This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
LY6D / Lymphocyte antigen 6D · IHC design guide
Design Immunohistochemistry for LY6D
Plan chromogenic LY6D IHC on paraffin sections using the catalog antibody at 2–5 μg/ml (datasheet A09680). Use esophageal squamous epithelium as a high-staining reference and assess membranous and cytoplasmic signal (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
LY6D 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 | Membranous and cytoplasmic in squamous epithelium (HPA tissue IHC) | |
| Staining pattern | Squamous epithelial cells show membranous and cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A09680) | |
| Positive control | Cervix+4 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 | Normal squamous epithelium can stain strongly (HPA tissue IHC) | |
| Regulation | Higher in squamous epithelia (HPA tissue IHC) | |
| Isoform / epitope | No isoforms annotated; mature chain spans aa 21–98 and is surface-facing, with no cytoplasmic tail (UniProt) |
Section 1
Recommended LY6D IHC & IF Protocols
The catalog antibody protocol (datasheet: A09680) is accompanied by published LY6D IHC protocols for bladder cancer tissue microarrays (PMC7539380) and pancreatic cancer tissue microarrays (PMC8715959).
| Sample | Paraffin-embedded human skin cancer tissue; fixative not specified (datasheet A09680) |
| Fixation | Image fixative and duration unreported (datasheet A09680); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A09680); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A09680) |
| Primary antibody | Rabbit anti-LY6D, 2-5 μg/ml (datasheet A09680) |
| Primary incubation | Overnight at 4 °C (datasheet A09680) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A09680) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | LY6D-positive staining in squamous epithelial cells of cervix (HPA tissue IHC: High). HPA tissue profile: Membranous and cytoplasmic expression in squamous epithelium. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet: A09680); compare the published pH 9 retrieval conditions if optimization is needed (PMC7539380; PMC8715959).
Section 2
What Is the Expected LY6D Staining Pattern?
LY6D is a cell membrane protein with no transmembrane segment; UniProt describes it at the outer surface of transitional and stratified squamous epithelia (UniProt Q14210 topology; tissue specificity). In paraffin-section IHC, expect membranous and some cytoplasmic staining in squamous epithelial cells, with medium staining reported in skin basal-layer cells and urothelium (HPA tissue IHC). HPA rates the tissue pattern Enhanced for agreement between staining and RNA expression (HPA tissue IHC).
| Clear membrane outlines, with some cytoplasmic color, in squamous epithelial cells. | This fits the observed IHC profile (HPA tissue IHC: membranous and cytoplasmic expression). High staining is reported in squamous epithelial cells of cervix, esophagus, oral mucosa, tonsil and vagina (HPA tissue IHC). Judge the pattern within the relevant cells as well as its intensity. |
| Predominantly nuclear staining, with no convincing epithelial membrane pattern. | Nuclear localization conflicts with the cell membrane assignment (UniProt Q14210 subcellular location) and the observed tissue profile (HPA tissue IHC). Treat it as a possible staining artefact and check controls and detection conditions before interpreting the nuclei as LY6D. |
| Strong color in cell types reported as negative, such as adipocytes or respiratory epithelial cells. | Those cells are reported as not detected in the sampled tissues (HPA tissue IHC: adipose tissue and bronchus). Unexpected staining raises possible cross-reactivity or endogenous detection activity; the slide alone cannot distinguish them. Compare with a no-primary control and the expected epithelial distribution. |
| Diffuse color across epithelium, stroma and empty areas, without distinct cell borders. | A field-wide haze does not establish the reported membranous and cytoplasmic epithelial pattern (HPA tissue IHC). In routine IHC, excess background can arise from detection reagents, inadequate blocking or overly concentrated primary antibody. Evaluate control sections before assigning weak diffuse color to LY6D. |
| No staining in a well-preserved, known-positive squamous epithelial section. | An absent signal conflicts with reported high staining in several squamous epithelia (HPA tissue IHC: cervix, esophagus, oral mucosa, tonsil and vagina). First verify tissue identity, slide quality, antibody performance and detection controls. This record does not establish LY6D-specific fixation sensitivity or a retrieval requirement. |
💡Expected LY6D appearanceCall positive when squamous epithelial cells show discernible membrane-associated staining with possible cytoplasmic signal, often high in the listed positive tissues (HPA tissue IHC); isolated nuclear color or widespread staining in reported-negative cell types is suspect (UniProt Q14210 subcellular location; HPA tissue IHC).
| Cellular compartment | UniProt assigns LY6D to the cell membrane and reports no transmembrane segment (UniProt Q14210 topology). HPA observes both membranous and cytoplasmic tissue IHC staining (HPA tissue IHC). Assess membrane localization alongside cell identity; cytoplasmic color alone is less specific than the combined reported pattern. |
| Choice of reference tissue | Squamous epithelial cells score High in cervix, esophagus, oral mucosa, tonsil and vagina; skin basal-layer cells and urinary bladder urothelial cells score Medium (HPA tissue IHC). Use the reported cell type and level when choosing a comparison section, and avoid requiring identical intensity across tissues. |
| Strength and limits of validation | HPA calls tissue IHC reliability Enhanced because staining is highly consistent with RNA expression (HPA tissue IHC). Three listed antibodies carry Enhanced IHC status, while their ICC fields are empty (HPA antibodies). These ratings support the reported tissue pattern but do not validate every unexpected compartment or cell type. |
| Protein processing and epitope uncertainty | UniProt lists a signal peptide at residues 1–20, a mature chain at 21–98 and a propeptide at 99–128 (UniProt Q14210 processing). The supplied record gives no antibody epitope. It therefore cannot establish which processed form the IHC-validated antibody recognizes or predict a retrieval condition. |
| IF/ICC Q&A: is a membrane signal expected? | Yes as a localization expectation: HPA summarizes LY6D as membrane-associated (HPA subcellular), consistent with UniProt (UniProt Q14210 subcellular location). HPA supplies no ICC-IF images or listed antibody ICC validation (HPA subcellular; HPA antibodies). Consult the separate IF/ICC guide for experimental design. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive squamous epithelium is blank. | This disagrees with reported High staining in several squamous epithelia (HPA tissue IHC). The cause remains undetermined from a blank slide. | Confirm that the section contains the specified epithelial cells, then review the catalog antibody's IHC-P instructions, detection reagents and positive control. Do not infer a LY6D-specific fixation or retrieval defect from this result. |
| Signal is cytoplasmic, with no discernible cell-border staining. | Cytoplasmic staining is reported, but the reference pattern also includes membranes (HPA tissue IHC). The isolated pattern has weaker localization support. | Compare a reported-positive section and no-primary control. Review staining strength and morphology before scoring; record the observed compartment rather than calling a membrane-positive result. |
| Nuclei are the strongest stained structures. | A dominant nuclear pattern differs from the membrane assignment and tissue IHC profile (UniProt Q14210 subcellular location; HPA tissue IHC). | Check whether counterstain or detection color is being mistaken for target signal. Inspect controls, then reassess epithelial borders at higher magnification. |
| Reported-negative cells stain as strongly as the epithelial positive control. | Unexpected staining could reflect cross-reactivity or endogenous detection activity; HPA reports no detection in several listed negative cell types (HPA tissue IHC). | Run a no-primary control on the same tissue and review blocking appropriate to the detection system. If color persists, resolve the background before attributing it to LY6D. |
| The whole section has weak, diffuse color. | Diffuse background obscures the cell-specific membrane and cytoplasmic pattern (HPA tissue IHC); routine IHC reagent or blocking issues are possible. | Inspect no-primary and negative-tissue controls. Check detection reagent handling, blocking and antibody dilution against the chosen IHC-P instructions; score only localized staining above background. |
| Skin or urinary bladder looks weaker than a squamous epithelial positive control. | HPA reports Medium staining in skin basal-layer cells and urothelium, versus High staining in several squamous epithelia (HPA tissue IHC). | Compare the correct cell populations and section quality before changing conditions. Medium staining can agree with the reference; a completely blank result still warrants control review. |
Experiment evidence
IHC & IF images showing LY6D staining
A09680 · IHCIHC analysis of LY6D using anti-LY6D antibody (A09680). LY6D was detected in a paraffin-embedded section of human skin cancer tissue. Heat m
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000167656-LY6D/tissue/cerebral+cortex#imid_6891096
HPA · EsophagusHuman Protein Atlas IHC, Esophagus. Source: https://www.proteinatlas.org/ENSG00000167656-LY6D/tissue/esophagus#imid_6891084
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000167656-LY6D/tissue/colon#imid_6895278
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000167656-LY6D/tissue/liver#imid_6895279
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for LY6D IHC & IF
🧪Run esophagus first: its squamous epithelial cells should show strong LY6D staining (HPA: High in esophageal squamous epithelial cells). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); on the esophagus slide, adjacent stromal cells should show background rather than specific cell-surface staining (UniProt Q14210: expression in transitional and stratified squamous epithelia; cell membrane location).
Positive control tissue: Cervix (Squamous epithelial cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for LY6D; derive a cell-line control from the positive tissue's cell type (Squamous epithelial cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only slide and a concentration-matched rabbit IgG control matched to the primary antibody’s clonality; confirm specificity with LY6D knockout material or peptide competition if the immunizing peptide is available (datasheet caption: rabbit anti-LY6D antibody; standard IHC practice). Block endogenous peroxidase before HRP/DAB detection in the esophagus section (datasheet caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A LY6D-specific fixation window or fixation effect is unreported, and the selected A09680 paraffin-section caption does not state its fixative (datasheet caption: fixative unreported). The caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required; use that condition as an IHC starting point (datasheet caption: EDTA heat retrieval). The supplied evidence does not establish whether frozen sections or IF are easier, and it provides no ICC-IF image-bearing cell lines; assess esophageal epithelial edge staining against stromal background when scoring (HPA subcellular: no ICC-IF image-bearing cell lines; UniProt Q14210: cell membrane location; standard IHC practice).
HPA tissue IHC evidence for LY6D
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 |
|---|---|---|---|---|
| Cervix | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Skin | Cells in basal layer | Medium | Protein (IHC) | HPA → |
| Tonsil | Squamous epithelial 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 LY6D IHC Tips
These LY6D questions focus on interpreting chromogenic staining in paraffin sections, using the catalog antibody’s tissue image and the supplied protein expression evidence.
Which retrieval condition should I try first for LY6D in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet A09680). The selected paraffin-section image used that condition before incubation with 2 μg/ml catalog antibody overnight at 4°C (datasheet A09680). If staining is weak, compare retrieval heating and cooling conditions on serial sections while keeping antibody concentration, detection, and exposure to DAB constant (standard IHC practice). Include a squamous epithelial positive control and inspect tissue preservation alongside signal, because excessive retrieval can make damaged areas difficult to score (HPA: high LY6D in squamous epithelial cells; standard IHC practice).
How should I troubleshoot weak staining when the tissue fixative is unknown?
Treat LY6D sensitivity to fixation as unknown: the selected image identifies a paraffin section but does not state its fixative (datasheet A09680). Record the fixative and fixation duration for each specimen, then compare similarly processed sections before changing the antibody concentration or DAB development time (standard IHC practice). Run a known positive squamous epithelial section in the same batch to distinguish a specimen problem from a failed staining run (HPA: high expression in squamous epithelial cells; standard IHC practice). Do not assign an optimal fixation duration to LY6D from its reported membrane location or tissue staining pattern (UniProt Q14210: cell membrane; HPA: tissue IHC profile).
Where should convincing LY6D staining appear within the section?
Prioritise staining at epithelial cell borders, since LY6D is assigned to the cell membrane and has no transmembrane segment (UniProt Q14210: subcellular location and topology). Some cytoplasmic signal can coexist with that pattern in squamous epithelium (HPA: membranous and cytoplasmic expression in squamous epithelium). Compare the distribution with adjacent intact epithelium: high staining is reported in squamous epithelial cells of cervix, esophagus, oral mucosa, tonsil, and vagina (HPA: tissue IHC). Diffuse signal confined to damaged tissue, without a corresponding epithelial pattern, warrants review of morphology and staining controls before it is called positive (standard IHC practice).
Could LY6D processing or isoforms explain inconsistent staining?
Check the antibody’s stated immunogen or epitope before attributing a negative section to biological absence; the supplied record does not locate the catalog antibody’s epitope (datasheet A09680; UniProt Q14210). LY6D has a signal sequence at residues 1–20 and a reported mature chain at 21–98, so epitope position matters when assessing processed protein (UniProt Q14210: processing). The record lists 0 isoforms and 0 annotated glycosylation sites; these annotations do not establish how this antibody responds to processing (UniProt Q14210: isoforms and glycosylation). Compare serial sections using the same retrieval and detection conditions before assigning a staining difference to epitope accessibility (standard IHC practice).
How can I assess LY6D in a multiplex fluorescence experiment?
Pair LY6D with a validated squamous epithelial marker and check whether signal follows epithelial cell borders, the expected membrane location (HPA: squamous epithelial expression; UniProt Q14210: cell membrane). Choose a fluorophore in a channel with low measured tissue autofluorescence, and include single-stain and no-primary controls to assess bleed-through and background (standard IF practice). If the antibody recognises an extracellular epitope, compare staining without permeabilisation; if its epitope is intracellular, test gentle permeabilisation while checking border integrity (standard IF practice). Treat this as an IF optimisation question because the supplied selected image documents chromogenic staining of a paraffin section, with no IF validation specified for this antibody (datasheet A09680).
What should I change when DAB appears throughout the tissue?
First compare a no-primary section with the stained section to assess background from detection reagents or endogenous peroxidase (standard chromogenic IHC practice). The selected image used 10% goat serum blocking, a peroxidase-conjugated secondary for 30 minutes at 37°C, and DAB development (datasheet A09680). Apply an appropriate peroxidase block, check washing and DAB development time, and titrate the primary around the documented 2 μg/ml condition on matched sections (standard IHC practice; datasheet A09680). Judge improvements against intact squamous epithelium and a no-primary control, retaining the tissue pattern as well as lower background (HPA: squamous epithelial expression; standard IHC practice).
How should I score LY6D across sections with different epithelial content? ⚠ ANSWER MARKED FOR VERIFICATION
Define the epithelial region of interest before scoring, because reported LY6D staining is membranous and cytoplasmic in squamous epithelium (HPA: tissue IHC profile). Record the percentage of positive epithelial cells and staining intensity separately, or calculate an H-score from intensity categories 0–3 and their cell percentages (standard IHC scoring practice). Normalise cell counts to the total number of evaluable epithelial cells, or report positive-cell density per mm² of intact epithelial area when cell segmentation is unreliable (standard quantitative IHC practice). Keep retrieval, DAB development, thresholds, and exclusion of damaged regions consistent across sections so that staining differences remain interpretable (standard IHC practice).
How can I distinguish true LY6D staining from an artefact?
Look for a reproducible epithelial pattern with cell-border staining, allowing for the cytoplasmic component reported in squamous epithelium (UniProt Q14210: cell membrane; HPA: tissue IHC profile). Compare with a positive squamous epithelial control and review unexpected staining in a reported negative population, such as adipocytes or bone-marrow hematopoietic cells (HPA: tissue IHC). Signal restricted to section edges or necrotic areas should prompt morphology review, while signal persisting in a no-primary control should prompt checks for endogenous enzyme activity and detection background (standard IHC practice). Do not identify a stained cell as a B-lineage cell solely from LY6D’s proposed developmental function; assess cell identity independently (UniProt Q14210: function; standard IHC interpretation).
Boster reagents
Best LY6D / Lymphocyte antigen 6D IHC Antibodies
A09680 has real IHC data from a paraffin-embedded human skin cancer section (catalog IHC image caption); the catalog lists human reactivity (catalog: reactivity). No IF data are supplied (catalog: IF images).
Real IHC data
Anti-LY6D Antibody ®
Cat # A09680
A09680 will render with its IHC figure from a paraffin-embedded human skin cancer section (catalog IHC image caption). It is listed for human IHC, WB and ELISA, with no IF application or image listed (catalog: applications, reactivity and IF images).
Which to pick: Choose A09680 for human paraffin-section IHC: it is a rabbit polyclonal antibody listed for human IHC (catalog: host, clonality, applications and reactivity), and its own figure shows EDTA pH 8.0 retrieval and 2 μg/ml primary antibody (A09680 IHC image caption). The caption does not report the fixative (A09680 IHC image caption). No SKU in the supplied catalog is listed for IF/ICC or cross-species use (catalog: applications and reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q14210 (LY6D_HUMAN, Lymphocyte antigen 6D).
- Human Protein Atlas. LY6D tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. LY6D subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. LY6D antibody validation summary (3 antibodies).
- OLFM4, LY6D and S100A7 as potent markers for distant metastasis in estrogen receptor-positive breast carcinoma. Cancer science 2018 — PMC6172070.
- Lymphocyte antigen 6 superfamily member D is a marker of urothelial and squamous differentiation: implications for risk stratification of bladder cancer. Biomarker research 2020 — PMC7539380.
- Single-cell RNA sequencing identifies a novel proliferation cell type affecting clinical outcome of pancreatic ductal adenocarcinoma. Frontiers in oncology 2023 — PMC10433893.
- Systematic Analysis of an Invasion-Related 3-Gene Signature and Its Validation as a Prognostic Model for Pancreatic Cancer. Frontiers in oncology 2021 — PMC8715959.
- PubMed PMID:7790363 — UniProt-cited evidence.
- PubMed PMID:9366413 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for LY6D (Lymphocyte antigen 6D). Expected localisation Membranous and cytoplasmic in squamous epithelium (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A09680). Validated IHC antibody: A09680 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Cervix (Squamous epithelial cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human skin cancer tissue; fixative not specified (datasheet A09680); 2. Fixation: Image fixative and duration unreported (datasheet A09680); 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: EDTA pH 8.0 (datasheet A09680); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A09680); 8. Primary antibody: Rabbit anti-LY6D, 2-5 μg/ml (datasheet A09680); 9. Primary incubation: Overnight at 4 °C (datasheet A09680). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: Normal squamous epithelium can stain strongly (HPA tissue IHC); Isoform / epitope: No isoforms annotated; mature chain spans aa 21–98 and is surface-facing, with no cytoplasmic tail (UniProt); Regulation: Higher in squamous epithelia (HPA tissue IHC). Evidence: UniProt Q14210, HPA tissue IHC · Enhanced, HPA ICC-IF · Membrane, Datasheet A09680, PMC7539380, PMC8715959. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/LY6D/immunohistochemistry-ihc-guide.