This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
PDPN / Podoplanin · IHC design guide
Design Immunohistochemistry for PDPN
Use lung type I alveolar cells and breast myoepithelial cells as positive controls for PDPN IHC (HPA tissue IHC). Assess staining with its expected membrane location (UniProt) and account for positive lymphatic vessels (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
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, including apical and basolateral surfaces (UniProt) | |
| Staining pattern | Alveoli, lymphatics and myoepithelium (HPA tissue IHC); membrane expected (UniProt) | |
| 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 | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image M01124-4) | |
| Caveat | Lymphatic vessels may complicate lesional scoring (HPA tissue IHC) | |
| Regulation | Up-regulated in colorectal tumors (UniProt) | |
| Isoform / epitope | 6 isoforms; check extracellular vs cytoplasmic epitope (UniProt) |
Section 1
Recommended PDPN IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by four published PDPN IHC protocols covering thoracic tumors, placenta, canine testicular tumors, and pancreatic cancer (PMC12109644; PMC11202497; PMC12771865; PMC5711987).
| 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 retrieval). Adjust for the chosen antibody: published D2-40 protocols also used no retrieval or pH 9 (PMC12109644; PMC12771865).
Section 2
What Is the Expected PDPN Staining Pattern?
PDPN is a transmembrane protein whose main observed location is the plasma membrane (UniProt Q86YL7 topology; HPA subcellular ICC-IF). In paraffin-section IHC, expect staining in breast myoepithelial cells and lung alveolar type I cells, both rated High by HPA (HPA tissue IHC). HPA rates tissue IHC reliability Enhanced, with medium consistency between staining and RNA expression (HPA tissue IHC).
| Crisp staining outlines breast myoepithelial cells or lung alveolar type I cells; nearby cells remain distinguishable. | This fits the main plasma-membrane location and HPA's High staining in those cell populations (HPA subcellular ICC-IF; HPA tissue IHC). Judge the named cells within their tissue architecture rather than treating every cell in the section as a positive control (general IHC practice). |
| Signal is chiefly nuclear or uniformly fills cells, with no convincing membrane outline. | That distribution does not match PDPN's main observed plasma-membrane location (HPA subcellular ICC-IF). UniProt also lists cytosol, so cytoplasmic signal alone is not proof of an artifact (UniProt Q86YL7 subcellular location). Recheck localization and controls before scoring it as specific (general IHC practice). |
| Strong staining appears in adipocytes or adrenal glandular cells while the expected cell populations are hard to identify. | HPA reports PDPN as Not detected in those named cell populations (HPA tissue IHC). Consider cross-reactivity or endogenous chromogenic activity, and compare with a control lacking primary antibody (general IHC practice). A negative HPA call applies to the specified cells, not every structure in that tissue. |
| Brown color spreads across the section or collects over many unrelated cells without clear borders. | Diffuse deposition cannot establish PDPN's expected membrane pattern (HPA subcellular ICC-IF; general IHC practice). Check the control lacking primary antibody, reagent background, blocking and washes before interpreting intensity (general IHC practice). |
| No staining is visible in breast myoepithelial cells or lung alveolar type I cells. | These are High-staining HPA cell populations, so their absence raises a technical or sampling question (HPA tissue IHC). Confirm that the relevant cells are present, then review the IHC-validated antibody's documented procedure, detection reagents and control performance (general IHC practice). |
💡Expected PDPN appearanceCall a positive result when clear membrane-associated chromogenic staining marks the expected breast myoepithelial or lung alveolar type I cells, where HPA reports High staining; diffuse color without cell borders or color in HPA-negative named cells is suspect (HPA tissue IHC; HPA subcellular ICC-IF; general IHC practice).
| Membrane topology (UniProt Q86YL7 topology) | PDPN has an extracellular region at 23–131, a transmembrane segment at 132–152 and a short cytoplasmic tail at 153–162 (UniProt Q86YL7 topology). These regions support a membrane-centered interpretation; they do not identify the catalog antibody's epitope. |
| Cell-specific tissue pattern (HPA tissue IHC) | HPA reports High staining in breast myoepithelial and lung alveolar type I cells, but Not detected in adipocytes and several named glandular cell populations (HPA tissue IHC). Select controls and score results by cell type. |
| Subcellular evidence (HPA subcellular ICC-IF) | Plasma membrane and cell-junction localization are supported in ICC-IF; mitochondrial localization is uncertain (HPA subcellular ICC-IF). ICC-IF localization helps interpret compartment, but does not establish a separate IF/ICC protocol. |
| IHC evidence strength (HPA tissue IHC) | The tissue IHC profile is rated Enhanced, although HPA reports medium consistency between antibody staining and RNA expression (HPA tissue IHC). Use the pattern as a reference, then evaluate the actual slide and controls. |
| Sequence variation and glycosylation (UniProt Q86YL7) | UniProt lists 6 isoforms and multiple glycosylation sites in the extracellular region (UniProt Q86YL7 isoforms; glycosylation; topology). Without an antibody epitope or isoform-specific validation, these annotations cannot predict which variants this stain detects. |
| Antigen retrieval choice (general IHC practice) | Use the IHC-validated antibody's documented paraffin-section conditions and assess retrieval with controls (general IHC practice). Neither the supplied HPA pattern nor UniProt topology establishes a PDPN-specific retrieval requirement or fixation sensitivity. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells are present, but the slide has no signal. | A failed detection step, unsuitable antibody conditions or ineffective retrieval can cause a negative IHC run (general IHC practice); the supplied sources do not identify a PDPN-specific failure mechanism. | Verify the run control and detection reagents, then follow the IHC-validated antibody's documented paraffin-section procedure (general IHC practice). Use HPA's High-staining breast myoepithelial or lung alveolar type I cells as a cell-specific reference (HPA tissue IHC). |
| Color is widespread, including over tissue spaces or unrelated cells. | Non-specific reagent deposition, incomplete blocking or insufficient washing may obscure cell boundaries (general IHC practice). This is inconsistent with a readable membrane-centered pattern (HPA subcellular ICC-IF). | Inspect the control lacking primary antibody; review blocking, washes and chromogen development, then reassess whether positive cell borders remain distinct (general IHC practice). |
| Adipocytes or adrenal glandular cells look strongly positive. | Those named cells are Not detected in HPA tissue IHC, so cross-reactivity or endogenous chromogenic activity is plausible (HPA tissue IHC; general IHC practice). | Compare the same cell type with a control lacking primary antibody and check endogenous enzyme blocking appropriate to the detection system (general IHC practice). Do not extend HPA's cell-specific negative call to the whole tissue. |
| Signal is mainly nuclear or diffuse cytoplasmic. | The result departs from HPA's supported main plasma-membrane location, although UniProt also annotates cytosol (HPA subcellular ICC-IF; UniProt Q86YL7 subcellular location). | Check the positive-control compartment, counterstain and control lacking primary antibody; score membrane staining separately from ambiguous intracellular color (general IHC practice). |
| A suspected positive field looks weak or entirely negative. | The sampled field may lack the specific cells HPA identifies as High, even if their tissue is present elsewhere on the section (HPA tissue IHC; general IHC practice). | Locate and assess breast myoepithelial cells or lung alveolar type I cells before calling the specimen negative; record the cell population scored (HPA tissue IHC; general IHC practice). |
| IF/ICC shows extra intracellular signal that seems to conflict with IHC. | HPA supports plasma-membrane and cell-junction ICC-IF localization, but labels mitochondrial localization uncertain; modalities and images require their own controls (HPA subcellular ICC-IF; general IHC practice). | Use the supported locations to interpret the image and treat mitochondrial assignment cautiously (HPA subcellular ICC-IF). Consult the separate IF/ICC guide for that application's procedure. |
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.
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 cells type I should stain (HPA: High in lung alveolar cells type I). Use adipose tissue adipocytes as the external negative (HPA: Not detected in adipocytes); on the lung slide, cells outside the identified type I alveolar staining pattern should show only background, but should not be assumed PDPN-negative without cell identification.
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 a no-primary (secondary-only) control and a host-species- and antibody-class-matched isotype control to assess detection background (standard IHC practice). Use a PDPN knockout specimen, if available, as a biological negative; peptide competition is an alternative only when the immunizing peptide is known (standard IHC practice). For chromogenic lung IHC, quench endogenous peroxidase and check that pigment is not mistaken for chromogen (standard IHC practice).
⚠️Feasibility: The selected M01124-4 mouse-lung IHC caption does not state a fixative (catalog caption: fixative unreported), and the supplied evidence reports no target-specific fixation window or retrieval dependence; optimize retrieval empirically for paraffin sections. The supplied evidence does not establish whether frozen sections or IF/ICC are easier, although plasma-membrane localization provides an IF interpretation reference (HPA subcellular: plasma membrane supported). Lung pigment can complicate chromogenic interpretation (standard IHC practice), so score staining in the identified type I alveolar cells (HPA: High in lung 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
These questions address PDPN staining in paraffin sections using chromogenic IHC, with one entry on related IF experiments.
How should I adjust retrieval when PDPN staining is weak in paraffin sections?
Use citrate buffer at pH 6.0 for heat-induced retrieval at 95–98 °C for 20 min (page retrieval rule). Compare weak sections with a lung control containing alveolar type I cells, which show high PDPN staining (HPA: lung tissue IHC). Keep section thickness, cooling, antibody incubation and chromogen development consistent across the comparison (standard IHC practice). If staining remains weak, test a different retrieval buffer or pH on matched sections, checking whether any gain in membrane staining also increases background or damages morphology; the catalog antibody’s epitope is not specified here (standard IHC practice; supplied antibody evidence).
Could fixation explain inconsistent PDPN staining between paraffin blocks?
Target-specific PDPN sensitivity to fixation is unknown from the supplied evidence; the selected catalog image identifies mouse lung staining but does not state its fixative (selected SKU M01124-4 caption). Record fixation duration, tissue thickness and processing history for each block before attributing a staining difference to PDPN biology (standard IHC practice). Compare blocks under the same citrate pH 6.0, 95–98 °C, 20 min retrieval conditions and the same detection conditions (page retrieval rule; standard IHC practice). Include a known positive lung section in each run, and interpret a weak test block cautiously if that control also loses staining (HPA: high in alveolar type I cells; standard IHC practice).
Which staining pattern should count as PDPN in lung sections?
Prioritize staining at the cell boundary because PDPN is mainly localized to the plasma membrane, including apical and basolateral regions and membrane projections (HPA: supported plasma membrane location; UniProt Q86YL7 subcellular location). In lung, examine the alveolar lining carefully: alveolar type I cells show high staining in the tissue atlas (HPA: lung tissue IHC). Score diffuse nuclear color separately from convincing membrane staining, and check whether the signal follows intact cell outlines rather than section folds or torn edges (standard IHC practice). A cytosolic annotation also exists, so investigate reproducible nonmembranous staining with controls before assigning it to PDPN (UniProt Q86YL7 subcellular location; standard IHC practice).
How could epitope location affect interpretation of a negative PDPN stain?
First establish whether the antibody recognizes an extracellular or cytoplasmic sequence; the supplied catalog evidence does not identify its epitope (supplied antibody evidence). Mature PDPN has an extracellular region at residues 23–131, a transmembrane segment at 132–152 and a cytoplasmic tail at 153–162 (UniProt Q86YL7 topology and processing). The record lists 6 isoforms and 24 glycosylation sites, so confirm which forms and epitopes the antibody detects before interpreting absence of staining as absence of PDPN (UniProt Q86YL7 isoforms and glycosylation; standard IHC practice). Compare the suspect section with a positive control processed in the same run (standard IHC practice).
How should I check PDPN localisation in a multiplex IF experiment?
Pair PDPN with an independently validated marker of the expected cell type, such as an alveolar type I cell marker in lung, and inspect colocalisation at cell boundaries (HPA: high in alveolar type I cells; HPA: supported plasma membrane location; standard IF practice). Choose a spectrally separated, longer wavelength fluorophore after checking unstained tissue for autofluorescence, then acquire single-color controls for bleed-through (standard IF practice). Set permeabilisation according to the antibody’s mapped epitope: extracellular residues 23–131 may be accessible without it, whereas the cytoplasmic tail at 153–162 requires membrane access (UniProt Q86YL7 topology; standard IF practice). Do not transfer the catalog lung IHC image into an IF fixation claim; its fixative is unreported (selected SKU M01124-4 caption).
What should I check when PDPN chromogenic staining spreads beyond cell boundaries?
Start with a no-primary control and a tissue control expected to have little signal, such as adipocytes, which are not detected in the tissue atlas (HPA: adipose tissue IHC; standard IHC practice). Check endogenous peroxidase blocking before DAB development, then shorten chromogen development or adjust antibody concentration if the no-primary control is clean but test sections look diffuse (standard chromogenic IHC practice). Examine pigment, trapped reagent, section folds and drying edges against an unstained or counterstained section before calling brown deposits PDPN (standard IHC practice). Genuine staining should be assessed against PDPN’s supported plasma membrane location and the expected cell population (HPA: subcellular location and tissue IHC).
How should I score PDPN across paraffin sections with different cell compositions? ⚠ ANSWER MARKED FOR VERIFICATION
Define the membrane-positive compartment and the cell population before scoring, because PDPN is mainly at the plasma membrane and tissue expression varies by cell type (HPA: subcellular location and tissue IHC). For a cell population, report the percentage of positive cells and an intensity-weighted H-score from 0–300, with thresholds set on controls before comparing groups (standard IHC scoring practice). For structures that are difficult to count as individual cells, report positive area or structure density per mm² within a defined region (standard IHC quantification practice). Normalize each measure to eligible cells or analyzed tissue area, and exclude folds, necrosis and empty space consistently (standard IHC practice).
How can I distinguish true PDPN positivity from an artefact?
Look for reproducible cell-boundary staining in an appropriate cell population: alveolar type I cells show high staining, and the plasma membrane is the supported principal location (HPA: lung tissue IHC; HPA: subcellular location). Treat isolated nuclear color, staining limited to cut edges or necrotic areas, and deposits unrelated to intact cell outlines as suspect until controls resolve them (standard IHC practice). Compare the section with a no-primary control to identify endogenous enzyme signal or nonspecific chromogen deposits (standard chromogenic IHC practice). A negative result in one cell population does not establish a failed run when a concurrently processed positive control stains as expected (standard IHC practice).
Boster reagents
Best PDPN / Podoplanin IHC Antibodies
M01124-4 has IHC images from mouse lung, rat lung and human lung squamous carcinoma; it is listed for IHC and IF in human, mouse and rat (catalog image captions, applications and reactivity).
Real IHC data
Anti-Podoplanin Rabbit Monoclonal Antibody
Cat # M01124-4
M01124-4 is the SKU that will render, with IHC and IF listed for human, mouse and rat (catalog applications and reactivity). Its IHC captions identify mouse lung, rat lung and human lung squamous carcinoma; no IF image is supplied (catalog image captions).
Which to pick: For tissue IHC, choose M01124-4 when a pictured tissue example is useful: it is a rabbit monoclonal with an IHC figure captioned mouse lung (catalog host, clonality and IHC caption). For IF planning and cross-species work, M01124-4 is listed for IF and for human, mouse and rat reactivity; ICC validation and an IF image are unreported (catalog applications, reactivity and IF image alts). M01124-2 is a human-reactive monoclonal IHC alternative without an IHC image (catalog clone, applications, reactivity and image alts); M01124-4’s IHC captions do not report paraffin processing or fixative (catalog IHC 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.
Immunohistochemistry protocol sheet for PDPN (Podoplanin). Expected localisation Plasma membrane, including apical and basolateral surfaces (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: M01124-4 at 1:200-1:1000 (the product's own IHC validation image is shown). Controls (HPA): positive Breast (Myoepithelial cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet M01124-4); 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-PDPN, 1:200-1:1000 (datasheet M01124-4); 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 M01124-4); Caveat: Lymphatic vessels may complicate lesional scoring (HPA tissue IHC); Isoform / epitope: 6 isoforms; check extracellular vs cytoplasmic epitope (UniProt); Regulation: Up-regulated in colorectal tumors (UniProt). Evidence: UniProt Q86YL7, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the plasma membrane. In addition localized to…, Datasheet M01124-4, PMC12109644, PMC11202497. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PDPN/immunohistochemistry-ihc-guide.