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NDFIP2 / NEDD4 family-interacting protein 2 · IHC design guide
Design Immunohistochemistry for NDFIP2
Plan chromogenic NDFIP2 IHC in paraffin sections with the catalog antibody’s documented workflow (datasheet M08384). Compare cytoplasmic staining with the tissue profile while accounting for its uncertain reliability (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NDFIP2 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 tissue staining (HPA tissue IHC); endosome and Golgi membranes (UniProt) | |
| Staining pattern | Cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M08384) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adrenal gland+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Presumed off-target staining; splice/transcript discrepancy (HPA tissue IHC) | |
| Regulation | Low tissue specificity (HPA tissue RNA) | |
| Isoform / epitope | No annotated isoforms; cytoplasmic versus extracellular epitopes matter (UniProt) |
Section 1
Recommended NDFIP2 IHC & IF Protocols
The catalog antibody's IHC-P protocol is paired with a published mouse kidney staining method (PMC5606929 Methods).
| Sample | Paraffin-embedded human placenta tissue; fixative not specified (datasheet M08384) |
| Fixation | Image fixative and duration unreported (datasheet M08384); 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 M08384); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M08384) |
| Primary antibody | Mouse monoclonal (clone 10D6D7) anti-NDFIP2, 2 μg/ml (datasheet M08384) |
| Primary incubation | Overnight at 4 °C (datasheet M08384) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet M08384) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NDFIP2-positive staining in lymphoid tissue of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet M08384).
Section 2
What Is the Expected NDFIP2 Staining Pattern?
NDFIP2 is an endosome, multivesicular body and Golgi membrane protein with three transmembrane segments (UniProt Q9NV92 topology). In paraffin sections, expect chiefly cytoplasmic staining in the relevant cells, including colon glandular cells and esophageal squamous epithelial cells (HPA: High in both). Treat the pattern as provisional: HPA rates tissue IHC Uncertain, reports presumed off-target binding, and notes a splice or transcript discrepancy (HPA: tissue IHC reliability).
| Cytoplasmic staining in colon glandular cells or esophageal squamous epithelial cells, with visible tissue structure. | These are useful positive patterns because HPA reports High staining in those cells (HPA: colon glandular cells High; esophagus squamous epithelial cells High). A granular appearance can fit intracellular membranes (UniProt Q9NV92 subcellular location), but granularity alone does not establish antibody specificity (HPA: tissue IHC Uncertain). |
| Predominantly nuclear staining or a sharp outline confined to the cell surface. | That distribution is difficult to reconcile with endosome and Golgi membrane localization (UniProt Q9NV92 subcellular location). Check whether the pattern persists with appropriate controls before interpreting it as NDFIP2; HPA reports presumed off-target binding in tissue IHC (HPA: reliability description). |
| Strong staining in a cell population listed as Not detected, such as adrenal gland glandular cells. | This conflicts with that specific HPA observation (HPA: adrenal gland glandular cells Not detected). Consider cross-reactivity or endogenous detection activity, then check a no-primary control. A Not detected result for one cell population does not classify every cell in that organ (HPA: tissue IHC cell-specific entries). |
| Widespread, nearly uniform color over cells and surrounding tissue, obscuring cell boundaries. | This is hard to score as the cytoplasmic pattern HPA describes (HPA: cytoplasmic expression in most tissues). General IHC causes can include excess antibody or detection background; compare a no-primary control and inspect whether known positive cell populations remain distinguishable. |
| No staining in otherwise interpretable colon glandular cells or appendix lymphoid tissue. | Both are reported High by HPA (HPA: colon glandular cells High; appendix lymphoid tissue High). Review the IHC detection workflow and tissue preservation before calling the target absent. HPA rates its tissue IHC evidence Uncertain, so a single negative section cannot resolve expression or assay specificity (HPA: reliability). |
💡Expected NDFIP2 appearanceA plausible positive is chiefly cytoplasmic color in colon glandular or appendix lymphoid cells at the reported High level (HPA: High in both; HPA: cytoplasmic profile); dominant nuclear, cell-surface-only or control-positive color calls for investigation (UniProt Q9NV92 subcellular location; HPA: tissue IHC Uncertain).
| Compartment and topology (UniProt Q9NV92 topology) | NDFIP2 has three transmembrane segments, a long cytoplasmic N-terminal region and endosome, multivesicular body and Golgi membrane locations (UniProt Q9NV92 topology and subcellular location). The expected tissue readout is cytoplasmic; the exact epitope of a selected antibody is not specified here. |
| Cell-specific tissue pattern (HPA: tissue IHC) | HPA reports High staining in appendix lymphoid, colon glandular, and esophagus and vagina squamous epithelial cells; it reports Medium staining in several neuronal populations (HPA: listed tissue cells). Choose comparisons by cell type, since HPA also lists Not detected populations within other organs. |
| Confidence in tissue interpretation (HPA: reliability) | HPA rates tissue IHC Uncertain and says presumed off-target binding was observed and disregarded; it also flags a splice or transcript discrepancy (HPA: reliability description). Use the reported pattern as a reference for comparison, not a standalone identity test. |
| IF/ICC pattern? (HPA: subcellular; HPA: HPA009160 ICC Supported) | For the separate IF/ICC guide, HPA reports supported localization to vesicles and lists A-431 and CACO-2 images (HPA: subcellular). That observation is compatible with intracellular membrane localization (UniProt Q9NV92), but it does not upgrade the Uncertain tissue IHC rating (HPA: tissue IHC reliability). |
| Processing and modification (UniProt Q9NV92) | UniProt lists the chain as residues 1–336, with no signal peptide, propeptide, annotated isoforms or glycosylation sites; it lists four SRC-related phosphotyrosines (UniProt Q9NV92 processing, isoforms and modified residues). These annotations alone do not predict staining strength or antigen retrieval behavior. |
| Fixation sensitivity (source scope) | Target-specific effects of fixation or antigen retrieval on NDFIP2 staining are unreported in the supplied UniProt and HPA records. Select and optimize those steps as general IHC workflow variables; do not infer a retrieval requirement from topology, phosphorylation or HPA staining levels. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells show little or no color (HPA: colon glandular cells High; appendix lymphoid tissue High). | Possible general IHC workflow failure, unsuitable tissue preservation or an antibody that does not perform in the chosen conditions; HPA tissue IHC is Uncertain (HPA: reliability). | Check the positive control and detection reagents, then optimize the IHC antibody dilution and retrieval conditions as general IHC practice. Record the cell types assessed before concluding that NDFIP2 is absent. |
| Signal is mainly nuclear, despite the expected cytoplasmic tissue profile (HPA: cytoplasmic expression in most tissues). | The distribution conflicts with the annotated endosome and Golgi membrane locations (UniProt Q9NV92 subcellular location); off-target staining is a live possibility (HPA: reliability description). | Compare the no-primary control and a reported positive cell population. If nuclear staining persists as the dominant result, treat target assignment as unresolved rather than scoring it as specific NDFIP2. |
| A listed Not detected population stains strongly, such as heart muscle cardiomyocytes (HPA: cardiomyocytes Not detected). | Cross-reactivity or endogenous detection activity may explain the disagreement; HPA has already flagged presumed off-target binding in tissue IHC (HPA: reliability description). | Run a no-primary control to assess the detection system, then compare staining with a reported High population. Restrict any conclusion to the named cell population, since tissue entries are cell specific (HPA: tissue IHC). |
| Color covers the section broadly and masks cell-level interpretation. | General IHC background may arise from antibody or detection conditions. The result cannot be matched confidently to the cytoplasmic profile (HPA: tissue IHC profile). | Inspect a no-primary control, revisit blocking and washing, and titrate the primary antibody as general IHC practice. Reassess only when cellular boundaries and the relevant positive population can be read. |
| The expected cytoplasmic color is faint beside the counterstain (HPA: cytoplasmic tissue profile). | General IHC counterstain or detection balance can reduce readability; faint color alone cannot resolve specificity while tissue IHC remains Uncertain (HPA: reliability). | Review positive and no-primary controls together, then adjust counterstain or detection conditions as general IHC practice. Score only interpretable cells and report the observed compartment and intensity separately. |
| Vesicular IF/ICC images and the paraffin-section result appear different (HPA: supported vesicle localization; HPA: tissue IHC Uncertain). | The HPA ICC and tissue IHC assessments have different validation statuses (HPA: HPA009160 ICC Supported, IHC Uncertain); paraffin-section color may also resolve less subcellular detail than IF. | Interpret each application against its own evidence. Use cytoplasmic cell-level tissue staining as the IHC comparison (HPA: tissue IHC profile), and reserve vesicle-level assessment for the separate IF/ICC guide (HPA: subcellular). |
Experiment evidence
IHC & IF images showing NDFIP2 staining
M08384 · IHCIHC analysis of NDFIP2 using anti-NDFIP2 antibody (M08384). NDFIP2 was detected in a paraffin-embedded section of human placenta tissue. Hea
M08384 · IHCIHC analysis of NDFIP2 using anti-NDFIP2 antibody (M08384). NDFIP2 was detected in a paraffin-embedded section of human squamous cell lung c
M08384 · IFIF analysis of NDFIP2 using anti-NDFIP2 antibody (M08384). NDFIP2 was detected in an immunocytochemical section of A431 cells. Enzyme antige
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000102471-NDFIP2/tissue/cerebral+cortex#imid_2665036
HPA · EsophagusHuman Protein Atlas IHC, Esophagus. Source: https://www.proteinatlas.org/ENSG00000102471-NDFIP2/tissue/esophagus#imid_2664992
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000102471-NDFIP2/tissue/colon#imid_2664962
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000102471-NDFIP2/tissue/liver#imid_2664947
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA009160; green = NDFIP2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA009160; green = NDFIP2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NDFIP2 IHC & IF
🧪Run colon first: glandular cells should stain (HPA: High in colon glandular cells). Use adrenal gland glandular cells as a negative tissue (HPA: Not detected); on the colon slide, treat adjacent cells as internal negatives only if their staining stays at background and their negative status is independently confirmed.
Positive control tissue: Appendix (Lymphoid tissue, HPA High)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NDFIP2 in A-431, CACO-2, with annotated localisation: Vesicles (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and concentration-matched mouse isotype controls, matching the catalog antibody host species (selected SKU M08384 tissue-IHC caption: mouse anti-NDFIP2), plus NDFIP2 knockout tissue or a validated peptide-block control. Quench endogenous peroxidase and check glandular background on the colon section before interpreting DAB staining (standard chromogenic IHC practice).
⚠️Feasibility: The selected SKU M08384 tissue-IHC caption reports a paraffin section with heat retrieval in EDTA at pH 8.0, but its fixative is unreported; no target-specific fixation window or fixation effect is reported (selected SKU M08384 tissue-IHC caption). Retrieval was used in that example, but its necessity has not been established; the supplied evidence does not establish that frozen sections or IF are easier (selected SKU M08384 tissue-IHC caption). For IF/ICC, vesicular localization is supported in A-431 and CACO-2 images (HPA subcellular); in colon IHC, assess glandular background and endogenous peroxidase as potential artefacts (standard IHC practice).
HPA tissue IHC evidence for NDFIP2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Presumed off target binding observed and disregarded. Caution, Splice and/or transcript discrepancy exists.). 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 |
|---|---|---|---|---|
| Appendix | Lymphoid tissue | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Vagina | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NDFIP2 IHC Tips
Troubleshoot NDFIP2 staining in paraffin sections by checking retrieval, compartment-specific signal, controls, and cell-level interpretation.
Which retrieval conditions should I try first for NDFIP2 in paraffin sections?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet M08384). The selected tissue image used this retrieval before overnight incubation at 4°C with 2 μg/ml primary antibody; its caption gives no retrieval duration (caption M08384). If staining is weak, keep the buffer and antibody conditions fixed while adjusting heating duration on matched sections, recording morphology and background at each setting (standard IHC practice). Only then consider another retrieval buffer as a fallback, because changing several variables together makes a gain in signal difficult to attribute (standard IHC practice).
How should I troubleshoot fixation-related loss of NDFIP2 staining?
The selected paraffin-section caption does not identify its fixative, so target-specific NDFIP2 fixation sensitivity is unknown (caption M08384). Record the fixative, fixation interval, tissue thickness, and processing history for each block before comparing staining across specimens (standard IHC practice). If a section has weak signal, compare it with a similarly processed section stained in the same run, beginning with the documented EDTA pH 8.0 retrieval and 2 μg/ml primary concentration (datasheet M08384; caption M08384). Preserve a morphology check during any fixation or retrieval trial, since tissue damage can make apparent staining differences unreliable (standard IHC practice).
What cellular pattern is plausible, and when should I question it?
Expect a predominantly cytoplasmic, potentially punctate pattern: NDFIP2 is annotated on endosome, multivesicular body, and Golgi membranes, while subcellular imaging supports vesicular localisation (UniProt Q9NV92 localisation; HPA subcellular: vesicles supported). The tissue survey describes cytoplasmic expression in most tissues, but rates its IHC reliability uncertain because presumed off-target binding was observed (HPA tissue IHC: profile and reliability). Evaluate staining within the relevant cells and against nearby negative areas under identical development conditions (standard IHC practice). Predominantly nuclear staining or a uniform outline of every cell warrants antibody and detection-control review before it is scored as NDFIP2 (UniProt Q9NV92 localisation; standard IHC practice).
How do topology and an unknown epitope affect interpretation?
NDFIP2 has three annotated transmembrane segments at residues 232–252, 258–278, and 288–308 (UniProt Q9NV92 topology). Its long 1–231 region is cytoplasmic, whereas residues 309–336 are annotated extracellular; the supplied product caption does not locate the antibody epitope (UniProt Q9NV92 topology; caption M08384). Check an available immunogen or epitope map before assigning a staining difference to epitope accessibility, and compare retrieval on matched sections (standard IHC practice). UniProt lists zero annotated isoforms and reports phosphorylation at Tyr-151, -167, -171, and -177; neither observation establishes what this antibody detects after tissue processing (UniProt Q9NV92 isoforms and modified residues).
How can IF help assess the pattern seen by chromogenic IHC?
Use IF as a follow-up localisation check for the IHC pattern, comparing vesicular signal with the chromogenic section rather than assuming identical sensitivity (HPA subcellular: vesicles supported; standard microscopy practice). In colon, multiplex NDFIP2 with an epithelial cell marker to identify glandular cells, where the tissue survey reports high staining (HPA tissue IHC: colon glandular cells high). Choose spectrally separated fluorophores after inspecting tissue autofluorescence, and include single-stain and no-primary controls (standard IF practice). If the antibody epitope maps to cytoplasmic residues 1–231, assess permeabilisation; if it maps to an extracellular segment, evaluate accessibility separately without assuming the same treatment is needed (UniProt Q9NV92 topology; standard IF practice).
How do I separate NDFIP2 signal from chromogenic background?
The selected image used 10% goat serum blocking, a mouse primary at 2 μg/ml, a peroxidase-conjugated secondary, and DAB development (caption M08384). For high diffuse staining, compare a no-primary section and shorten chromogen development before changing the primary concentration; include a peroxidase block to assess endogenous enzyme signal (standard IHC practice). Check whether staining follows tissue edges, damaged regions, or exposed surfaces rather than cellular structures (standard IHC practice). Treat a clean negative control as necessary but insufficient evidence of NDFIP2 specificity, because the tissue survey explicitly notes presumed off-target binding (HPA tissue IHC: uncertain reliability).
How should I score NDFIP2 across sections with different cell composition? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and viable tissue region before scoring, then keep illumination, chromogen development, and intensity thresholds consistent across the comparison (standard quantitative IHC practice). For a defined cell population, report the percentage positive and an H-score calculated as the sum of percentages at intensities 0–3 multiplied by their intensity grades, giving a possible range of 0–300 (standard quantitative IHC practice). For discrete positive cells, report counts per mm² of viable tissue within the same compartment (standard quantitative IHC practice). Record compartment and cell identity alongside each score, since the tissue survey reports different NDFIP2 staining levels among cell types (HPA tissue IHC: positive and negative cell listings).
What would distinguish credible NDFIP2 staining from an artefact?
Give greater weight to intracellular, vesicle-compatible staining in identified cells than to a signal confined to nuclei or section margins (UniProt Q9NV92 localisation; HPA subcellular: vesicles supported; standard IHC practice). The selected caption reports detection in a human placenta section, while the tissue survey lists placental decidual cells as not detected; identify the stained cell population before treating those observations as conflicting (caption M08384; HPA tissue IHC: placenta decidual cells not detected). Exclude necrotic and edge regions, and check no-primary and peroxidase-block controls for staining from the detection workflow (standard IHC practice). Report residual uncertainty when interpreting a positive result because the tissue survey flags presumed off-target binding (HPA tissue IHC: uncertain reliability).
Boster reagents
Best NDFIP2 / NEDD4 family-interacting protein 2 IHC Antibodies
M08384 has IHC images from human paraffin sections of placenta, squamous cell lung carcinoma, breast cancer, and spleen, plus ICC/IF data in A431 cells (catalog image captions).
Real IHC data
Anti-NDFIP2 Antibody ® (monoclonal, 10D6D7)
Cat # M08384
M08384 is listed for human IHC, with images from paraffin sections of placenta, squamous cell lung carcinoma, breast cancer, and spleen (catalog applications; IHC image captions). The same SKU has an ICC/IF image in A431 cells (catalog IF image caption).
Which to pick: Choose M08384 for human paraffin-section IHC: it is a mouse monoclonal, clone 10D6D7, with IHC listed and a paraffin-section image (catalog antibody record; IHC image captions); the fixative is unreported (catalog IHC image captions). For IF/ICC, M08384 is also listed and has an A431-cell image (catalog applications; IF image caption); use the separate IF/ICC guide for that application. No cross-species choice is supported here: M08384 lists human reactivity only (catalog antibody record).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9NV92 (NFIP2_HUMAN, NEDD4 family-interacting protein 2).
- Human Protein Atlas. NDFIP2 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. NDFIP2 subcellular location (ICC-IF): Localized to vesicles..
- Human Protein Atlas. NDFIP2 antibody validation summary (1 antibodies).
- NDFIP allows NEDD4/NEDD4L-induced AQP2 ubiquitination and degradation. PloS one 2017 — PMC5606929.
- Ndfip Proteins Target Robo Receptors for Degradation and Allow Commissural Axons to Cross the Midline in the Developing Spinal Cord. Cell reports 2019 — PMC6913780.
- Ndfip2 is a potential regulator of the iron transporter DMT1 in the liver. Scientific reports 2016 — PMC4822147.
- PubMed PMID:12796489 — UniProt-cited evidence.
- PubMed PMID:12761501 — UniProt-cited evidence.
- PubMed PMID:15057823 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NDFIP2 (NEDD4 family-interacting protein 2). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC); endosome and Golgi membranes (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M08384). Validated IHC antibody: M08384 at 2 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Lymphoid tissue); negative Adrenal gland. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human placenta tissue; fixative not specified (datasheet M08384); 2. Fixation: Image fixative and duration unreported (datasheet M08384); 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: EDTA pH 8.0 (datasheet M08384); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M08384); 8. Primary antibody: Mouse monoclonal (clone 10D6D7) anti-NDFIP2, 2 μg/ml (datasheet M08384); 9. Primary incubation: Overnight at 4 °C (datasheet M08384). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Presumed off-target staining; splice/transcript discrepancy (HPA tissue IHC); Isoform / epitope: No annotated isoforms; cytoplasmic versus extracellular epitopes matter (UniProt); Regulation: Low tissue specificity (HPA tissue RNA). Evidence: UniProt Q9NV92, HPA tissue IHC · Uncertain, HPA ICC-IF · vesicles., Datasheet M08384, PMC5606929. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NDFIP2/immunohistochemistry-ihc-guide.