LYPD3 / Ly6/PLAUR domain-containing protein 3 · IHC design guide

Design Immunohistochemistry for LYPD3

Plan LYPD3 IHC in paraffin sections using squamous epithelium as a positive tissue reference (HPA tissue IHC). This guide covers membrane localization expected from UniProt and practical controls for interpreting tissue staining (UniProt; HPA tissue IHC).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for LYPD3 (IHC for LYPD3): expected localisation Cell membrane expected (UniProt); squamous epithelia stain (HPA tissue IHC), antibody A08396, validated IHC image, and IHC protocol steps — Printable **LYPD3** IHC protocol sheet — expected localisation Cell membrane expected (UniProt); squamous epithelia stain (HPA tissue IHC), antibody A08396, controls and protocol steps. Open the full LYPD3 IHC guide →

Navigate to: Protocols Staining pattern Controls Antibodies

LYPD3 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	Cell membrane expected (UniProt); squamous epithelia stain (HPA tissue IHC)
	Staining pattern	Selective squamous epithelial staining; compartment unspecified (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A08396)
	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	Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A08396)
	Caveat	Presumed off-target staining was observed and disregarded (HPA tissue IHC)
	Regulation	Present in chronic-wound keratinocytes (UniProt)
	Isoform / epitope	No isoforms listed; check epitope against mature chain 31–326 (UniProt)

Section 1

Recommended LYPD3 IHC & IF Protocols

The catalog antibody’s IHC-P protocol (datasheet A08396) is accompanied by published LYPD3 protocols for HNSCC and breast cancer tissue microarrays (PMC11250566; PMC6874102).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human skin cancer tissue; fixative not specified (datasheet A08396)
Fixation	Image fixative and duration unreported (datasheet A08396); 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 A08396); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A08396)
Primary antibody	Rabbit anti-LYPD3, 1-2 μg/ml (datasheet A08396)
Primary incubation	Overnight at 4 °C (datasheet A08396)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A08396)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	LYPD3-positive staining in squamous epithelial cells of cervix (HPA tissue IHC: High). HPA tissue profile: Selective expression in squamous epithelia. No signal in the no-primary control.

💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A08396); use citrate pH 6.0 when reproducing either published protocol (PMC11250566; PMC6874102).

Section 2

What Is the Expected LYPD3 Staining Pattern?

In paraffin-section IHC, expect LYPD3 chiefly along the cell membrane of squamous epithelial cells; the protein has no transmembrane segment and is GPI anchored (UniProt O95274 topology; UniProt O95274 keywords). HPA reports high staining in several squamous epithelia and rates its tissue IHC profile Enhanced, while noting presumed off-target binding that was disregarded (HPA tissue IHC). Interpret the staining by its cell type and location together.

What am I looking at on my slide?
Strong staining outlines squamous epithelial cells in cervix, esophagus, oral mucosa, tonsil or vagina (HPA tissue IHC).	This fits the reported high epithelial staining and the expected membrane location (HPA tissue IHC; UniProt O95274 subcellular location). Compare stained cells with the section's morphology before scoring intensity (general IHC practice).
Predominantly nuclear staining appears in squamous epithelium.	A nuclear-only pattern conflicts with the reported cell-membrane location (UniProt O95274 subcellular location). Treat it as suspect IHC signal and review controls and antibody specificity before assigning it to LYPD3 (general IHC practice).
Cells outside the reported epithelial pattern stain, particularly in a comparison tissue.	Compare the exact cell type with HPA's tissue entries: adipocytes in adipose tissue and respiratory epithelial cells in bronchus were not detected (HPA tissue IHC). Unexpected staining can reflect antibody cross-reactivity or endogenous detection activity (general IHC practice).
Color spreads across epithelium, stroma and blank areas without clear cell borders.	This is diffuse background rather than an interpretable membrane pattern (UniProt O95274 subcellular location; general IHC practice). Review blocking, washes, detection chemistry and the no-primary control before scoring (general IHC practice).
No staining appears in an adequately sampled HPA high-staining squamous epithelium.	This conflicts with HPA's reported high staining in the specified epithelial cell types (HPA tissue IHC). First verify tissue identity and the positive control, then check primary-antibody use and detection steps (general IHC practice).

💡Expected LYPD3 appearanceA convincing positive is high staining associated with squamous epithelial cells, consistent with LYPD3's membrane location; isolated nuclear color or widespread cell-independent deposit is suspect (HPA tissue IHC; UniProt O95274 subcellular location; general IHC practice).

How each factor affects the staining
Cell location and processing	LYPD3 is a GPI-anchored membrane protein with no transmembrane segment; its annotated mature chain spans residues 31–326 (UniProt O95274 topology; UniProt O95274 processing). Use membrane association as the IHC interpretation anchor.
Epitope location	UniProt lists a signal peptide at residues 1–30 and a propeptide at 327–346 (UniProt O95274 processing). Antibody epitope information is absent here, so those annotations cannot predict this antibody's staining.
Glycosylation	Four glycosylation sites are annotated at residues 118, 163, 176 and 183 (UniProt O95274 glycosylation). Their effect on this IHC antibody or antigen retrieval is unreported in the supplied sources.
Tissue and cell-type controls	HPA reports high staining in squamous epithelial cells at several sites and no detection in adipocytes from adipose tissue or respiratory epithelial cells from bronchus (HPA tissue IHC). These are cell-specific comparisons, not whole-tissue guarantees.
Esophagus evidence	HPA reports high esophageal squamous-cell IHC staining, whereas UniProt describes esophageal expression as weak (HPA tissue IHC; UniProt O95274 tissue specificity). Keep the source and measurement context attached to each claim.
IF/ICC Q: Should intracellular fluorescence be scored like IHC membrane staining?	A: No. HPA's ICC-IF summary localizes LYPD3 to approved endoplasmic-reticulum and vesicle sites (HPA subcellular ICC-IF), while UniProt lists the cell membrane (UniProt O95274 subcellular location). Assess IF/ICC on its own guide page.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
The expected squamous epithelium is blank.	Tissue selection or an IHC workflow step may have failed; the listed squamous epithelial cells stain high in HPA IHC (HPA tissue IHC; general IHC practice).	Confirm the epithelial cells are present and inspect a concurrently stained positive section, primary-antibody step and detection controls (general IHC practice).
Only nuclei stain in a nominally positive section.	That compartment conflicts with LYPD3's reported membrane location (UniProt O95274 subcellular location).	Compare with a positive squamous epithelial section and a no-primary control; withhold a positive call if the nuclear pattern persists (general IHC practice).
Color appears in HPA non-detected comparison cells.	Unexpected cell staining can arise from antibody cross-reactivity or endogenous detection activity (HPA tissue IHC; general IHC practice).	Check cell identity, a no-primary control and endogenous-enzyme blocking appropriate to the chromogenic system (general IHC practice).
Diffuse color obscures epithelial borders.	Background may reflect insufficient blocking, washing or overly strong detection conditions (general IHC practice).	Review blocking and washes, and compare the no-primary control before adjusting assay conditions (general IHC practice).
Staining is weak or uneven across a section.	Section handling or staining steps may vary across the slide (general IHC practice); the supplied sources give no LYPD3-specific fixation sensitivity.	Check section coverage and reagent delivery, then assess antigen retrieval with routine assay controls; do not infer an LYPD3-specific retrieval requirement (general IHC practice).
A tumor stains while adjacent tissue has little signal.	UniProt reports LYPD3 in transitional cell carcinomas and breast cancer tissue, with absent staining in adjacent normal tissue in that account (UniProt O95274 tissue specificity).	Score tumor and adjacent cells separately, record membrane-associated staining, and avoid treating one tissue compartment as the control for every other compartment (UniProt O95274 subcellular location; general IHC practice).

Source: UniProt O95274 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing LYPD3 staining

IHC analysis of LYPD3 using anti-LYPD3 antibody (A08396). LYPD3 was detected in a paraffin-embedded section of human skin cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-LYPD3 Antibody (A08396) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A08396 · IHCIHC analysis of LYPD3 using anti-LYPD3 antibody (A08396). LYPD3 was detected in a paraffin-embedded section of human skin cancer tissue. Hea

IHC analysis of LYPD3 using anti-LYPD3 antibody (A08396). LYPD3 was detected in a paraffin-embedded section of rat stomach tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-LYPD3 Antibody (A08396) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

A08396 · IHCIHC analysis of LYPD3 using anti-LYPD3 antibody (A08396). LYPD3 was detected in a paraffin-embedded section of rat stomach tissue. Heat medi

IF analysis of LYPD3 using anti-LYPD3 antibody (A08396). LYPD3 was detected in a paraffin-embedded section of human skin cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 5 μg/mL rabbit anti-LYPD3 Antibody (A08396) overnight at 4°C. Cy3 Conjugated Goat Anti-Rabbit IgG (BA1032) was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A08396 · IFIF analysis of LYPD3 using anti-LYPD3 antibody (A08396). LYPD3 was detected in a paraffin-embedded section of human skin cancer tissue. Heat

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000124466-LYPD3/tissue/cerebral+cortex#imid_10685727

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000124466-LYPD3/tissue/colon#imid_10685365

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000124466-LYPD3/tissue/liver#imid_10685366

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000124466-LYPD3/tissue/kidney#imid_10685375

HPA · ICC-IF HaCaTHuman Protein Atlas immunofluorescence, HaCaT, antibody HPA077859; green = LYPD3, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for LYPD3 IHC & IF

🧪Run cervix first: squamous epithelial cells should stain (HPA: High in cervix squamous epithelial cells). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); on the cervix slide, use adjacent nonsquamous cells to assess background against the no-primary control, without assuming they are target-negative.

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 ICC-IF images show LYPD3 in HaCaT, RT-4, U2OS, with annotated localisation: Endoplasmic reticulum (approved), Vesicles (approved) (HPA subcellular).

Technical controls: Include no-primary (secondary-only) and host- and isotype-matched rabbit IgG controls, matching clonality if known (caption: rabbit primary antibody; standard IHC controls). Use a validated LYPD3 knockout specimen as a biological negative if available, and block endogenous peroxidase before HRP/DAB detection (caption: HRP/DAB; standard IHC practice).

⚠️Feasibility: The selected A08396 paraffin-section caption does not state a fixative, and no target-specific fixation window or fixation effect is reported in the supplied evidence (caption: fixative not stated). Heat retrieval with EDTA at pH 8.0 is documented for that IHC example, but retrieval dependence and whether frozen sections or IF are easier are unreported (caption: EDTA retrieval at pH 8.0). In cervix, check for nonspecific signal from endogenous peroxidase in blood-containing areas when interpreting DAB staining (standard IHC practice).

HPA tissue IHC evidence for LYPD3

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded.). 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 corneal layer	High	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 LYPD3 IHC Tips

Troubleshoot LYPD3 staining in paraffin sections by checking retrieval, compartment, cell type, and controls before comparing signal intensity (UniProt O95274; HPA tissue IHC).

Which retrieval conditions should I try first when LYPD3 staining is weak?

Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A08396). The selected LYPD3 tissue image used that retrieval before incubation with 2 μg/ml antibody overnight at 4°C, so reproduce those conditions when investigating weak staining (datasheet A08396). If signal remains weak, compare shorter and longer heating under the same EDTA condition while holding section preparation and detection constant (standard IHC practice). Include a squamous epithelial positive control and assess tissue preservation alongside staining, because damaged morphology can make an apparent gain in signal difficult to interpret (HPA tissue IHC; standard IHC practice).

Could fixation explain inconsistent LYPD3 staining between paraffin samples?

Target-specific fixation sensitivity for LYPD3 is unknown from the supplied evidence, and the selected paraffin-section caption does not state a fixative (datasheet A08396). Record the fixative, fixation duration, processing history, and section age for each sample before comparing staining intensity (standard IHC practice). Run samples together with the same EDTA retrieval at pH 8.0, 2 μg/ml primary concentration, and overnight incubation at 4°C used in the selected image (datasheet A08396). If staining differs, compare matched control tissue processed with each sample and report the preparation difference as a possible confounder, rather than assigning a LYPD3-specific fixation mechanism (standard IHC practice).

Should LYPD3 appear only at the cell surface in chromogenic IHC?

Interpret a crisp cell-boundary pattern in the appropriate epithelium in light of LYPD3's annotated cell-membrane location and GPI-anchor keyword (UniProt O95274). Intracellular staining also deserves review because approved cell-imaging locations include endoplasmic reticulum and vesicles (HPA subcellular). Score membrane-associated and cytoplasmic DAB separately rather than treating every brown deposit as equivalent target signal (standard IHC practice). Compare the pattern with squamous epithelial cells in cervix, esophagus, oral mucosa, tonsil, or vagina, where high tissue-IHC staining is reported (HPA tissue IHC). Check boundaries at higher magnification and use the same pH 8.0 EDTA retrieval across compared sections (datasheet A08396; standard IHC practice).

How do processing and glycosylation affect my reading of an LYPD3 stain?

The annotated mature LYPD3 chain spans residues 31–326, following a 1–30 signal peptide and preceding a 327–346 propeptide (UniProt O95274). Four glycosylation sites are annotated at residues 118, 163, 176, and 183, within or near the two annotated UPAR/Ly6 domains (UniProt O95274). The record lists 0 isoforms, but the supplied caption does not identify the catalog antibody's epitope (UniProt O95274; datasheet A08396). Therefore, do not explain a changed IHC pattern by a particular isoform or masked epitope without additional antibody-mapping evidence; compare the same retrieval and controls first (standard IHC practice).

How should I investigate LYPD3 by multiplex IF after seeing an IHC pattern?

For multiplex IF, pair LYPD3 with a validated squamous epithelial marker and check whether their positive cells coincide in the tissue being examined (HPA tissue IHC; standard IF practice). Choose a fluorophore channel with low measured tissue autofluorescence, and include an unstained section to assess that background (standard IF practice). LYPD3 is annotated at the cell membrane, while approved cell-imaging locations include endoplasmic reticulum and vesicles (UniProt O95274; HPA subcellular). Because the catalog antibody's epitope side is unspecified, compare staining without permeabilisation to a gently permeabilised condition before interpreting intracellular signal (datasheet A08396; standard IF practice). The pH 8.0 EDTA condition documents paraffin IHC retrieval, not an IF validation (datasheet A08396).

How can I reduce diffuse brown staining without losing true LYPD3 signal?

Check a section lacking primary antibody to identify detection-system staining, and inspect an unstained section for tissue pigment before changing the primary concentration (standard IHC practice). Block endogenous peroxidase before HRP/DAB development and use an appropriate serum or protein block to limit nonspecific staining (standard IHC practice). The selected image used 10% goat serum, 2 μg/ml rabbit primary overnight at 4°C, and a peroxidase-linked secondary for 30 minutes at 37°C (datasheet A08396). If background persists, titrate primary antibody against a squamous epithelial positive control while preserving the same EDTA pH 8.0 retrieval (HPA tissue IHC; datasheet A08396; standard IHC practice).

What should I measure when comparing LYPD3 IHC across sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the epithelial or tumor region before scoring, and exclude folds, necrosis, and section edges from measurements (standard IHC practice). Report the percentage of positive cells and staining intensity separately, or calculate an H-score from intensity categories 0–3 and their percentages, yielding 0–300 (standard IHC practice). Score membrane-associated and cytoplasmic signal separately because LYPD3 has a membrane annotation and approved intracellular imaging locations (UniProt O95274; HPA subcellular). Normalize positive-cell counts to the total evaluable cells in the same compartment, or report positive area per mm² of evaluable tissue (standard IHC practice). Keep retrieval, detection, exposure, and scoring thresholds consistent across compared sections (standard IHC practice).

When should an apparent LYPD3-positive area be treated as artefact?

Give greatest weight to reproducible staining in the expected cell compartment and cell type: LYPD3 is membrane annotated, and high tissue-IHC signal occurs in several squamous epithelia (UniProt O95274; HPA tissue IHC). Intracellular staining can still merit investigation because approved cell-imaging locations include endoplasmic reticulum and vesicles (HPA subcellular). Treat staining concentrated at section edges, folds, or necrosis as suspect, and compare it with nearby intact tissue (standard IHC practice). If DAB appears in unexpected cells, check a no-primary control for endogenous peroxidase or detection background before calling them positive (standard IHC practice). HPA reports presumed off-target binding despite its Enhanced tissue-IHC reliability, so corroborate unusual patterns independently (HPA tissue IHC).

Boster reagents

Best LYPD3 / Ly6/PLAUR domain-containing protein 3 IHC Antibodies

A08396 has IHC images from human skin cancer and rat stomach paraffin sections, plus an IF image from human skin cancer paraffin sections (A08396 image captions).

Real IHC data

Anti-LYPD3 Antibody ®

Cat # A08396

View product →

A08396 will render with its human skin cancer paraffin-section IHC figure; its captions also document rat stomach IHC and human skin cancer IF (A08396 image captions). M08396-1 will render as a human-reactive IHC antibody, although no IHC image is supplied for it (M08396-1 catalog applications, reactivity and image list).

Which to pick: For tissue IHC, choose A08396 when an illustrated paraffin-section workflow is useful; its IHC captions document human skin cancer and rat stomach sections, and do not report the fixative (A08396 IHC image captions). For IF, choose A08396 based on its human paraffin-section IF image; ICC validation is not documented (A08396 IF image caption and applications). For work across species, A08396 lists human, mouse and rat reactivity, while the rabbit monoclonal M08396-1 lists human reactivity and IHC without a supplied figure (catalog reactivity, host, clonality, applications and image lists).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry O95274 (LYPD3_HUMAN, Ly6/PLAUR domain-containing protein 3).
Human Protein Atlas. LYPD3 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. LYPD3 subcellular location (ICC-IF): Localized to the endoplasmic reticulum and vesicles..
Human Protein Atlas. LYPD3 antibody validation summary (3 antibodies).
m6A-dependent mature miR-151-5p accelerates the malignant process of HNSCC by targeting LYPD3. Molecular biomedicine 2024 — PMC11250566.
Identification and validation of cellular senescence patterns to predict clinical outcomes and immunotherapeutic responses in lung adenocarcinoma. Cancer cell international 2021 — PMC8647370.
β2-Adrenergic Signalling Promotes Cell Migration by Upregulating Expression of the Metastasis-Associated Molecule LYPD3. Biology 2020 — PMC7168268.
The Lineage Determining Factor GRHL2 Collaborates with FOXA1 to Establish a Targetable Pathway in Endocrine Therapy-Resistant Breast Cancer. Cell reports 2019 — PMC6874102.
PubMed PMID:11245483 — UniProt-cited evidence.
PubMed PMID:11179665 — UniProt-cited evidence.
PubMed PMID:12975309 — UniProt-cited evidence.