PDPN / Podoplanin · IHC design guide

Design Immunohistochemistry for PDPN

Plan PDPN IHC on paraffin sections using membrane localization and alveolar type I cells in lung as guides (UniProt; HPA tissue IHC). Start the IHC-validated antibody at 1:200–1:1000 and score staining by cell type (datasheet: 1:200–1:1000; HPA tissue IHC).

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

Immunohistochemistry protocol sheet for PDPN (IHC for PDPN): expected localisation Plasma membrane expected; apical or basolateral staining possible (UniProt), antibody M01124-4, validated IHC image, and IHC protocol steps — Printable **PDPN** IHC protocol sheet — expected localisation Plasma membrane expected; apical or basolateral staining possible (UniProt), antibody M01124-4, controls and protocol steps. Open the full PDPN IHC guide →

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PDPN 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	Plasma membrane expected; apical or basolateral staining possible (UniProt)
	Staining pattern	Alveolar, lymphatic, stromal and basal-cell staining; compartment unreported (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen)
	Positive control ⓘ	Breast+2 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 sections. (standard IHC practice; not target-specific)
	Caveat	PDPN-positive stroma can complicate tumor-cell scoring (HPA tissue IHC)
	Regulation	Up-regulated in colorectal tumors (UniProt)
	Isoform / epitope	Six isoforms; check whether the epitope is extracellular or cytoplasmic (UniProt)

Section 1

Recommended PDPN IHC & IF Protocols

Compare the catalog antibody’s IHC-P protocol with three published PDPN protocols for paraffin sections (PMC12109644; PMC11202497; PMC12771865).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Tissue sections; selected-image fixative not specified (standard IHC workflow)
Fixation	Image fixative and duration unreported (datasheet M01124-4); 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-PDPN, 1:200-1:1000 (datasheet M01124-4)
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	PDPN-positive staining in myoepithelial cells of breast (HPA tissue IHC: High). HPA tissue profile: Distinct expression in alveoli, lymphatic vessels, placental and ovarian stroma, myoepithelium and basal cells of squamous epithelia. No signal in the no-primary control.

💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen-retrieval rule); the published alternatives used different retrieval conditions with clone D2-40 (PMC12109644; PMC12771865).

Section 2

What Is the Expected PDPN Staining Pattern?

PDPN should chiefly outline the plasma membrane of positive cells, consistent with its single transmembrane segment and extracellular domain (UniProt Q86YL7 topology; HPA subcellular). In paraffin sections, expect strong staining in breast myoepithelial cells and lung alveolar type I cells (HPA tissue IHC: High). HPA rates its tissue profile Enhanced, while describing only medium consistency between antibody staining and RNA expression (HPA tissue IHC).

What am I looking at on my slide?
Crisp cell borders in breast myoepithelium or lung alveolar type I cells.	This fits the expected membrane compartment and two documented High cell populations (UniProt Q86YL7 topology; HPA tissue IHC: High). Judge the relevant cells, since neighboring cells need not share their staining level (HPA tissue IHC).
Predominantly nuclear, uniformly cytoplasmic, or punctate intracellular staining without a membrane outline.	Treat this as a compartment mismatch and investigate artefact (HPA subcellular: plasma membrane supported; UniProt Q86YL7 topology). UniProt also lists cytosol, so a cytoplasmic component alone does not prove artefact; HPA calls mitochondrial localization uncertain (UniProt Q86YL7 subcellular; HPA subcellular).
Strong staining in adipocytes or bone marrow hematopoietic cells.	These particular cell populations are listed as Not detected; assess cross-reactivity or endogenous detection activity before calling them PDPN-positive (HPA tissue IHC: Not detected; general IHC practice). The HPA entries do not make every cell in those tissues a negative control (HPA tissue IHC).
Broad, hazy color across cells and surrounding tissue, obscuring cell borders.	Interpret the diffuse signal cautiously because it cannot establish the membrane pattern supported for PDPN (HPA subcellular; UniProt Q86YL7 topology). Check detection and blocking controls and whether the signal tracks tissue structure (general IHC practice).
No staining in the expected cells of a breast or lung positive-control section.	An absent signal where HPA reports High expression raises an assay-performance question, although a single section does not establish why it failed (HPA tissue IHC: High). Review control tissue identity and the validated IHC conditions before interpreting a study specimen as negative (general IHC practice).

💡Expected PDPN appearanceCall a positive result when distinct membrane staining marks breast myoepithelial or lung alveolar type I cells, where HPA reports High staining; diffuse haze or isolated staining of HPA Not detected cell populations is suspect (HPA tissue IHC; HPA subcellular).

How each factor affects the staining
Membrane topology and cellular location (UniProt Q86YL7 topology; HPA subcellular).	PDPN has one transmembrane segment, with residues 23–131 extracellular and 153–162 cytoplasmic (UniProt Q86YL7 topology). HPA supports plasma-membrane localization and cell junctions; its mitochondrial assignment is uncertain (HPA subcellular). Use a membrane outline as the principal morphology check (HPA subcellular).
Cell-specific reference pattern and evidence strength (HPA tissue IHC).	HPA reports High staining in breast myoepithelial and lung alveolar type I cells, Medium in round or early spermatids, and Low in several listed epithelial populations (HPA tissue IHC). Its overall Enhanced tissue reliability carries a medium RNA–staining consistency description, so match the named cell type and expected level (HPA tissue IHC).
Antibody-specific IHC validation (HPA antibodies).	HPA lists IHC Enhanced for HPA007534 and CAB008376; this category means the pattern was reproduced by independent antibodies or orthogonal data (HPA antibodies). That supports their reported IHC patterns, while assay behavior must still be checked for the antibody and section actually used (general IHC practice).
Sequence processing, glycosylation, and isoforms (UniProt Q86YL7).	UniProt lists a cleaved signal peptide at residues 1–22, a mature chain at 23–162, 24 glycosylation sites, and 6 isoforms (UniProt Q86YL7). These annotations do not identify this assay's epitope or establish antigen-retrieval needs, shedding, or fixation sensitivity (UniProt Q86YL7).
IF/ICC Q: Does the cell-imaging evidence change the IHC call? (HPA subcellular).	A: HPA ICC-IF supports plasma membrane and cell-junction localization, with mitochondrial localization uncertain (HPA subcellular). These images inform the compartment check; they do not establish a paraffin-IHC protocol or make an uncertain mitochondrial signal an expected positive pattern (HPA subcellular).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Positive-control breast myoepithelial or lung alveolar type I cells are blank (HPA tissue IHC: High).	The control may be unsuitable or the staining run may have failed; the blank slide alone cannot distinguish these causes (general IHC practice).	Confirm that the expected cells are present, then check the run control and the IHC-validated antibody's stated conditions (HPA tissue IHC; general IHC practice).
Signal is weak only in oral, esophageal, or vaginal squamous epithelium (HPA tissue IHC: Low).	HPA lists these squamous epithelial populations as Low; their weak signal need not indicate assay failure (HPA tissue IHC).	Compare a documented High population on the same run before changing the assay; score the specified cells separately (HPA tissue IHC: High and Low; general IHC practice).
Staining is chiefly nuclear or diffusely cytoplasmic (HPA subcellular: plasma membrane supported).	The compartment conflicts with the principal HPA location, though UniProt also lists cytosol (HPA subcellular; UniProt Q86YL7 subcellular).	Recheck morphology against a membrane-positive control and examine detection and background controls before assigning PDPN specificity (HPA subcellular; general IHC practice).
Adipocytes or marrow hematopoietic cells stain strongly (HPA tissue IHC: Not detected).	Cross-reactivity or endogenous detection activity is possible; a stained negative-reference population alone does not identify which (HPA tissue IHC; general IHC practice).	Inspect appropriate reagent and detection controls, and compare the staining with known positive cell morphology (HPA tissue IHC; general IHC practice).
Diffuse color obscures an otherwise plausible membrane signal (HPA subcellular).	Background from detection, blocking, or excess reagent is possible; appearance alone cannot isolate the source (general IHC practice).	Review the controls, blocking, washing, and antibody dilution used in the run; reassess whether cell borders remain distinguishable (general IHC practice).
A mitochondrial-looking punctate pattern dominates an IF/ICC image (HPA subcellular: uncertain).	HPA labels mitochondrial localization uncertain, while its main supported location is the plasma membrane (HPA subcellular).	Treat puncta as unresolved and check membrane and cell-junction signal against appropriate imaging controls; keep the IHC interpretation anchored to tissue morphology (HPA subcellular; general IF/IHC practice).

Source: UniProt Q86YL7 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing PDPN staining

M01124-4 · IHCMosue lung was stained with anti-Podoplanin rabbit antibody

M01124-4 · IHCRat lung was stained with anti-Podoplanin rabbit antibody

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000162493-PDPN/tissue/cerebral+cortex#imid_2752028

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000162493-PDPN/tissue/colon#imid_2751962

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000162493-PDPN/tissue/liver#imid_2751955

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000162493-PDPN/tissue/kidney#imid_2751948

HPA · ICC-IF EFO-21Human Protein Atlas immunofluorescence, EFO-21, antibody HPA007949; green = PDPN, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for PDPN IHC & IF

🧪Run lung first: alveolar type I cells should stain (HPA: High in lung alveolar cells type I). Use adipose tissue as the negative, focusing on adipocytes (HPA: Not detected in adipocytes); on the lung slide, cells outside the expected alveolar type I staining pattern provide an internal background check, but should not be assumed PDPN-negative without cell identification (HPA: High in alveolar cells type I).

Positive control tissue: Breast (Myoepithelial cells, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show PDPN in EFO-21, HAP1, U2OS, with annotated localisation: Plasma membrane (supported) (HPA subcellular).

Technical controls: Include no-primary (secondary-only) and host-species-, isotype- and clonality-matched irrelevant immunoglobulin controls, plus PDPN-knockout material as a biological negative (standard IHC practice). For lung chromogenic IHC, quench endogenous peroxidase and assess background in the no-primary section (standard IHC practice).

⚠️Feasibility: A target-specific fixation window and the effect of fixation are unreported in the supplied evidence; the selected M01124-4 lung caption does not state a fixative (selected product caption). Retrieval dependence is also unreported, so optimize antigen retrieval using the catalog antibody’s IHC-P instructions; the supplied evidence does not establish whether frozen sections or IF would be easier (supplied IHC evidence). In lung, assess endogenous peroxidase background carefully when scoring alveolar staining (standard IHC practice; HPA: High in alveolar cells type I).

HPA tissue IHC evidence for PDPN

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium 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
Breast	Myoepithelial cells	High	Protein (IHC)	HPA →
Lung	Alveolar cells type I	High	Protein (IHC)	HPA →
Testis	Round or early spermatids	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Adrenal gland	Glandular cells	Not detected	Protein (IHC)	HPA →
Appendix	Glandular cells	Not detected	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Not detected	Protein (IHC)	HPA →
Caudate	Glial cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced PDPN IHC Tips

Troubleshoot PDPN staining in paraffin section IHC, with one Q&A on IF multiplexing.

How should I adjust retrieval when PDPN staining is weak?

Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min, for PDPN paraffin IHC (page retrieval setting). If staining remains weak, compare a longer retrieval interval on adjacent sections while keeping the buffer and detection conditions constant; excessive heating can damage tissue morphology (standard IHC practice). Use lung alveolar type I cells as a positive reference and compare their staining with nearby structures on the same section (HPA: High in lung alveolar type I cells). Record both signal and tissue preservation before adopting a changed condition, because the supplied retrieval setting has no stated antibody-specific optimization result (page retrieval setting).

Could fixation explain weak or uneven PDPN staining?

PDPN-specific sensitivity to fixation is unknown from the supplied evidence; the selected lung IHC caption does not state a fixative (selected IHC caption: M01124-4). For paraffin IHC, document fixative, fixation duration, processing, and section age before comparing specimens, since these variables can affect antigen accessibility (standard IHC practice). Compare sections processed together using the same citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval setting). If the expected alveolar type I cell staining differs across batches, repeat staining with a common reference section before attributing the difference to PDPN expression (HPA: High in lung alveolar type I cells; standard IHC practice).

Which staining pattern should count as PDPN positive?

Score a crisp cell surface pattern as the primary finding, because PDPN is a membrane protein and HPA supports plasma membrane localisation (UniProt Q86YL7 topology; HPA subcellular: plasma membrane supported). Staining may outline apical or basolateral surfaces and cell projections, so assess the entire cell boundary where tissue orientation permits (UniProt Q86YL7 subcellular localisation). Compare suspected positive cells with lung alveolar type I cells or breast myoepithelial cells as documented tissue references (HPA: High in lung alveolar type I and breast myoepithelial cells). Treat diffuse nuclear staining as discordant with the supplied localisation evidence and investigate background, counterstain, and section quality before scoring it as PDPN (UniProt Q86YL7 subcellular localisation; standard IHC practice).

Could the antibody epitope change which PDPN cells stain?

Check the catalog antibody's stated immunogen and epitope before interpreting discordant paraffin IHC results; no epitope is specified in the supplied evidence (catalog antibody: epitope unspecified). PDPN has 6 annotated isoforms, an extracellular segment at residues 23–131, a transmembrane segment at 132–152, and a cytoplasmic segment at 153–162 (UniProt Q86YL7 isoforms and topology). Its extracellular region has 24 annotated glycosylation sites, so an extracellular antibody's recognition may depend on the actual epitope and specimen processing; that dependence has not been established here (UniProt Q86YL7 glycosylation; standard IHC practice). Compare documented epitope information and staining controls before claiming that a negative cell population lacks every PDPN isoform (UniProt Q86YL7 isoforms; standard IHC practice).

How can IF help check a disputed PDPN IHC pattern?

On a separate IF section, pair PDPN with a marker identifying the expected cell population, then inspect colocalisation at cell boundaries rather than relying on overlapping field-level signal (HPA: High in lung alveolar type I cells; UniProt Q86YL7 subcellular localisation; standard IF practice). Choose a fluorophore channel after examining unstained tissue autofluorescence, and include single-stain controls when comparing channels (standard IF practice). If the antibody recognises extracellular residues 23–131, test staining without permeabilisation; a cytoplasmic epitope at 153–162 generally requires permeabilisation after fixation (UniProt Q86YL7 topology; standard IF practice). Confirm the catalog antibody's epitope first, because none is specified here, and interpret IF alongside the paraffin IHC result rather than assuming equivalent antigen access (catalog antibody: epitope unspecified; standard IF/IHC practice).

How do I reduce diffuse or vessel-associated background?

First compare the staining with a no-primary control and an appropriate negative tissue compartment, then check whether the signal follows cell membranes (standard IHC practice; HPA subcellular: plasma membrane supported). Block endogenous peroxidase before chromogenic detection and use a matched detection control to identify enzyme-related color unrelated to primary antibody binding (standard chromogenic IHC practice). Titrate the catalog antibody and adjust blocking or washing one variable at a time, recording whether alveolar type I cell outlines remain visible (standard IHC practice; HPA: High in lung alveolar type I cells). Do not call every vessel-associated signal background: HPA describes PDPN expression in lymphatic vessels, so evaluate cellular location and control staining together (HPA tissue profile).

How should I quantify PDPN across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the compartment and cell population before scoring, then measure membrane staining as percentage of positive cells and intensity, or calculate an H-score using consistent thresholds (HPA subcellular: plasma membrane supported; standard IHC practice). For alveolar regions, normalise the positive cell count to the number of assessable alveolar type I cells; for lymphatic structures, report positive profiles per mm² of evaluable tissue when cell counting is impractical (HPA: High in lung alveolar type I cells; HPA tissue profile: lymphatic vessels; standard IHC practice). Exclude folds, necrosis, and damaged edges using the same criteria for every section (standard IHC practice). Keep retrieval, imaging, and scoring settings consistent, and report how background controls informed the positivity threshold (standard IHC practice).

When is a PDPN positive result biologically credible?

A credible PDPN result combines membrane-associated staining with the expected cellular context, such as lung alveolar type I cells or breast myoepithelial cells (HPA subcellular: plasma membrane supported; HPA: High in both cell types). Compare suspicious staining with a no-primary control and with compartments reported as not detected, such as adipocytes, while accounting for tissue composition (HPA: Not detected in adipocytes; standard IHC practice). Nuclear-only color, section-edge staining, and signal concentrated in necrosis warrant investigation before interpretation (UniProt Q86YL7 subcellular localisation; standard IHC practice). Residual endogenous peroxidase can also create chromogenic signal, so confirm peroxidase blocking and preserve cell-level localisation when deciding whether a section is positive (standard chromogenic IHC practice).

Boster reagents

Best PDPN / Podoplanin IHC Antibodies

The catalog antibody has IHC images from lung samples in mouse, rat and human, and lists IF for all three species (catalog: M01124-4 applications, reactivity and IHC image captions).

Real IHC data Mosue lung was stained with anti-Podoplanin rabbit antibody

Anti-Podoplanin Rabbit Monoclonal Antibody

Cat # M01124-4

View product →

M01124-4 is the SKU rendered with an IHC figure; its captions describe mouse lung, rat lung and human lung squamous carcinoma (catalog: M01124-4 IHC image captions). It lists IHC and IF applications and Human, Mouse and Rat reactivity; no IF image is supplied (catalog: M01124-4 applications, reactivity and image alts).

Which to pick: Choose M01124-4 for tissue IHC across human, mouse or rat samples: it is a rabbit monoclonal with IHC listed, and its own IHC captions document staining in all three species (catalog: M01124-4 applications, reactivity, description and IHC image captions). For IF, M01124-4 lists a 1:200–1:1000 dilution, but the payload supplies no IF image or separate ICC validation (catalog: M01124-4 dilution and image alts). M01124-2 is a human reactive rabbit monoclonal IHC option without a supplied IHC image; tissue processing and fixative are unreported for both SKUs (catalog: M01124-2 applications, reactivity and image alts; M01124-4 IHC image captions).

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

References

UniProt Consortium. UniProt entry Q86YL7 (PDPN_HUMAN, Podoplanin).
Human Protein Atlas. PDPN tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. PDPN subcellular location (ICC-IF): Mainly localized to the plasma membrane. In addition localized to the cell junctions and mitochondria..
Human Protein Atlas. PDPN antibody validation summary (3 antibodies).
Diagnostic Utility of Podoplanin Immunohistochemistry Combined with the NanoSuit-Correlative Light and Electron Microscopy Method for Thoracic Malignant Tumors. Diagnostics (Basel, Switzerland) 2025 — PMC12109644.
Combining RNAscope, Immunohistochemistry (IHC) and Digital Image Analysis to Assess Podoplanin (PDPN) Protein and PDPN_mRNA Expression on Formalin-Fixed Paraffin-Embedded Normal Human Placenta Tissues. Current issues in molecular biology 2024 — PMC11202497.
Expression of periostin and podoplanin in canine testicular tumours. BMC veterinary research 2025 — PMC12771865.
Expression of podoplanin in stromal fibroblasts plays a pivotal role in the prognosis of patients with pancreatic cancer. Surgery today 2018 — PMC5711987.
PubMed PMID:9651190 — UniProt-cited evidence.
PubMed PMID:10393083 — UniProt-cited evidence.
PubMed PMID:16710414 — UniProt-cited evidence.