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FLT4 / Vascular endothelial growth factor receptor 3 · IHC design guide
Design Immunohistochemistry for FLT4
Plan paraffin-section FLT4 IHC around the cytoplasmic and membranous tissue pattern, including endothelial cells (HPA tissue IHC). This guide covers fixation consistency, epitope selection and interpretation of staining from the secreted isoform (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
FLT4 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 and membranous staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Membranous and cytoplasmic signal, including endothelial cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A01276-2) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Bone marrow+3 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 A01276-2) | |
| Caveat | Secreted isoform 3 may complicate tissue staining interpretation (UniProt) | |
| Regulation | FLT4 expression regulation is unspecified (UniProt) | |
| Isoform / epitope | 3 isoforms; check extracellular vs cytoplasmic epitope coverage (UniProt) |
Section 1
Recommended FLT4 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published chromogenic FLT4 staining methods for colorectal tissue (PMC4413649) and Kaposi sarcoma biopsies (PMC10093722).
| Sample | Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A01276-2) |
| Fixation | Image fixative and duration unreported (datasheet A01276-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet A01276-2) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01276-2) |
| Primary antibody | Rabbit anti-FLT4, 0.5-1μg/ml (datasheet A01276-2) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A01276-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | FLT4-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and membranous expression in most tissues, including endothelial cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate retrieval at pH 6 (datasheet: A01276-2); the Kaposi sarcoma protocol reports 15 min at 98 °C (PMC10093722).
Section 2
What Is the Expected FLT4 Staining Pattern?
FLT4 is a transmembrane receptor with extracellular and cytoplasmic regions (UniProt P35916 topology). In paraffin-section IHC, expect cytoplasmic and membranous staining in many tissues, including endothelial cells (HPA tissue IHC: Approved; cytoplasmic and membranous profile). Assess the stained cell type alongside its compartment: HPA also reports high staining in selected epithelial, glandular, kidney tubular, and heart muscle cells (HPA tissue IHC).
| Membranous staining with cytoplasmic signal in endothelial cells and other tissue-appropriate cells. | This fits the approved tissue pattern (HPA tissue IHC: Approved). The membrane signal is consistent with FLT4 topology, while intracellular signal is plausible because ligand binding can trigger internalization (UniProt P35916). |
| Strong nuclear-only staining dominates the section, with little membranous or cytoplasmic signal. | Treat the result as questionable against the usual tissue pattern (HPA tissue IHC). Nuclear localization is listed for FLT4, so nuclear signal alone does not prove an artifact; check controls and staining distribution (UniProt P35916 subcellular; standard IHC practice). |
| Hematopoietic cells in bone marrow or germinal center cells stain strongly. | These populations were reported as not detected (HPA tissue IHC). Consider cross-reactivity or endogenous detection activity, then inspect negative controls; a whole tissue cannot be judged negative from those cell-specific entries (standard IHC practice). |
| Diffuse staining covers tissue, blank regions, and multiple unrelated cell types. | A uniform haze is difficult to reconcile with the reported cell-associated pattern (HPA tissue IHC). Check the no-primary control for detection background and review blocking, washing, and chromogen development (standard IHC practice). |
| No signal appears in a section containing an expected high-staining population. | Check that the population is present: kidney tubule cells, bronchial respiratory epithelial cells, and heart cardiomyocytes are listed as high (HPA tissue IHC). If present, review the assay with a positive control (standard IHC practice). |
💡Expected FLT4 appearanceCall the slide positive when tissue-appropriate cells show distinct membranous and cytoplasmic staining, with strong signal plausible in HPA high populations; diffuse haze or staining confined to reported not-detected populations needs control review (HPA tissue IHC; standard IHC practice).
| Cell type and tissue context | FLT4 staining is broad, but intensity differs by annotated population: selected glandular cells and kidney tubule cells are high, while bone marrow hematopoietic cells are not detected (HPA tissue IHC). |
| Compartment and receptor trafficking | FLT4 spans the membrane, and ligand-mediated autophosphorylation leads to rapid internalization (UniProt P35916 topology; subcellular). Interpret cytoplasmic staining with the observed tissue profile; compartment alone cannot establish receptor activation (HPA tissue IHC). |
| Isoforms and epitope location | Three isoforms are listed, including secreted isoform 3; isoform 2 has a truncated C-terminus (UniProt P35916). Without the antibody epitope, the record cannot establish which isoforms its staining detects. |
| Secreted protein and tissue correlation | HPA cautions that at least one variant is secreted and RNA–protein tissue correlation is complex (HPA tissue IHC: reliability description). Score the observed IHC pattern by cell and compartment rather than inferring it from RNA alone. |
| IHC antibody evidence | Both listed antibodies have Approved IHC status (HPA antibodies: HPA067906, HPA074389). Neither is identified here as IHC Enhanced; Approved status does not by itself establish identical staining across antibodies (HPA antibodies). |
| IF/ICC comparison | On its separate IF/ICC guide page, the relevant comparison is mainly cytosolic signal, with supported plasma-membrane and approved centrosome localization (HPA ICC-IF). That cell-image pattern does not replace the tissue IHC readout (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known high-staining cells are present but remain unstained. | The cause is unresolved by the tissue record; weak detection or unsuitable retrieval is possible (HPA tissue IHC; standard IHC practice). | Run a known-positive section and review the antibody's IHC-P instructions, retrieval, dilution, and detection controls before interpreting absence (standard IHC practice). |
| No-primary control develops color. | Endogenous detection activity or nonspecific secondary detection may contribute to color (standard chromogenic IHC practice). | Review the detection-system block and no-primary control, then adjust blocking or detection conditions as appropriate for that system (standard chromogenic IHC practice). |
| Staining forms a diffuse haze across the section. | Background may reflect insufficient blocking or washing, or excessive antibody or chromogen development (standard IHC practice). | Compare controls and titrate antibody and development conditions while preserving signal in an HPA high-staining cell population (standard IHC practice; HPA tissue IHC). |
| Only nuclei stain strongly. | The result conflicts with the usual cytoplasmic and membranous tissue profile, though nuclear FLT4 is listed (HPA tissue IHC; UniProt P35916 subcellular). | Inspect positive and no-primary controls and confirm whether appropriate cells also show the expected compartments before scoring the result (standard IHC practice; HPA tissue IHC). |
| Cells listed as not detected show convincing color. | Cross-reactivity, endogenous detection activity, or cell-identification error may explain the mismatch (HPA tissue IHC; standard IHC practice). | Verify cell identity against the counterstain and compare a no-primary control; report the precise cell population rather than declaring the whole tissue positive (standard IHC practice). |
| Staining differs between antibody preparations. | The supplied record lists multiple isoforms but does not specify antibody epitopes; different isoform recognition remains unproven (UniProt P35916; HPA antibodies). | Check each antibody's documented epitope and IHC validation, and compare the same annotated cell populations under matched scoring criteria (standard IHC practice; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing FLT4 staining
A01276-2 · IHCIHC analysis of VEGF Receptor 3 using anti-VEGF Receptor 3 antibody (A01276-2). VEGF Receptor 3 was detected in paraffin-embedded section of
A01276-2 · IHCIHC analysis of VEGF Receptor 3 using anti-VEGF Receptor 3 antibody (A01276-2). VEGF Receptor 3 was detected in paraffin-embedded section of
A01276-2 · IFIF analysis of VEGF Receptor 3 using anti-VEGF Receptor 3 antibody (A01276-2). VEGF Receptor 3 was detected in immunocytochemical section of
A01276-3 · IHCIHC analysis of VEGF Receptor 3 using anti-VEGF Receptor 3 antibody (A01276-3). VEGF Receptor 3 was detected in paraffin-embedded section of
A01276-3 · IHCIHC analysis of VEGF Receptor 3 using anti-VEGF Receptor 3 antibody (A01276-3). VEGF Receptor 3 was detected in paraffin-embedded section of
A01276-3 · IFIF analysis of VEGF Receptor 3 using anti-VEGF Receptor 3 antibody (A01276-3). VEGF Receptor 3 was detected in immunocytochemical section of
M01276-1 · IHCImmunohistochemical analysis of paraffin-embedded H. testis section using VEGFR3. was diluted at 1:25 dilution. A peroxidase-conjugated goat
M01276-1 · IHCImmunohistochemical analysis of paraffin-embedded H. kidney section using VEGFR3. was diluted at 1:25 dilution. A peroxidase-conjugated goat
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000037280-FLT4/tissue/cerebral+cortex#imid_18987261
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000037280-FLT4/tissue/colon#imid_18987187
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000037280-FLT4/tissue/liver#imid_18987188
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000037280-FLT4/tissue/kidney#imid_18987173
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA074389; green = FLT4, blue = nucleus, red = microtubules.
HPA · ICC-IF HEK293Human Protein Atlas immunofluorescence, HEK293, antibody HPA074389; green = FLT4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for FLT4 IHC & IF
🧪Run adrenal gland first: glandular cells should stain (HPA: High in adrenal gland glandular cells). Use bone marrow hematopoietic cells as the negative tissue (HPA: Not detected in bone marrow hematopoietic cells); on the adrenal slide, cells without specific staining should remain at background, but vascular endothelial cells should not be presumed negative (UniProt P35916: protein detected in endothelial cells).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show FLT4 in HEK293, MCF-7, U2OS, with annotated localisation: Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, a rabbit IgG isotype control matched to the catalog primary (selected A01276-2 caption: rabbit primary), and an FLT4 knockout specimen as a biological negative when available. For adrenal DAB sections, check intrinsic pigment and block endogenous peroxidase; assess endogenous biotin if using the caption’s biotin-based detection (selected A01276-2 caption: biotinylated secondary and SABC–DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected SKU caption does not state a fixative (selected A01276-2 caption: fixative not stated). Heat retrieval in citrate buffer at pH 6 for 20 minutes provides a paraffin-section starting point (selected A01276-2 caption); the supplied evidence does not establish that frozen sections or IF are easier, although ICC-IF images are available (HPA subcellular: HEK293, MCF-7 and U2OS). Inspect adrenal sections for pigment that could be mistaken for brown DAB signal.
HPA tissue IHC evidence for FLT4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — At least one protein variant secreted, tissue location of RNA and protein might differ and correlation is complex. Antibody staining in cells/structures not annotated, view images.). 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced FLT4 IHC Tips
Troubleshoot FLT4 staining in paraffin sections by checking retrieval, compartment, isoform coverage, controls and cell-specific scoring before interpreting chromogenic signal.
How should I optimize retrieval when FLT4 staining is weak?
Start with heat-mediated citrate retrieval at pH 6 for 20 minutes (datasheet A01276-2). The selected paraffin-section example used that treatment before staining with 1 µg/mL catalog antibody overnight, so preserve the documented conditions while checking whether section adhesion, heating and cooling were consistent across slides (datasheet A01276-2; standard IHC practice). If staining remains weak, test a second retrieval condition on adjacent sections as a fallback, alongside the documented citrate condition and a no-primary control (standard IHC practice). Compare signal in intact cells with background and tissue damage before adopting a stronger treatment, because retrieval can change both staining and morphology (standard IHC practice).
Can I attribute weak FLT4 staining to fixation?
The selected FLT4 image identifies a paraffin-embedded section but does not state its fixative, so target-specific fixation sensitivity is unknown (datasheet A01276-2). Record the fixative, fixation duration, processing history and section age for each specimen before comparing staining intensity (standard IHC practice). If weak staining tracks with a processing batch, stain comparable sections together using the documented citrate retrieval at pH 6 for 20 minutes and include a specimen with established staining as a run control (datasheet A01276-2; standard IHC practice). Treat any improvement after changing fixation or processing as an observed result for those specimens, rather than an established FLT4-specific effect (standard IHC practice).
Which FLT4 staining compartments should I expect in tissue sections?
Assess membranous and cytoplasmic staining together: FLT4 has an extracellular region at residues 25–775, one transmembrane segment at 776–796 and a cytoplasmic region at 797–1363 (UniProt P35916 topology). HPA reports cytoplasmic and membranous tissue staining, including endothelial cells, while UniProt also lists nuclear and secreted locations (HPA tissue IHC; UniProt P35916 subcellular location). Ligand-mediated autophosphorylation leads to rapid internalization, so cytoplasmic signal need not exclude receptor staining (UniProt P35916 subcellular location). Score compartments separately, and investigate isolated nuclear or extracellular staining with controls before treating it as the same signal as cell-associated staining (standard IHC practice).
Could the antibody miss an FLT4 isoform or processed epitope?
FLT4 has 3 reported isoforms, including a secreted isoform 3; isoform 2 has a truncated C-terminus with fewer phosphorylation sites (UniProt P35916 isoforms and function). The supplied caption does not identify this catalog antibody’s epitope, so staining alone cannot establish which isoforms it detects (datasheet A01276-2). Before comparing specimens, obtain the immunogen or epitope mapping and determine whether it lies in the extracellular region, residues 25–775, or cytoplasmic region, residues 797–1363 (UniProt P35916 topology; standard IHC practice). Interpret discordant cell-associated and extracellular staining cautiously until epitope coverage and assay specificity are established with appropriate controls (standard IHC practice).
How can IF help resolve ambiguous FLT4 staining in the IHC section?
Use IF on a comparable section to ask whether FLT4 signal overlaps an independently validated endothelial marker, since FLT4 protein is detected in endothelial cells (UniProt P35916 tissue specificity; standard IF practice). Choose fluorophores after checking the specimen’s autofluorescence in an unstained section, and include single-label controls to assess bleed-through (standard IF practice). Match permeabilisation to the antibody’s mapped epitope: access to the cytoplasmic region, residues 797–1363, generally requires permeabilisation, whereas an accessible extracellular epitope may be assessed without it (UniProt P35916 topology; standard IF practice). Keep IF compartment findings distinct from the documented chromogenic paraffin-section result, whose fixative and IF performance are unreported (datasheet A01276-2).
How do I reduce diffuse or vessel-adjacent DAB background?
Run a no-primary section and inspect staining after the peroxidase block to identify detection-system or endogenous-enzyme signal (standard IHC practice). The selected example used 10% goat serum, a biotinylated secondary, streptavidin-biotin detection and DAB, so include detection-only controls when background follows tissue structures (datasheet A01276-2; standard IHC practice). If background persists, check blocking, wash stringency and primary concentration while retaining the documented 1 µg/mL condition as the comparison (datasheet A01276-2; standard IHC practice). Judge a reduction by whether intact cell-associated membrane or cytoplasmic staining remains distinguishable from diffuse deposit, since both compartments are reported in tissue (HPA tissue IHC; standard IHC practice).
How should I quantify FLT4 when several cell types stain? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because HPA reports cytoplasmic and membranous FLT4 staining in most tissues, including endothelial cells (HPA tissue IHC). For a defined cell population, report the percentage of positive cells and an H-score calculated as 1 × % weak + 2 × % moderate + 3 × % strong, with membrane and cytoplasm scored separately (standard IHC practice). For stained vascular profiles, report profile density per mm² of viable tissue and normalise stained profiles to the total evaluable profiles in that area (standard IHC practice). Apply one threshold across batches and exclude folds, necrosis and tissue edges from the denominator (standard IHC practice).
What distinguishes credible FLT4 staining from artefact?
Look for reproducible cell-associated membranous or cytoplasmic staining in intact cells, consistent with the tissue pattern reported for FLT4 (HPA tissue IHC; standard IHC practice). Endothelial staining is biologically plausible, but HPA also reports high staining in glandular cells of appendix and rectum, so vessel location alone cannot establish identity or specificity (UniProt P35916 tissue specificity; HPA tissue IHC). Question signal confined to tissue edges, necrotic areas or folds, and compare it with no-primary and detection-only sections to assess nonspecific DAB or endogenous-enzyme staining (standard IHC practice). Investigate predominantly nuclear staining separately because HPA tissue IHC describes cytoplasmic and membranous expression, although UniProt also lists a nuclear location (HPA tissue IHC; UniProt P35916 subcellular location).
Boster reagents
Best FLT4 / Vascular endothelial growth factor receptor 3 IHC Antibodies
Catalog anti-FLT4 antibodies have IHC images from human colon cancer, mouse liver, and human testis paraffin sections, plus IF/ICC images from A431 cells (catalog image captions).
Real IHC data
Anti-VEGF Receptor 3/FLT4 Antibody ®
Cat # A01276-2
Real IHC data
Anti-VEGF Receptor 3/FLT4 Antibody ®
Cat # A01276-3
Real IHC data
Anti-VEGFR3 Antibody
Cat # M01276-1
A01276-2 has human colon cancer paraffin IHC and A431 cell IF images; A01276-3 has mouse liver paraffin IHC and A431 cell IF images (catalog image captions). M01276-1 has a human testis paraffin IHC image and lists IHC-P as an application (M01276-1 IHC caption; catalog applications).
Which to pick: For tissue IHC, choose A01276-2 for human colon cancer paraffin sections, A01276-3 for mouse liver paraffin sections, or monoclonal M01276-1 for human testis paraffin sections (each SKU’s IHC caption; catalog clone 818CT12.1.1). For IF/ICC, choose A01276-2 or A01276-3; both list those applications and show A431 cell IF images (catalog applications; IF captions). For cross-species IHC, A01276-3 lists human, mouse, and rat reactivity and has paraffin IHC images in all three species; its captions do not report the fixative (catalog reactivity; A01276-3 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P35916 (VGFR3_HUMAN, Vascular endothelial growth factor receptor 3).
- Human Protein Atlas. FLT4 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. FLT4 subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the plasma membrane and centrosome..
- Human Protein Atlas. FLT4 antibody validation summary (2 antibodies).
- Genistein suppresses FLT4 and inhibits human colorectal cancer metastasis. Oncotarget 2015 — PMC4413649.
- Upregulation of Cell Surface Glycoproteins in Correlation with KSHV LANA in the Kaposi Sarcoma Tumor Microenvironment. Cancers 2023 — PMC10093722.
- FLT4 Mutations Are Associated with Segmental Lymphatic Dysfunction and Initial Lymphatic Aplasia in Patients with Milroy Disease. Genes 2021 — PMC8535675.
- PubMed PMID:1327515 — UniProt-cited evidence.
- PubMed PMID:1319394 — UniProt-cited evidence.
- PubMed PMID:8386825 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for FLT4 (Vascular endothelial growth factor receptor 3). Expected localisation Cytoplasmic and membranous staining in most tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A01276-2). Validated IHC antibody: A01276-2 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Bone marrow. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A01276-2); 2. Fixation: Image fixative and duration unreported (datasheet A01276-2); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: Citrate pH 6 , 20 min (datasheet A01276-2); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01276-2); 8. Primary antibody: Rabbit anti-FLT4, 0.5-1μg/ml (datasheet A01276-2); 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 A01276-2); Caveat: Secreted isoform 3 may complicate tissue staining interpretation (UniProt); Isoform / epitope: 3 isoforms; check extracellular vs cytoplasmic epitope coverage (UniProt); Regulation: FLT4 expression regulation is unspecified (UniProt). Evidence: UniProt P35916, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the plasma…, Datasheet A01276-2, PMC4413649, PMC10093722. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/FLT4/immunohistochemistry-ihc-guide.