (UniProt P10747 location; HPA tissue IHC). Check localization against a positive tissue control and examine the omitted-primary control before interpreting it as CD28 (standard IHC practice). |
| Strong staining of adipocytes or respiratory epithelial cells (HPA tissue IHC). | HPA reports CD28 as not detected in adipocytes of adipose tissue or respiratory epithelial cells of bronchus (HPA tissue IHC). Widespread staining there raises possible cross-reactivity or endogenous detection activity; assess the omitted-primary and detection controls (standard IHC practice). |
| Diffuse color across cells and tissue spaces, obscuring cell boundaries (standard IHC practice). | This is background rather than a scorable selective pattern (standard IHC practice; HPA tissue IHC). Compare an omitted-primary section, inspect blocking and washing, and review detection development under the same imaging conditions (standard IHC practice). |
| No signal in lymph-node or tonsil non-germinal-center cells (HPA tissue IHC). | An absent result conflicts with the reported medium staining in those cells (HPA tissue IHC). Verify that the control section contains the relevant cell population, then review antibody dilution, retrieval, and detection using the catalog antibody's IHC-P instructions (standard IHC practice). |
💡Expected CD28 appearanceCall positive a selective, approximately medium-intensity signal in lymph-node or tonsil non-germinal-center immune-cell subsets, including the cytoplasmic pattern documented for tissue IHC; diffuse staining of unrelated cells is suspect (HPA tissue IHC).
| Tissue IHC versus ICC-IF localization (HPA tissue IHC; HPA subcellular). | HPA describes selective cytoplasmic tissue IHC staining, while its ICC-IF summary supports plasma-membrane localization (HPA tissue IHC; HPA subcellular). Preserve that distinction when scoring paraffin sections; ICC-IF images do not establish the exact chromogenic tissue pattern (HPA tissue IHC; HPA subcellular). |
| CD28 topology (UniProt P10747 topology). | The extracellular region spans residues 19–152, the transmembrane segment 153–179, and the cytoplasmic tail 180–220 (UniProt P10747 topology). Membrane-associated signal is biologically plausible, but the supplied record does not identify the catalog antibody's epitope or predict its IHC appearance (UniProt P10747 topology). |
| Tissue-dependent abundance (HPA tissue IHC). | HPA reports medium staining in lymph-node and tonsil non-germinal-center cells, but low staining in appendix lymphoid tissue, splenic red-pulp cells, and marrow hematopoietic cells (HPA tissue IHC). A weak result in a low-staining tissue is less decisive than failure in the reported medium-staining controls (HPA tissue IHC). |
| Antibody-specific validation (HPA antibodies). | HPA lists IHC validation as Enhanced for HPA070003 and ICC validation as Supported for HPA041233 (HPA antibodies). These statuses apply to their respective antibodies and applications; they do not establish IHC-P performance for another antibody (HPA antibodies). |
| Reported negative cell populations (HPA tissue IHC). | HPA lists several unstained populations, including adipocytes in adipose tissue and glandular cells in adrenal gland (HPA tissue IHC). Use a documented negative population alongside a positive tissue to recognize widespread nonspecific color (HPA tissue IHC; standard IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Positive tissue has no interpretable staining (HPA tissue IHC). | The relevant non-germinal-center cells may be absent from the section, or the staining run may have failed (HPA tissue IHC; standard IHC practice). | Confirm the cell population on the counterstain; check the IHC-P dilution, retrieval, and detection steps against the catalog antibody instructions, then repeat with a positive control (standard IHC practice). |
| Low signal in appendix, spleen, or bone marrow (HPA tissue IHC). | These are reported as low-staining sites, with the specified positive populations differing by tissue (HPA tissue IHC). | Include lymph node or tonsil as a medium-staining comparator and score the specified cells in each section before treating a weak field as a failed run (HPA tissue IHC; standard IHC practice). |
| Staining appears mainly nuclear (UniProt P10747 location; HPA tissue IHC). | A nuclear-only pattern conflicts with the supplied CD28 localization and tissue IHC description; its source remains unresolved (UniProt P10747 location; HPA tissue IHC). | Compare the positive tissue and omitted-primary sections, then review staining specificity and detection background before assigning the signal to CD28 (standard IHC practice). |
| Many unrelated cell types stain strongly (HPA tissue IHC). | Cross-reactivity or endogenous detection activity is possible when cells HPA calls not detected show signal (HPA tissue IHC; standard IHC practice). | Inspect a documented negative population, run an omitted-primary control, and review the blocking and detection steps under matched conditions (HPA tissue IHC; standard IHC practice). |
| Diffuse chromogen obscures the selective pattern (HPA tissue IHC). | Excess background can make the reported subset staining impossible to distinguish (standard IHC practice; HPA tissue IHC). | Compare matched controls and review primary-antibody dilution, blocking, washes, and chromogen development using the catalog antibody's IHC-P instructions (standard IHC practice). |
| Can ICC-IF membrane images define the expected IHC-P result? (HPA subcellular). | HPA supports plasma-membrane localization by ICC-IF, but reports selective cytoplasmic staining for tissue IHC; the antibody validation entries also differ by application (HPA subcellular; HPA tissue IHC; HPA antibodies). | Judge the paraffin-section result against tissue IHC controls and its own antibody validation; consult the separate IF/ICC guide for fluorescence interpretation (HPA tissue IHC; HPA antibodies; standard IHC practice). |
Experiment evidence
IHC & IF images showing CD28 staining
A00065 · IHCImmunohistochemistry of CD28 in human spleen tissue with CD28 antibody at 2 μg/ml.
A00065 · IFImmunofluorescence of CD28 in human spleen tissue with CD28 antibody at 20 μg/ml.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000178562-CD28/tissue/cerebral+cortex#imid_19992101
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000178562-CD28/tissue/colon#imid_19992019
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000178562-CD28/tissue/liver#imid_19992028
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000178562-CD28/tissue/kidney#imid_19992021
HPA · ICC-IF JURKATHuman Protein Atlas immunofluorescence, JURKAT, antibody HPA041233; green = CD28, blue = nucleus, red = microtubules.
HPA · ICC-IF JURKATHuman Protein Atlas immunofluorescence, JURKAT, antibody HPA041233; green = CD28, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CD28 IHC & IF
🧪Run lymph node first: non-germinal center cells should stain at Medium level (HPA: lymph node, non-germinal center cells, Medium). Use adipose tissue adipocytes as the biological tissue negative (HPA: adipocytes, Not detected); other cells on the lymph node slide should remain unstained unless they express CD28 (UniProt P10747: expressed in T-cells and plasma cells).
Positive control tissue: Lymph node (Non-germinal center cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CD28 in JURKAT, U2OS, with annotated localisation: Plasma membrane (supported) (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 CD28-knockout material if available (standard IHC practice). For chromogenic lymph node IHC, quench endogenous peroxidase and check background from endogenous biotin if using biotin-based detection (standard IHC practice).
⚠️Feasibility: CD28 is a cell-surface protein, so assess staining at the cell membrane (UniProt P10747: cell surface; HPA: plasma membrane supported). A target-specific fixation window, fixation effect, and retrieval dependency are unreported in the supplied evidence; the A00065 spleen IHC caption does not state a fixative (A00065 caption: fixative not stated). The supplied evidence does not establish whether frozen sections or IF are easier; in lymph node, distinguish staining of CD28-expressing cells from background in surrounding cells (UniProt P10747: expressed in T-cells and plasma cells; standard IHC practice).
HPA tissue IHC evidence for CD28
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data. Pending external verification.). 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 |
|---|---|---|---|---|
| Lymph node | Non-germinal center cells | Medium | Protein (IHC) | HPA → |
| Tonsil | Non-germinal center cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| 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 CD28 IHC Tips
Troubleshoot CD28 staining in paraffin sections by checking retrieval, cellular distribution, controls, and scoring; use the IF guidance for a separate fluorescence workflow.
How should I retrieve CD28 in paraffin sections when staining is weak?
Use citrate pH 6.0 heat-induced antigen retrieval at 95–98 °C for 20 min for CD28 paraffin-section IHC (page retrieval setting). If staining remains weak, compare a longer or shorter retrieval time on adjacent sections while keeping the antibody and detection conditions constant (standard IHC practice). Include lymph node or tonsil as a tissue comparison, where non-germinal center cells show medium staining (HPA tissue IHC). Assess improvement in the expected immune-cell population without accepting a simultaneous rise in diffuse background or tissue damage (HPA tissue IHC; standard IHC practice).
Could fixation explain weak or uneven CD28 staining?
CD28-specific sensitivity to fixation is unknown because no target-specific fixation evidence is supplied for this IHC workflow (supplied evidence). The selected human spleen image reports staining at 2 µg/ml, but its caption does not state a fixative (catalog antibody caption, A00065). Compare sections with documented, consistent processing and run them through the same citrate pH 6.0 retrieval cycle before changing antibody concentration (page retrieval setting; standard IHC practice). If staining varies across a section, inspect preservation and retrieval uniformity alongside a positive control before attributing the difference to CD28 expression (standard IHC practice).
Should CD28 appear on the membrane or in the cytoplasm?
Prioritize staining along the cell surface: CD28 is a cell-membrane protein, with an extracellular segment at residues 19–152 and a transmembrane segment at 153–179 (UniProt P10747 topology). Supported subcellular staining is at the plasma membrane, although tissue IHC reports selective cytoplasmic staining in subsets of immune cells (HPA subcellular; HPA tissue IHC). Evaluate membrane outlining and any cytoplasmic signal separately in morphologically suitable immune cells, rather than combining them into one positive category (standard IHC practice). Diffuse staining of unrelated parenchymal cells warrants a control review before it is scored as CD28 (UniProt P10747 tissue specificity; standard IHC practice).
How can isoforms and epitope location affect CD28 IHC interpretation?
CD28 has 7 annotated isoforms, but the supplied evidence does not map this antibody's epitope to any isoform (UniProt P10747 isoforms; supplied antibody evidence). Its extracellular region spans residues 19–152, while residues 180–220 are cytoplasmic; epitope location therefore matters when interpreting accessible surface signal (UniProt P10747 topology; standard IHC practice). Five annotated glycosylation sites lie in the extracellular region, and cytoplasmic modified residues include phosphorylation sites at 189, 191, and 209 (UniProt P10747 modifications). If staining differs between preparations, check antibody epitope documentation and processing conditions before claiming isoform-specific expression or a modification-dependent effect (standard IHC practice).
How should I assess CD28 in a separate multiplex IF experiment?
For a separate IF experiment, pair CD28 with a T-cell marker such as CD3 and inspect whether the signals occur in the same cells (UniProt P10747 tissue specificity; standard IF practice). Choose a fluorophore channel after checking the tissue's autofluorescence, and include single-stain and no-primary controls to assess bleed-through and background (standard IF practice). Match permeabilisation to the mapped epitope: an extracellular epitope may be assessed without permeabilisation, whereas a cytoplasmic epitope requires access across the membrane (UniProt P10747 topology; standard IF practice). The supplied antibody evidence does not identify its epitope or establish an IF fixation condition, so optimize those choices within the IF workflow (supplied antibody evidence).
What should I check when chromogenic CD28 staining looks diffuse?
Compare the section with a no-primary control to identify staining from the detection reagents, and check the peroxidase block when using a peroxidase-based chromogen (standard IHC practice). Reassess antibody concentration and washing if color extends across cells that lack convincing immune-cell morphology (standard IHC practice). The catalog image shows human spleen staining at 2 µg/ml, which is a documented image condition rather than a validated concentration for every specimen (catalog antibody caption, A00065). Use a matched comparison section and inspect whether background rises outside the expected immune-cell distribution before interpreting faint color as CD28 (UniProt P10747 tissue specificity; standard IHC practice).
How should I quantify CD28 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the region and eligible immune-cell population before scoring, because CD28 is expressed in T-cells and plasma cells according to the protein record (UniProt P10747 tissue specificity). Report the percentage of positive eligible cells and staining intensity, or use an H-score when intensity categories are reproducible (standard IHC practice). If counting cells per mm², normalize to the analyzed tissue area and report immune-cell density so changes in cellular composition are visible (standard IHC practice). Apply the same compartment rule, threshold, counterstain review, and exclusion criteria to every section; record membrane and cytoplasmic staining separately where both occur (HPA subcellular; HPA tissue IHC; standard IHC practice).
How can I distinguish true CD28 staining from artefact?
Look for staining in plausible immune cells and assess its distribution against CD28's cell-surface location and reported tissue-IHC cytoplasmic pattern (UniProt P10747 tissue specificity and location; HPA tissue IHC). Lymph node and tonsil non-germinal center cells offer documented positive comparisons, while several non-immune cell types are reported as not detected (HPA tissue IHC). Treat strong color confined to section edges, necrotic areas, or a no-primary control as suspect; review retrieval, washing, and endogenous peroxidase blocking (standard IHC practice). HPA rates tissue staining as Enhanced but pending external verification, so corroborate a surprising cell-type assignment with morphology and an independent marker (HPA tissue IHC; standard IHC practice).
Boster reagents
Best CD28 / T-cell-specific surface glycoprotein CD28 IHC Antibodies
A00065 has IHC and IF images from human spleen tissue (catalog IHC and IF image captions); Human is its only listed reactivity (catalog reactivity: Human).
Real IHC data
Anti-CD28 Antibody
Cat # A00065
A00065 will render with an IHC image from human spleen tissue at 2 μg/mL (catalog IHC image caption). Its IF application is supported by a human spleen image at 20 μg/mL (catalog applications; catalog IF image caption).
Which to pick: Choose A00065 for human tissue IHC; its image documents human spleen, but section processing and fixative are unreported, so paraffin compatibility cannot be confirmed from the caption (catalog IHC image caption). For IF, A00065 has a human spleen image; ICC validation and clonality are unreported. No cross-species option is listed (catalog reactivity: Human).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P10747 (CD28_HUMAN, T-cell-specific surface glycoprotein CD28).
- Human Protein Atlas. CD28 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CD28 subcellular location (ICC-IF): Localized to the plasma membrane..
- Human Protein Atlas. CD28 antibody validation summary (2 antibodies).
- Clinicopathological significance of CD28 overexpression in adult T-cell leukemia/lymphoma. Cancer science 2022 — PMC8748252.
- Immunohistochemical biomarkers and distribution of telocytes in ApoE(-/-) mice. Cell biology international 2019 — PMC6851738.
- GLUT5-Mediated Disruption of the Gut-Testis Axis Deteriorates Olanzapine-Induced Testicular Fibrosis and is Ameliorated by Dapagliflozin. Drug design, development and therapy 2026 — PMC13600974.
- Tumor CTLA-4 overexpression predicts poor survival in patients with nasopharyngeal carcinoma. Oncotarget 2016 — PMC4914341.
- PubMed PMID:2825196 — UniProt-cited evidence.
- PubMed PMID:2162892 — UniProt-cited evidence.
- PubMed PMID:11916166 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CD28 (T-cell-specific surface glycoprotein CD28). Expected localisation Observed immune-cell cytoplasm (HPA tissue IHC); expected cell surface (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A00065 at 2 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Lymph node (Non-germinal center cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A00065); 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 anti-CD28, 2 μg/mL (datasheet A00065); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Tissue IHC shows cytoplasm despite surface topology (HPA tissue IHC; UniProt); Isoform / epitope: 7 isoforms; check extracellular versus cytoplasmic epitope (UniProt); Regulation: Regulation of staining intensity unreported (UniProt). Evidence: UniProt P10747, HPA tissue IHC · Enhanced, HPA ICC-IF · plasma membrane., Datasheet A00065, PMC8748252. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CD28/immunohistochemistry-ihc-guide.