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CD109 / CD109 antigen · IHC design guide
Design Immunohistochemistry for CD109
Plan chromogenic CD109 IHC on paraffin sections using parathyroid glandular cells and hair follicle external root sheath cells as positive controls (HPA tissue IHC). This guide covers fixation consistency, membranous staining assessment (HPA tissue IHC), and epitope planning for 4 annotated isoforms (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CD109 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 | Membrane in parathyroid gland and hair follicle (HPA tissue IHC) | |
| Staining pattern | Membranous in glandular and external root sheath cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Hair+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 paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Endothelial cell expression may add tissue signal (UniProt) | |
| Regulation | Expressed in activated platelets (UniProt) | |
| Isoform / epitope | 4 isoforms; mature chain 22–1420; map the epitope (UniProt) |
Section 1
Recommended CD109 IHC & IF Protocols
Compare the catalog antibody's IHC-P protocol (catalog datasheet) with 4 published CD109 IHC methods (PMC6639710; PMC7926025; PMC5929441; PMC5808841).
| Sample | Paraffin-embedded human liver tissue; fixative not specified (datasheet A03248) |
| Fixation | Image fixative and duration unreported (datasheet A03248); 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-CD109, 1:50 recommended; image 1:200 (datasheet A03248) |
| 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 | CD109-positive staining in cells in external root sheath of hair (HPA tissue IHC: High). HPA tissue profile: Membranous expression in parathyroid gland and hair follicle. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page antigen retrieval). Tris–EDTA pH 9.0 strengthened staining with KU42.33C (PMC5929441).
Section 2
What Is the Expected CD109 Staining Pattern?
CD109 is a cell-surface protein without a transmembrane segment (UniProt Q6YHK3 topology; UniProt Q6YHK3: cell membrane). In paraffin-section IHC, expect predominantly membranous staining in parathyroid glandular cells and cells of the hair follicle external root sheath (HPA tissue IHC: High; membranous profile). HPA rates the tissue pattern Enhanced, citing consistency between antibody staining and RNA expression (HPA tissue IHC: Enhanced).
| Clear cell-edge staining in parathyroid glandular cells or the hair follicle external root sheath. | This matches the reported membranous pattern in two High-staining cell populations (HPA tissue IHC: profile; High in both populations). Judge the relevant cells and compartment together; a dark deposit elsewhere does not establish the expected CD109 pattern. |
| Predominantly nuclear or broadly cytoplasmic staining, with little staining at cell edges. | Reassess this as an IHC result because HPA describes membranous tissue staining (HPA tissue IHC: membranous profile). Cytosolic localization is additionally reported by ICC-IF, so cytoplasmic signal alone is not proof of artefact (HPA subcellular: Cytosol, approved). |
| Strong staining in adipocytes or respiratory epithelial cells instead of the expected positive cells. | These cell types were Not detected in the listed HPA tissue images (HPA tissue IHC: adipose adipocytes; bronchus respiratory epithelium). Check antibody specificity and endogenous detection activity before calling them CD109-positive; an unexpected stain alone cannot distinguish those causes (general IHC practice). |
| Haze across cells and tissue spaces obscures their edges. | The compartment cannot be scored reliably against the membranous reference pattern (HPA tissue IHC: membranous profile). Background may arise from nonspecific antibody binding or the detection system; compare controls before interpreting faint cell-associated color (general IHC practice). |
| No visible staining in parathyroid glandular cells or the hair follicle external root sheath. | This conflicts with the reported High staining in those cells (HPA tissue IHC: High in both populations). First check that the expected cells are present, then assess the staining run; one negative section does not establish absent CD109 expression (general IHC practice). |
💡Expected CD109 appearanceCall a positive result when High, predominantly membranous staining outlines parathyroid glandular cells or hair follicle external root sheath cells (HPA tissue IHC: High; membranous profile); diffuse haze or isolated staining in HPA-listed Not detected cell types warrants review rather than a CD109-positive call (HPA tissue IHC: Not detected; general IHC practice).
| Surface location and topology | A cell-edge pattern fits CD109's cell-membrane annotation and absence of a transmembrane segment (UniProt Q6YHK3: cell membrane; topology). Its GPI-anchor keyword supports a surface-associated interpretation but does not specify how it will appear after paraffin processing (UniProt Q6YHK3: GPI-anchor keyword). |
| Processed protein and epitope | UniProt annotates a signal peptide at residues 1–21, a CD109 chain at 22–1420, and a propeptide at 1421–1445 (UniProt Q6YHK3: processing). Those boundaries matter when interpreting a known antibody epitope; no epitope position is supplied here, so reactivity with each processed form cannot be predicted. |
| Isoforms | Four isoforms are listed; isoform 1 is reported in keratinocytes and placenta, and isoform 4 in placenta (UniProt Q6YHK3: isoforms; tissue specificity). These expression notes do not establish which isoforms the IHC antibody recognizes or guarantee staining in every cell of those tissues. |
| Tissue evidence and antibody validation | The HPA tissue pattern is Enhanced, and HPA015723 has an Enhanced IHC designation (HPA tissue IHC: reliability; HPA antibodies: HPA015723 IHC Enhanced). Use the reported cell-level pattern as the comparison; the designation does not validate an unexpected compartment or cell type in a new specimen. |
| Antigen retrieval | Retrieval conditions are an assay variable for paraffin-section IHC (general IHC practice). No CD109-specific retrieval or fixation response is supplied by UniProt or HPA; optimize against a reported positive cell population without claiming that a particular treatment exposes a CD109 epitope. |
| IF/ICC Q&A: where should CD109 appear? | Mainly at the plasma membrane, with an additional cytosolic location (HPA subcellular: Plasma membrane, supported; Cytosol, approved). This ICC-IF observation can inform localization, but it does not replace the separate membranous tissue-IHC reference pattern (HPA tissue IHC: profile). |
| Situation | Likely cause | Next action |
|---|---|---|
| The known-positive tissue gives no signal. | The expected cells may be absent from the section, or the staining run may have failed; HPA reports High staining only for the specified cell populations (HPA tissue IHC: High in parathyroid glandular cells and external root sheath cells). | Confirm those cells are present and compare a run control. Review primary-antibody application, retrieval settings, and detection steps as general IHC checks; no CD109-specific retrieval setting is established here (general IHC practice). |
| Color appears mainly inside nuclei. | Nuclear dominance conflicts with the reported membranous IHC profile (HPA tissue IHC: membranous profile). The appearance alone does not identify which staining step caused it. | Compare the same run with its controls and inspect whether cell-edge staining is also present. Recheck the detection and counterstain steps before interpreting nuclear color as CD109 (general IHC practice). |
| A HPA-listed Not detected cell type stains strongly. | Unexpected staining may reflect antibody cross-reactivity or endogenous detection activity; the slide alone cannot separate them (HPA tissue IHC: Not detected cell types; general IHC practice). | Compare with a no-primary control and, where applicable, an endogenous-activity control. Confirm cell identity and compartment before assigning CD109 positivity (general IHC practice). |
| Diffuse color makes cell borders hard to distinguish. | Nonspecific binding or detection background may obscure the membranous pattern (general IHC practice; HPA tissue IHC: membranous profile). | Check no-primary and run controls, then review blocking, primary-antibody concentration, washes, and chromogen development within the assay workflow (general IHC practice). Score localization only when cell edges can be resolved. |
| Cytoplasmic staining accompanies weak membrane staining. | HPA reports an additional cytosolic location by ICC-IF, while its tissue-IHC summary emphasizes membrane staining (HPA subcellular: Cytosol, approved; HPA tissue IHC: membranous profile). The two assay observations should be interpreted in their own contexts. | Record membrane and cytoplasmic staining separately, compare positive tissue and controls, and avoid treating cytoplasmic color alone as the expected paraffin-section pattern (HPA tissue IHC: profile; general IHC practice). |
| A tissue described as expressing CD109 by UniProt is IHC-negative. | UniProt's broad tissue-expression description and HPA's cell-specific IHC observations answer different questions (UniProt Q6YHK3: tissue specificity; HPA tissue IHC: cell-level results). Expression in a tissue does not guarantee a visible stain in every sampled cell. | Identify the cells actually present and compare them with HPA's named positive populations. Verify assay performance using one of those populations before interpreting the negative result (HPA tissue IHC: High in parathyroid glandular cells and external root sheath cells; general IHC practice). |
Experiment evidence
IHC & IF images showing CD109 staining
A03248 · IHCImmunohistochemical analysis of paraffin-embedded human-liver, antibody was diluted at 1:200
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000156535-CD109/tissue/cerebral+cortex#imid_20368685
HPA · Parathyroid glandHuman Protein Atlas IHC, Parathyroid gland. Source: https://www.proteinatlas.org/ENSG00000156535-CD109/tissue/parathyroid+gland#imid_20368560
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000156535-CD109/tissue/colon#imid_20368595
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000156535-CD109/tissue/liver#imid_20368604
HPA · ICC-IF ASC52teloHuman Protein Atlas immunofluorescence, ASC52telo, antibody HPA008992; green = CD109, blue = nucleus, red = microtubules.
HPA · ICC-IF ASC52teloHuman Protein Atlas immunofluorescence, ASC52telo, antibody HPA008992; green = CD109, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CD109 IHC & IF
🧪Run hair first and require staining in external root sheath cells (HPA: High); use adipose tissue adipocytes as the negative comparison (HPA: Not detected). On the hair slide, use visibly unstained neighboring cells as an internal background reference without assuming that every cell outside the external root sheath lacks CD109.
Positive control tissue: Hair (Cells in external root sheath, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CD109 in ASC52telo, HaCaT, U2OS, with annotated localisation: Plasma membrane (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a nonimmune control matched to the primary antibody’s host species, isotype and clonality; and CD109-knockout material or immunizing-peptide competition, if available, to assess specificity (standard IHC practice). Quench endogenous peroxidase and check hair pigment against the no-primary slide when interpreting chromogenic signal (standard IHC practice).
⚠️Feasibility: A CD109-specific fixation window and retrieval dependency are unreported in the supplied evidence; optimize antigen retrieval empirically. The selected A03248 paraffin-section caption reports a 1:200 dilution but does not report the fixative, so it does not establish fixation (A03248 caption). The evidence does not establish whether frozen sections or IF are easier; hair pigment can complicate chromogenic scoring (standard IHC practice), while ICC-IF images support plasma-membrane localization with additional cytosolic signal (HPA subcellular). The selected A03248 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image A03248).
HPA tissue IHC evidence for CD109
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Hair | Cells in external root sheath | High | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CD109 IHC Tips
Troubleshoot CD109 chromogenic IHC in paraffin sections using the catalog antibody’s tissue image, the page retrieval setting, and independent localisation evidence.
How should I retrieve CD109 when paraffin sections stain weakly?
Use citrate pH 6.0 heat induced antigen retrieval at 95–98 °C for 20 min (page retrieval rule: membrane antigen). Keep heating, cooling, section thickness, and antibody incubation consistent while comparing sections, so a change in signal can be attributed to retrieval (standard IHC practice). The catalog antibody has a paraffin embedded human liver IHC image at 1:200, but its caption does not report retrieval conditions or fixative (A03248 caption). Judge recovery by a crisp membrane pattern in an appropriate control, using HPA’s membranous hair follicle and parathyroid staining as biological references (HPA tissue IHC).
Could fixation explain absent or patchy CD109 staining?
Target specific CD109 fixation sensitivity is unknown from the supplied evidence; the catalog image describes paraffin embedded human liver but does not state its fixative (A03248 caption). Record the actual fixative and fixation duration for each specimen, then compare matched sections processed together with the same pH 6.0, 20 min retrieval (standard IHC practice; page retrieval rule). Uneven fixation can produce patchy chromogenic staining in paraffin sections, so inspect tissue preservation before changing antibody concentration (standard IHC practice). Use a suitable positive control alongside the specimen and interpret any fixation effect as an observed comparison, not an established CD109 property (standard IHC practice).
Where should convincing CD109 staining appear in a tissue section?
Prioritise cell border staining: CD109 is annotated at the cell membrane, without a transmembrane segment, and has a GPI anchor annotation (UniProt Q6YHK3 topology and keywords). HPA reports membranous staining in parathyroid gland and hair follicle, including high signal in glandular cells and external root sheath cells (HPA tissue IHC). Some cytosolic signal can occur because HPA also reports an approved additional cytosolic location, but assess it separately from the supported plasma membrane signal (HPA subcellular). Compare borders between intact neighbouring cells and review the counterstain to assign signal to cells rather than nearby extracellular material (standard IHC practice).
Can this antibody distinguish CD109 isoforms or a glycosylation dependent epitope?
Do not assign an isoform from chromogenic staining alone: CD109 has 4 annotated isoforms, while the supplied catalog image does not map the antibody epitope (UniProt Q6YHK3 isoforms; A03248 caption). Isoform 4 is reported in placenta, and isoform 1 in keratinocytes and placenta; expression context cannot establish which isoform produced a stained section (UniProt Q6YHK3 tissue specificity). The precursor has 10 annotated glycosylation sites and a processed chain spanning residues 22–1420, so epitope accessibility warrants checking against antibody documentation when available (UniProt Q6YHK3 processing and glycosylation). Describe results as CD109 immunoreactivity unless an independently validated isoform specific reagent or orthogonal assay supports a narrower claim (standard IHC interpretation).
How can I check CD109 localisation by multiplex IF?
Use IF as a complementary localisation check, pairing CD109 with a validated marker for the expected cell type, such as external root sheath or parathyroid glandular cells (HPA tissue IHC; standard IF practice). Place the weaker signal in a bright, spectrally separated channel and choose fluorophores after inspecting tissue autofluorescence and single colour controls (standard IF practice). CD109 is membrane associated without a transmembrane segment; test staining without permeabilisation for an accessible extracellular epitope, and use controlled mild permeabilisation only if epitope information calls for intracellular access (UniProt Q6YHK3 topology; standard IF practice). Include single stain and secondary only controls before interpreting colocalisation; HPA reports a supported plasma membrane location and an additional cytosolic location (standard IF practice; HPA subcellular).
What should I change when CD109 IHC has diffuse brown background?
First distinguish diffuse chromogen from cell associated membrane staining using the counterstain and a matched section without primary antibody (standard IHC practice; HPA tissue IHC). For peroxidase detection, verify the routine endogenous peroxidase block and inspect the no primary control before assigning brown deposits to CD109 (standard chromogenic IHC practice). Titrate from the catalog image’s 1:200 antibody dilution using matched sections, and adjust blocking, washes, or detection exposure one variable at a time (A03248 caption; standard IHC practice). Compare with a negative cell population in the same run where feasible; HPA reports no detection in adipocytes and several other listed cell types (HPA tissue IHC).
How should I score CD109 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because the strongest supported localisation is the plasma membrane and HPA’s tissue signal is cell type dependent (HPA subcellular; HPA tissue IHC). Report the percentage of positive cells and membrane intensity, or calculate an H score from intensity categories 0–3 within a prespecified region (standard IHC scoring practice). If cells are sparse, report positive cells per mm² of viable tissue and also record the total cells or area assessed (standard IHC quantification practice). Normalise comparisons to the same cell type, viable area, section processing, and staining run; keep cytosolic signal separate from membrane scores (standard IHC practice; HPA subcellular).
How can I tell true CD109 staining from an IHC artefact?
A credible result follows intact cell borders in an appropriate population: HPA reports high membranous signal in external root sheath and parathyroid glandular cells (HPA tissue IHC). Review an unexpected cell type or nuclear pattern against morphology and controls, since HPA supports plasma membrane localisation and also allows additional cytosolic signal (HPA subcellular; standard IHC interpretation). Exclude section edges, folds, necrotic areas, and deposits also present without primary antibody before counting positive cells (standard chromogenic IHC practice). If brown signal persists in the no primary control, investigate endogenous peroxidase or detection background before interpreting it as CD109 (standard chromogenic IHC practice).
Boster reagents
Best CD109 / CD109 antigen IHC Antibodies
A03248 has real IHC data from paraffin-embedded human liver (catalog IHC image caption). IF is listed for human and mouse, with no IF image supplied (catalog applications, reactivity and IF image list).
Real IHC data
Anti-CD109 antigen CD109 Antibody
Cat # A03248
A03248 will render with an IHC image of paraffin-embedded human liver (catalog IHC image caption). It lists IHC and IF applications and human and mouse reactivity (catalog applications and reactivity).
Which to pick: For tissue IHC, pick A03248: its own image shows a paraffin-embedded human liver section, and the fixative is unreported (catalog IHC image caption). For IF, A03248 is listed, but no IF image or ICC application is supplied (catalog applications and IF image list). For human and mouse work, A03248 lists reactivity with both species; its shown IHC section is human, and clonality is unreported (catalog reactivity, IHC image caption and clone field).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q6YHK3 (CD109_HUMAN, CD109 antigen).
- Human Protein Atlas. CD109 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CD109 subcellular location (ICC-IF): Mainly localized to the plasma membrane. In addition localized to the cytosol..
- Human Protein Atlas. CD109 antibody validation summary (2 antibodies).
- Prognostic Significance of CD109 Expression in Patients with Ovarian Epithelial Cancer. Journal of pathology and translational medicine 2019 — PMC6639710.
- Serum CD109 levels reflect the node metastasis status in head and neck squamous cell carcinoma. Cancer medicine 2021 — PMC7926025.
- Development of novel monoclonal antibodies against CD109 overexpressed in human pancreatic cancer. Oncotarget 2018 — PMC5929441.
- Myxofibrosarcoma primary cultures: molecular and pharmacological profile. Therapeutic advances in medical oncology 2017 — PMC5808841.
- PubMed PMID:11861284 — UniProt-cited evidence.
- PubMed PMID:14980714 — UniProt-cited evidence.
- PubMed PMID:16754747 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CD109 (CD109 antigen). Expected localisation Membrane in parathyroid gland and hair follicle (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A03248 at 1:50 recommended; image 1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Hair (Cells in external root sheath); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human liver tissue; fixative not specified (datasheet A03248); 2. Fixation: Image fixative and duration unreported (datasheet A03248); 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-CD109, 1:50 recommended; image 1:200 (datasheet A03248); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Endothelial cell expression may add tissue signal (UniProt); Isoform / epitope: 4 isoforms; mature chain 22–1420; map the epitope (UniProt); Regulation: Expressed in activated platelets (UniProt). Evidence: UniProt Q6YHK3, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the plasma membrane. In addition localized to…, Datasheet A03248, PMC6639710, PMC7926025. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CD109/immunohistochemistry-ihc-guide.