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LAYN / Layilin · IHC design guide
Design Immunohistochemistry for LAYN
Plan chromogenic IHC for LAYN using the reported cytoplasmic tissue pattern and high staining in breast myoepithelial cells, smooth muscle cells and stomach glandular cells (HPA tissue IHC). Interpret that staining alongside LAYN’s annotated membrane location and extracellular and cytoplasmic regions (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
LAYN Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic staining across tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in cells across tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Breast+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A10853) | |
| Caveat | IHC and RNA show medium consistency; verify the pattern (HPA tissue IHC) | |
| Regulation | Low tissue specificity (HPA tissue RNA) | |
| Isoform / epitope | 3 isoforms; check whether the epitope is extracellular or cytoplasmic (UniProt) |
Section 1
Recommended LAYN IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by two published LAYN IHC methods (PMC10908921; PMC9747853).
| Sample | Paraffin-embedded Rat Brain tissue; fixative not specified (datasheet A10853) |
| Fixation | Image fixative and duration unreported (datasheet A10853); 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-LAYN, 1:100 (datasheet A10853) |
| 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 | LAYN-positive staining in myoepithelial cells of breast (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in all tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min for this membrane protein (page retrieval; UniProt Q6UX15 topology).
Section 2
What Is the Expected LAYN Staining Pattern?
LAYN is a transmembrane protein with an extracellular domain and cytoplasmic tail; UniProt reports colocalization with TLN1 at membrane ruffles (UniProt Q6UX15 topology). In tissue IHC, HPA reports cytoplasmic staining across tissues, with high staining in breast myoepithelial cells, smooth muscle cells, and stomach glandular cells (HPA tissue IHC). HPA rates its tissue staining Approved, with medium consistency against RNA data and external verification pending (HPA tissue IHC).
| Cytoplasmic staining is strongest in breast myoepithelial cells, smooth muscle cells, or stomach glandular cells. | This matches HPA's high staining in those cell types (HPA tissue IHC). Judge the signal in its tissue context; HPA also reports cytoplasmic expression across tissues, so staining elsewhere is not automatically unexpected (HPA tissue IHC). |
| A membrane edge or ruffle is visible alongside cytoplasmic staining. | Membrane staining is biologically plausible because LAYN spans the membrane and colocalizes with TLN1 at membrane ruffles (UniProt Q6UX15 topology). HPA's tissue IHC description is cytoplasmic, however, so a membrane-only result needs cautious interpretation (HPA tissue IHC). |
| Signal is confined to nuclei, with no convincing cytoplasmic or membrane staining. | A nuclear-only pattern conflicts with HPA's cytoplasmic tissue profile and UniProt's membrane topology (HPA tissue IHC; UniProt Q6UX15 topology). Treat it as a possible artefact and check controls before assigning it to LAYN. |
| Unexpected cells stain more strongly than the documented high-staining cell types. | The mismatch raises possible cross-reactivity or endogenous detection activity; neither can be established from appearance alone (HPA tissue IHC; standard IHC practice). HPA's low-staining examples include skeletal muscle myocytes and lymph-node germinal-center cells (HPA tissue IHC). |
| Color spreads across tissue or appears where cellular boundaries cannot be judged. | Diffuse background prevents a reliable call on LAYN localization (standard IHC practice). Compare a no-primary control and review blocking, washes, and detection conditions before interpreting weak staining (standard IHC practice). |
💡Expected LAYN appearanceA positive IHC result shows predominantly cytoplasmic staining, strongest in the documented high-staining cell types (HPA tissue IHC); nuclear-only signal or widespread color without cellular definition warrants investigation (HPA tissue IHC; standard IHC practice).
| Cellular compartment | HPA describes cytoplasmic tissue staining, while UniProt places LAYN at the membrane, with an extracellular region at residues 22–235 and a cytoplasmic region at 257–382 (HPA tissue IHC; UniProt Q6UX15 topology). Interpret membrane accent in that context. |
| Tissue and cell choice | Breast myoepithelial cells, smooth muscle cells, and stomach glandular cells are high-staining examples (HPA tissue IHC). Salivary glandular cells and skeletal muscle myocytes are low-staining examples; low means limited signal, not a guaranteed blank control (HPA tissue IHC). |
| Evidence strength | HPA rates the tissue IHC profile Approved, reports medium consistency with RNA expression, and notes that external verification is pending (HPA tissue IHC). The listed antibody, HPA040087, is IHC Approved; the supplied record does not label its IHC validation Enhanced (HPA antibodies). |
| Epitope and isoforms | UniProt records 3 LAYN isoforms, a signal peptide at residues 1–21, and one glycosylation site at residue 117 (UniProt Q6UX15). Without an antibody epitope map, these facts do not predict which isoforms or regions the IHC antibody detects. |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in a documented high-staining tissue | The result conflicts with HPA's high staining in the relevant cell type (HPA tissue IHC); failed detection or unsuitable assay conditions are possibilities (standard IHC practice). | Confirm that the expected cells are present, then review the no-primary and detection controls, antigen retrieval conditions, and antibody dilution (standard IHC practice). No LAYN-specific retrieval condition is supplied. |
| Only a sharp membrane outline stains | Membrane localization is plausible from LAYN topology, but HPA describes tissue IHC staining as cytoplasmic (UniProt Q6UX15 topology; HPA tissue IHC). | Check whether cytoplasmic signal is also present in documented high-staining cells and compare controls before calling an isolated outline a confirmed LAYN pattern (HPA tissue IHC; standard IHC practice). |
| An unexpected cell population dominates the slide | Possible explanations include antibody cross-reactivity or endogenous detection activity (standard IHC practice). HPA reports low rather than absent staining in several cell types (HPA tissue IHC). | Check a no-primary control and compare the cell type with HPA's tissue profile; do not treat low-staining examples as definitively negative (standard IHC practice; HPA tissue IHC). |
| Diffuse color obscures cell boundaries | Background from blocking, washing, or detection can obscure specific cellular staining (standard IHC practice). | Review blocking and washes, inspect a no-primary control, and adjust detection conditions before scoring localization or intensity (standard IHC practice). |
| Nuclear-only signal persists | A nuclear-only result conflicts with the reported cytoplasmic tissue pattern and membrane topology (HPA tissue IHC; UniProt Q6UX15 topology). | Verify the counterstain and inspect controls; score LAYN only where a convincing cellular pattern supports the call (standard IHC practice). |
| What should IF/ICC show? | HPA reports an approved cytosolic ICC-IF location, with images from A-431, U-251MG, and U2OS cells (HPA subcellular). | Use cytosolic staining as the HPA ICC-IF comparison and interpret any membrane accent alongside UniProt's membrane localization (HPA subcellular; UniProt Q6UX15 topology). |
Experiment evidence
IHC & IF images showing LAYN staining
A10853 · IHCImmunohistochemistry validation of LAYN using Anti-Layilin LAYN Antibody (A10853). Immunohistochemistry (IHC) analysis of paraffin-embedded
A10853 · IHCImmunohistochemistry validation of LAYN using Anti-Layilin LAYN Antibody (A10853). Immunohistochemistry (IHC) analysis of paraffin-embedded
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000204381-LAYN/tissue/cerebral+cortex#imid_11540923
HPA · Thyroid glandHuman Protein Atlas IHC, Thyroid gland. Source: https://www.proteinatlas.org/ENSG00000204381-LAYN/tissue/thyroid+gland#imid_11540891
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000204381-LAYN/tissue/colon#imid_11540849
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000204381-LAYN/tissue/liver#imid_11540858
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA040087; green = LAYN, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA040087; green = LAYN, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for LAYN IHC & IF
🧪Run breast first and look for LAYN staining in myoepithelial cells (HPA: High in breast myoepithelial cells). HPA detects LAYN in all 45 scored tissues, so use no-primary and isotype controls as negatives; treat other cells on the breast slide as internal comparators only when they show background-level staining, since no cell type is established here as LAYN-negative (HPA: no negative tissue rows).
Positive control tissue: Breast (Myoepithelial cells, HPA High)
Negative control tissue: None in HPA: LAYN is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show LAYN in A-431, U-251MG, U2OS, with annotated localisation: Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only section and a concentration-matched control immunoglobulin from the primary antibody’s host species, matching isotype for a monoclonal antibody or using nonimmune IgG for a polyclonal antibody. A LAYN knockout specimen, if available, provides a biological negative; for chromogenic breast IHC, quench endogenous peroxidase when using HRP detection and check for background around the myoepithelial layer (HPA: High in breast myoepithelial cells).
⚠️Feasibility: No supplied target-specific evidence reports a fixation window or retrieval effect for LAYN; the selected A10853 paraffin-section caption does not state its fixative (A10853 tissue-IHC caption). Optimize antigen retrieval empirically, and do not assume frozen sections or IF/ICC will be easier: HPA reports approved cytosolic ICC-IF localization, while UniProt annotates LAYN at the membrane (HPA subcellular; UniProt Q6UX15 subcellular). In breast, distinguish cell-associated myoepithelial staining from duct-edge background (HPA: High in breast myoepithelial cells).
HPA tissue IHC evidence for LAYN
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium 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 |
|---|---|---|---|---|
| Breast | Myoepithelial cells | High | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | High | Protein (IHC) | HPA → |
| Stomach | Glandular cells | High | Protein (IHC) | HPA → |
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: LAYN is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced LAYN IHC Tips
Troubleshoot LAYN staining in paraffin section IHC by checking retrieval, compartment, cell identity, controls, and scoring (UniProt Q6UX15; HPA tissue IHC).
How should I retrieve LAYN in paraffin sections when staining is weak?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min, as this page’s LAYN retrieval setting (page retrieval setting). Let slides cool in buffer and keep section thickness, heating, and detection conditions consistent when comparing runs (standard IHC practice). If staining remains weak, test a more alkaline retrieval buffer on matched sections as a fallback, while checking whether background or tissue damage increases (standard IHC practice). Assess membrane-associated and cytoplasmic staining separately, because LAYN has an extracellular domain at residues 22–235 and a cytoplasmic region at 257–382 (UniProt Q6UX15 topology).
Could fixation explain variable LAYN staining between paraffin blocks?
The selected A10853 image shows paraffin-embedded rat brain, but its caption does not state the fixative; LAYN-specific fixation sensitivity is therefore unknown from the supplied evidence (A10853 tissue-IHC caption). Record fixative, fixation duration, tissue thickness, and processing history for each block before comparing staining intensity (standard IHC practice). For new specimens, use a consistent routine fixation and processing schedule, then compare matched sections if a fixation variable needs testing (standard IHC practice). Judge any change against tissue preservation, background, and the same retrieval and detection conditions, rather than attributing an intensity difference to LAYN alone (standard IHC practice).
Should I expect membranous or cytoplasmic LAYN staining in IHC?
LAYN is annotated as a membrane protein with a transmembrane segment at residues 236–256 and colocalisation with TLN1 at membrane ruffles (UniProt Q6UX15). Tissue IHC reports cytoplasmic expression across tissues, while the approved subcellular profile places LAYN in the cytosol (HPA tissue IHC; HPA subcellular). Examine membrane-associated and cytoplasmic staining as separate observations, recording the cell type and whether the pattern recurs away from section edges (standard IHC practice). A cytoplasmic pattern is compatible with the supplied observations, but compartment alone cannot establish antibody specificity; compare negative controls and independent biological evidence (HPA tissue IHC; standard IHC practice).
How could LAYN isoforms or epitope placement change IHC results?
LAYN has 3 annotated isoforms, but the supplied material does not map the catalog antibody’s epitope or establish which isoforms it detects (UniProt Q6UX15; supplied antibody evidence). The extracellular region spans residues 22–235, whereas residues 257–382 are cytoplasmic, so epitope placement would affect accessibility and how staining is interpreted (UniProt Q6UX15 topology; standard IHC practice). A glycosylation site at residue 117 and phosphoserines at 286 and 299 identify modifications to consider when evaluating a mapped epitope (UniProt Q6UX15). Until an epitope is documented, avoid assigning an absent IHC signal to a particular isoform or modification; compare controls under identical retrieval conditions (standard IHC practice).
How can IF help assess LAYN localisation alongside cell identity?
On a separate IF/ICC workflow, multiplex LAYN with a marker appropriate to the expected cell type, since tissue IHC reports high staining in breast myoepithelial and smooth muscle cells (HPA tissue IHC). Choose spectrally distinct fluorophores and favor a red or far-red LAYN channel when the specimen has strong short-wavelength autofluorescence (standard IF practice). If the antibody epitope is cytoplasmic, test mild permeabilisation, such as 0.1% Triton X-100 for 5 min; for an accessible extracellular epitope, first assess staining without detergent (UniProt Q6UX15 topology; standard IF practice). Interpret overlap against single-label and secondary-only controls, while recognizing that HPA’s approved IF localisation is cytosolic (HPA subcellular; standard IF practice).
What should I check when LAYN IHC has diffuse brown background?
Include a no-primary control and inspect whether brown signal follows tissue structure, section edges, or areas with poor preservation (standard IHC practice). For peroxidase detection, a 3% hydrogen peroxide block for about 10 min is a general starting step to suppress endogenous enzyme activity, not LAYN-specific evidence (standard IHC practice). Titrate the catalog antibody and detection reagent on adjacent sections, keeping retrieval and DAB development time matched so the background comparison is interpretable (standard IHC practice). Because tissue IHC describes widespread cytoplasmic LAYN staining, use cell morphology and controls before calling every diffuse cytoplasmic signal nonspecific (HPA tissue IHC; standard IHC practice).
How should I score LAYN IHC across samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and viable tissue area before scoring, because HPA reports different staining levels across cell types and tissues (HPA tissue IHC; standard IHC practice). For cell staining, record the percentage at intensity grades 0–3 and calculate an H-score from 0–300, or report percent-positive cells with a prespecified positivity threshold (standard IHC practice). If spatial abundance matters, report positive cells per mm² of viable tissue and normalize cell-level positivity to the number of eligible cells, rather than total section area (standard IHC practice). Keep compartment scoring, exposure, counterstain, and analysis thresholds consistent across batches, and report membrane-associated and cytoplasmic results separately (UniProt Q6UX15; HPA tissue IHC; standard IHC practice).
How do I distinguish credible LAYN staining from artefact?
Check whether staining occurs in plausible cells: HPA reports high LAYN staining in breast myoepithelial cells, smooth muscle cells, and stomach glandular cells (HPA tissue IHC). Membrane-associated staining fits the annotated transmembrane topology, while cytoplasmic staining also has support from tissue IHC and the approved subcellular profile (UniProt Q6UX15; HPA tissue IHC; HPA subcellular). Treat signal limited to section edges, necrotic areas, or a no-primary control as suspect, and evaluate endogenous peroxidase when brown precipitate appears without primary antibody (standard IHC practice). The HPA tissue profile is approved with medium RNA-staining consistency and pending external verification, so require reproducible controls before making a biological claim (HPA tissue IHC).
Boster reagents
Best LAYN / Layilin IHC Antibodies
A10853 has paraffin-section IHC images from rat and mouse brain (image captions); IF is listed without an image (catalog). Human, mouse and rat reactivity are listed (catalog).
Real IHC data
Anti-Layilin LAYN Antibody
Cat # A10853
A10853 will render with IHC images captioned as paraffin-embedded rat and mouse brain (image captions). Its listed applications include IHC and IF, with human, mouse and rat reactivity; no IF image is supplied (catalog).
Which to pick: For tissue IHC, choose A10853: its own images show paraffin-embedded rat and mouse brain, while the fixative is unreported (image captions); its listed IHC dilution is 1:100 (catalog). For IF/ICC, A10853 lists IF at 1:50, but the payload supplies no IF image or ICC validation (catalog). For cross-species work, A10853 lists human, mouse and rat reactivity, though its pictured IHC evidence covers only rat and mouse brain; clonality is unspecified (catalog; image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q6UX15 (LAYN_HUMAN, Layilin).
- Human Protein Atlas. LAYN tissue IHC expression (reliability: Approved).
- Human Protein Atlas. LAYN subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. LAYN antibody validation summary (1 antibodies).
- The prognostic value of LAYN in HPV-related head and neck squamous cell carcinoma and its influence on immune cell infiltration. Discover oncology 2024 — PMC10908921.
- Low-dose anti-VEGFR2 therapy promotes anti-tumor immunity in lung adenocarcinoma by down-regulating the expression of layilin on tumor-infiltrating CD8(+)T cells. Cellular oncology (Dordrecht, Netherlands) 2022 — PMC9747853.
- LAYN Serves as a Prognostic Biomarker and Downregulates Tumor-Infiltrating CD8(+) T Cell Function in Hepatocellular Carcinoma. Journal of hepatocellular carcinoma 2024 — PMC11164088.
- PubMed PMID:12975309 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16554811 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for LAYN (Layilin). Expected localisation Cytoplasmic staining across tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A10853 at 1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Breast (Myoepithelial cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded Rat Brain tissue; fixative not specified (datasheet A10853); 2. Fixation: Image fixative and duration unreported (datasheet A10853); 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-LAYN, 1:100 (datasheet A10853); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A10853); Caveat: IHC and RNA show medium consistency; verify the pattern (HPA tissue IHC); Isoform / epitope: 3 isoforms ; check whether the epitope is extracellular or cytoplasmic (UniProt); Regulation: Low tissue specificity (HPA tissue RNA). Evidence: UniProt Q6UX15, HPA tissue IHC · Approved, HPA ICC-IF · cytosol., Datasheet A10853, PMC10908921, PMC9747853. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/LAYN/immunohistochemistry-ihc-guide.