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CD248 / Endosialin · IHC design guide
Design Immunohistochemistry for CD248
Plan CD248 chromogenic IHC using the catalog antibody’s formaldehyde-fixed, paraffin-section validation at 2.5 μg/mL (datasheet A04735). Compare staining with the low cytoplasmic tissue profile and high staining in duodenal and small-intestinal Paneth cells (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CD248 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 | Low cytoplasmic tissue staining (HPA tissue IHC); membrane expected (UniProt) | |
| Staining pattern | Low cytoplasmic staining across several tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A04735) | |
| Positive control | Duodenum+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Use formaldehyde-fixed sections (selected-SKU IHC image A04735); keep fixation consistent. (standard IHC practice; not target-specific) | |
| Caveat | Staining in unannotated cells may complicate scoring (HPA tissue IHC) | |
| Regulation | Expressed during angiogenesis (UniProt) | |
| Isoform / epitope | 2 isoforms; check extracellular vs cytoplasmic epitope (UniProt) |
Section 1
Recommended CD248 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published CD248 staining methods for IPF lung (PMC4832513) and renal cell carcinoma tissue (PMC8006336).
| Sample | Formaldehyde-fixed, paraffin-embedded Human Colon tissue (datasheet A04735) |
| Fixation | Image formalin-fixed; duration unreported (datasheet A04735); 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 (datasheet A04735); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% serum (datasheet A04735) |
| Primary antibody | Rabbit anti-CD248, 2.5 μg/mL (datasheet A04735) |
| 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 | CD248-positive staining in paneth cells of duodenum (HPA tissue IHC: High). HPA tissue profile: Low cytoplasmic expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate pH 6 retrieval (datasheet A04735); the renal cell carcinoma study also used heat-mediated citrate retrieval (PMC8006336).
Section 2
What Is the Expected CD248 Staining Pattern?
CD248 is a membrane protein with an extracellular region at residues 18–687 and a transmembrane segment at 688–708 (UniProt Q9HCU0 topology). Expect cell-surface staining in pericytes and stromal fibroblasts (UniProt Q9HCU0 tissue specificity). Interpret that pattern alongside HPA tissue IHC, which reports high Paneth-cell staining and low cytoplasmic expression in several tissues, with medium consistency between staining and RNA data (HPA: tissue IHC, Enhanced).
| Cell borders stain in perivascular or stromal cells, while nearby structures remain comparatively faint. | This fits the plasma-membrane location (HPA: approved ICC-IF location) and reported expression in pericytes and stromal fibroblasts (UniProt Q9HCU0). Score the identified cells and their compartment; vessel-adjacent staining alone does not identify the stained cell. |
| Nuclei stain prominently, or cytoplasm stains without a discernible cell-border component. | Prominent nuclear staining conflicts with the membrane assignment (UniProt Q9HCU0 topology; HPA: plasma membrane). Cytoplasmic staining needs more care: HPA reports low cytoplasmic expression in several tissues (HPA: tissue IHC profile). Check morphology and controls before treating either pattern as specific. |
| Strong staining appears in an unexpected cell population, such as normal vascular endothelium. | Widespread normal-endothelial staining conflicts with the UniProt expression description and warrants a cross-reactivity or endogenous-detection check (UniProt Q9HCU0 tissue specificity; general IHC practice). Unexpected epithelial staining is not automatically false: HPA reports high Paneth-cell staining and medium goblet-cell staining (HPA: tissue IHC). |
| Color spreads across tissue or appears similarly in cells with and without expected CD248 expression. | A diffuse deposit does not establish cell-surface localization (UniProt Q9HCU0 topology). As a general IHC check, compare a detection-only control and inspect blocking, washes, and chromogen development; background can obscure a real cell-border signal. |
| No staining appears in Paneth cells of a duodenum or small-intestine section. | Those cells are reported as High in HPA tissue IHC, so an absent result calls for a run-level review (HPA: duodenum and small intestine). Their staining is an empirical tissue reference; HPA's medium staining–RNA consistency limits its use as a stand-alone specificity test. |
💡Expected CD248 appearanceCall a result convincing when staining outlines pericytes or stromal fibroblasts (UniProt Q9HCU0; HPA: plasma membrane), with tissue-dependent intensity including High Paneth-cell staining in HPA IHC; prominent nuclear or uniform diffuse color is a warning pattern (HPA: tissue IHC; general IHC practice).
| Topology and compartment | The extracellular region spans residues 18–687; the membrane segment spans 688–708 (UniProt Q9HCU0 topology). These support a cell-border expectation but do not identify the antibody epitope or prove that every stained compartment is specific. |
| Cell and tissue context | UniProt reports strong pericyte expression, stromal fibroblasts, and tumor-associated vascular expression (UniProt Q9HCU0 tissue specificity). HPA records High Paneth cells and Medium endometrial stromal cells, among other findings (HPA: tissue IHC). Interpret intensity by the cell scored, not the tissue name alone. |
| Evidence limits | HPA labels tissue IHC Enhanced while describing medium consistency with RNA and staining in some unannotated structures (HPA: tissue IHC reliability). Its listed antibody, HPA051856, has Enhanced IHC validation (HPA: antibodies). Neither designation makes every unexpected stained cell definitively CD248-positive. |
| IF/ICC Q: Where should signal localize? | A: HPA assigns an approved plasma-membrane location in ICC-IF images from ASC52telo and BJ fibroblasts (HPA: subcellular ICC-IF). This supports compartment interpretation; IF/ICC has a separate guide and is not an IHC-P protocol option here. |
| Isoforms and modifications | UniProt lists 2 isoforms, a cleaved signal peptide at residues 1–17, and 27 glycosylation sites (UniProt Q9HCU0). Without an epitope map or isoform-specific staining evidence, these annotations cannot explain a particular positive or negative slide. |
| Situation | Likely cause | Next action |
|---|---|---|
| Paneth cells are unstained in a duodenum or small-intestine control. | A weak run is plausible because these cells are High in HPA tissue IHC (HPA: duodenum and small intestine); the image alone cannot identify which IHC step failed. | Check control section quality, antibody dilution, retrieval, detection reagents, and chromogen timing as general IHC practice. Target-specific fixation sensitivity is unreported in the supplied sources. |
| Expected perivascular or stromal staining is weak. | The field may contain few CD248-expressing cells; UniProt reports pericyte and stromal-fibroblast expression (UniProt Q9HCU0), and HPA reports tissue-dependent staining levels (HPA: tissue IHC). | Identify the cells morphologically and compare a tissue area with an HPA-reported positive population. Review the general IHC run controls before changing antibody dilution or retrieval. |
| Strong color appears in nuclei or broadly throughout cytoplasm. | Nuclear prominence conflicts with the membrane assignment (UniProt Q9HCU0 topology; HPA: subcellular ICC-IF). HPA also reports low cytoplasmic staining, so cytoplasmic color alone has limited specificity (HPA: tissue IHC). | Compare cell borders and detection-only controls, then adjust blocking, washing, or chromogen development as general IHC practice. Do not score nuclear color as CD248 without independent support. |
| Most cells or extracellular spaces show a similar diffuse deposit. | General IHC background or endogenous detection activity may be contributing; diffuse color does not show the expected membrane localization (HPA: subcellular ICC-IF). | Inspect a detection-only control and review blocking, washes, and development time as general IHC practice. Score CD248 only where individual stained cells and their compartment can be resolved. |
| Normal-vessel endothelium appears strongly positive. | That pattern conflicts with UniProt's description of absent or barely detectable expression in normal endothelial cells (UniProt Q9HCU0 tissue specificity); a neighboring pericyte may also be mistaken for an endothelial cell. | Recheck vessel-wall morphology and the precise stained cell, then compare controls. Treat persistent endothelial color as unresolved possible cross-reactivity or endogenous activity, rather than assigning it to CD248. |
| A cell type outside the expected stromal or perivascular pattern stains. | HPA reports staining outside that pattern, including High Paneth cells and Medium goblet cells (HPA: tissue IHC), while its tissue-level consistency with RNA is medium (HPA: reliability). | Check whether HPA records that exact tissue and cell type, inspect localization, and compare controls. If the finding remains discordant, report it as an observed stain without claiming confirmed CD248 expression. |
Experiment evidence
IHC & IF images showing CD248 staining
A04735 · IHCImmunohistochemistry Validation of TEM-1 in Human Colon Tissue Immunohistochemical analysis of paraffin-embedded Human Colon tissue using an
A04735 · IFImmunofluorescence Validation of TEM-1 in Human Colon Tissue Immunofluorescent analysis of 4% paraformaldehyde-fixed Human Colon Tissue labe
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000174807-CD248/tissue/cerebral+cortex#imid_14794056
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000174807-CD248/tissue/colon#imid_14781166
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000174807-CD248/tissue/liver#imid_14781167
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000174807-CD248/tissue/kidney#imid_14781160
HPA · ICC-IF ASC52teloHuman Protein Atlas immunofluorescence, ASC52telo, antibody HPA051856; green = CD248, blue = nucleus, red = microtubules.
HPA · ICC-IF ASC52teloHuman Protein Atlas immunofluorescence, ASC52telo, antibody HPA051856; green = CD248, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CD248 IHC & IF
🧪Run duodenum first: Paneth cells should stain strongly (HPA: High in duodenal Paneth cells). Use adipose tissue as the negative, assessing adipocytes (HPA: Not detected in adipocytes); on the duodenal slide, neighboring non-Paneth epithelial cells should show only background staining, but stromal cells cannot be assumed negative (UniProt Q9HCU0 tissue specificity).
Positive control tissue: Duodenum (Paneth cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CD248 in ASC52telo, BJ [Human fibroblast], 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 a knockout specimen or immunizing-peptide block if available (standard IHC practice). Block endogenous peroxidase for chromogenic detection, assess endogenous biotin if using biotin-based detection, and check intestinal autofluorescence if using IF (standard IHC/IF practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported; the selected-SKU metadata lists the fixative as unreported, although its caption says the tissue was fixed with formaldehyde (A04735 caption and fixative field). The caption uses heat-mediated citrate retrieval at pH 6, but does not establish whether retrieval is required (A04735 caption). Frozen sections are not shown to be easier; ICC-IF supports plasma membrane localization in ASC52telo and BJ cells without validating frozen tissue (HPA subcellular).
HPA tissue IHC evidence for CD248
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 |
|---|---|---|---|---|
| Duodenum | Paneth cells | High | Protein (IHC) | HPA → |
| Small intestine | Paneth cells | High | Protein (IHC) | HPA → |
| Appendix | Germinal center cells | Medium | Protein (IHC) | HPA → |
| Colon | Goblet cells | Medium | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | 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 → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CD248 IHC Tips
Troubleshoot CD248 staining in paraffin sections by checking retrieval, compartment, controls, and cell-specific scoring before interpreting the chromogenic signal.
What retrieval should I try first when CD248 staining is weak?
Start with heat-mediated citrate retrieval at pH 6 (datasheet A04735). The selected paraffin-section image used this retrieval before overnight primary incubation at 4°C (datasheet A04735). If staining remains weak, compare a modest range of heating times on adjacent sections while keeping antibody concentration and detection conditions constant (standard IHC practice). Excessive heating can damage tissue morphology and increase nonspecific staining, so judge signal alongside intact cell boundaries and a negative control (standard IHC practice). CD248 has an extracellular region spanning residues 18–687; retrieval performance still depends on the antibody’s actual epitope (UniProt Q9HCU0 topology; standard IHC practice).
Could fixation explain weak or uneven CD248 staining?
Target-specific fixation sensitivity for CD248 is unknown from the supplied evidence; do not assign weak staining to a particular fixative without a controlled comparison. For paraffin IHC, compare sections with documented fixation and processing histories, then keep retrieval and detection conditions matched (standard IHC practice). Uneven fixation or processing can produce uneven antigen access, so inspect tissue preservation before changing the primary antibody concentration (standard IHC practice). Run a known staining control in the same batch and include a primary-antibody omission control to distinguish a batch failure from detection background (standard IHC practice). Record fixation duration when available, because it helps make repeat experiments comparable (standard IHC practice).
Where should a convincing CD248 signal appear in tissue?
Expect CD248-associated staining at cell surfaces because its approved subcellular location is the plasma membrane (HPA: plasma membrane, approved). Its extracellular region spans residues 18–687, followed by a transmembrane segment at 688–708 and a cytoplasmic tail at 709–757 (UniProt Q9HCU0 topology). Examine stromal and perivascular cells carefully: CD248 is reported in stromal fibroblasts and strongly in pericytes (UniProt Q9HCU0 tissue specificity). HPA also reports low cytoplasmic staining across several tissues, so a cytoplasmic-only pattern needs corroboration rather than automatic rejection (HPA tissue IHC profile). Use morphology and an appropriate cell-type marker to identify stained cells within vascular structures (standard IHC practice).
How could the antibody epitope affect my CD248 IHC result?
CD248 has 2 annotated isoforms, so check which sequence region the antibody recognizes before comparing results across antibodies (UniProt Q9HCU0 isoforms; standard IHC practice). The protein has an extracellular region at residues 18–687 and a cytoplasmic tail at 709–757 (UniProt Q9HCU0 topology). Multiple extracellular glycosylation sites are annotated, but their effect on this antibody’s staining has not been established here (UniProt Q9HCU0 glycosylation). Ask for the immunogen or mapped epitope and verify whether it is present in both isoforms before interpreting a negative section (standard IHC practice). If epitope information is unavailable, compare independently validated antibodies and retain appropriate controls (standard IHC practice).
How should I adapt CD248 localisation checks for multiplex IF?
For multiplex IF, pair CD248 with a marker that identifies the expected stromal or perivascular cell population, then assess whether signals occupy the same cells (UniProt Q9HCU0 tissue specificity; standard IF practice). Choose fluorophores after checking unstained tissue for autofluorescence, and place the weaker signal in a channel with less background (standard IF practice). CD248 spans the plasma membrane, with extracellular residues 18–687 and cytoplasmic residues 709–757 (UniProt Q9HCU0 topology). For an extracellular epitope, begin without permeabilisation; for a cytoplasmic-tail epitope, test gentle permeabilisation against an intact-cell control (standard IF practice). Include single-stain controls to assess spectral bleed-through (standard IF practice).
How can I reduce diffuse chromogenic background around CD248-positive structures?
First compare the stained section with primary-antibody omission and detection-only controls to locate background from the detection system (standard IHC practice). In the selected paraffin-section example, the primary concentration was 2.5 µg/ml and blocking used 10% serum for 1 hour at room temperature (datasheet A04735). If diffuse staining persists, titrate the primary downward and review washing and blocking while preserving the same retrieval conditions (standard IHC practice). Apply a peroxidase block for HRP detection and interpret residual vascular or cellular pigment against the controls (standard chromogenic IHC practice). Prefer crisp cell-associated signal over broad staining at folds or section edges (standard IHC practice).
What should I score when comparing CD248 IHC across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the region of interest before scoring and keep acquisition, threshold, and counterstain settings consistent across sections (standard IHC practice). Report an H-score based on staining intensity and the percentage of positive cells, or report positive-cell density per mm² of viable tissue (standard IHC practice). Because CD248 is reported in stromal fibroblasts and pericytes, score identified compartments separately when the study asks about their contribution (UniProt Q9HCU0 tissue specificity). Normalise stromal counts to viable stromal area and perivascular counts to a defined vascular area or vessel count (standard IHC practice). Record excluded folds, necrosis, and edge regions before comparing specimens (standard IHC practice).
How do I distinguish true CD248 staining from artefact?
A convincing result links cell-associated staining to the plasma membrane and to morphologically identified stromal or perivascular cells (HPA: plasma membrane, approved; UniProt Q9HCU0 tissue specificity). Do not assign every stained endothelial-looking profile to CD248-positive endothelium: the record reports strong pericyte expression and limited expression in normal endothelial cells (UniProt Q9HCU0 tissue specificity). Treat staining confined to section edges, folds, or necrotic areas as suspect and compare it with well-preserved interior tissue (standard IHC practice). Check omission controls for endogenous enzyme or detection-system signal before accepting a chromogenic deposit (standard IHC practice). HPA tissue staining includes cytoplasmic patterns, so resolve unexpected compartments with controls and cell identification (HPA tissue IHC profile).
Boster reagents
Best CD248 / Endosialin IHC Antibodies
The catalog antibody A04735 has human colon paraffin-section IHC data and human colon tissue IF data (A04735 image captions); its listed reactivity is human (catalog: A04735).
Real IHC data
Anti-TEM1 CD248 Antibody
Cat # A04735
A04735 will render with an IHC figure from formaldehyde-fixed, paraffin-embedded human colon tissue (A04735 IHC image caption). Its IF validation shows paraformaldehyde-fixed human colon tissue (A04735 IF image caption).
Which to pick: Choose A04735 for human tissue IHC on paraffin sections: its own IHC image caption reports formaldehyde fixation, citrate-buffer heat retrieval at pH 6, and a primary concentration of 2.5 μg/mL (A04735 IHC image caption). For tissue IF, A04735 has a human colon example at 20 μg/mL; ICC is not listed among its applications (A04735 IF image caption; catalog: A04735 applications). No cross-species option is supported by the listed reactivity, which is human only (catalog: A04735 reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9HCU0 (CD248_HUMAN, Endosialin).
- Human Protein Atlas. CD248 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CD248 subcellular location (ICC-IF): Localized to the plasma membrane..
- Human Protein Atlas. CD248 antibody validation summary (1 antibodies).
- Role of CD248 as a potential severity marker in idiopathic pulmonary fibrosis. BMC pulmonary medicine 2016 — PMC4832513.
- Pan-cancer analysis identified CD248 as a potential target for multiple tumor types. Frontiers in pharmacology 2025 — PMC12018388.
- The Diagnostic and Immunotherapeutic Value of CD248 in Renal Cell Carcinoma. Frontiers in oncology 2021 — PMC8006336.
- Characterisation of fibroblast-like synoviocytes from a murine model of joint inflammation. Arthritis research & therapy 2013 — PMC3672796.
- PubMed PMID:10947988 — UniProt-cited evidence.
- PubMed PMID:11084048 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CD248 (Endosialin). Expected localisation Low cytoplasmic tissue staining (HPA tissue IHC); membrane expected (UniProt). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A04735). Validated IHC antibody: A04735 at 2.5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Duodenum (Paneth cells); negative Adipose tissue. IHC-P workflow on paraffin sections. Protocol steps: 1. Sample: Formaldehyde-fixed, paraffin-embedded Human Colon tissue (datasheet A04735); 2. Fixation: Image formalin-fixed; duration unreported (datasheet A04735); 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 (datasheet A04735); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% serum (datasheet A04735); 8. Primary antibody: Rabbit anti-CD248, 2.5 μg/mL (datasheet A04735); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Use formaldehyde-fixed sections (selected-SKU IHC image A04735); keep fixation consistent. (standard IHC practice; not target-specific); Caveat: Staining in unannotated cells may complicate scoring (HPA tissue IHC); Isoform / epitope: 2 isoforms; check extracellular vs cytoplasmic epitope (UniProt); Regulation: Expressed during angiogenesis (UniProt). Evidence: UniProt Q9HCU0, HPA tissue IHC · Enhanced, HPA ICC-IF · plasma membrane., Datasheet A04735, PMC4832513, PMC8006336. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CD248/immunohistochemistry-ihc-guide.