NEDD9 / Enhancer of filamentation 1 · IHC design guide

Design Immunohistochemistry for NEDD9

Plan chromogenic NEDD9 IHC on paraffin sections with the catalog antibody at 2–5 μg/ml (datasheet PB9289). Compare staining with the cytoplasmic, membranous and nuclear tissue profile, bearing in mind its uncertain reliability (HPA tissue IHC).

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

Immunohistochemistry protocol sheet for NEDD9 (IHC for NEDD9): expected localisation Cytoplasmic, membranous and nuclear tissue staining (HPA tissue IHC), antibody PB9289, validated IHC image, and IHC protocol steps — Printable **NEDD9** IHC protocol sheet — expected localisation Cytoplasmic, membranous and nuclear tissue staining (HPA tissue IHC), antibody PB9289, controls and protocol steps. Open the full NEDD9 IHC guide →

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NEDD9 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, membranous and nuclear tissue staining (HPA tissue IHC)
	Staining pattern	Cytoplasmic, membranous and nuclear staining across tissues (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet PB9289)
	Positive control ⓘ	Adipose tissue+4 more · see all
	Negative control ⓘ	None in HPA (detected in all 45 tissues); use no-primary + isotype controls

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific)
	Caveat	Antibody staining has low concordance with RNA expression (HPA tissue IHC)
	Regulation	SMAD3–ITCH promotes NEDD9 degradation (UniProt)
	Isoform / epitope	Three isoforms and p55; epitope coverage unreported (UniProt)

Section 1

Recommended NEDD9 IHC & IF Protocols

The catalog antibody IHC-P protocol (datasheet PB9289) is accompanied by published NEDD9 IHC methods for lung, cervical, and gastric specimens (PMC3493698; PMC3776827; PMC4360801).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet PB9289)
Fixation	Image fixative and duration unreported (datasheet PB9289); 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 PB9289); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet PB9289)
Primary antibody	Rabbit anti-NEDD9, 2-5μg/ml (datasheet PB9289)
Primary incubation	Overnight at 4 °C (datasheet PB9289)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet PB9289)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	NEDD9-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: Widely expressed cytoplasmic, membranous and nuclear expression. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet PB9289); these published excerpts do not specify retrieval conditions (PMC3493698; PMC3776827; PMC4360801).

Section 2

What Is the Expected NEDD9 Staining Pattern?

NEDD9 can appear in the cytoplasm, at the cell cortex or focal adhesions, and in nuclei; it has no transmembrane segment (UniProt Q14511 localization and topology). HPA reports widespread cytoplasmic, membranous and nuclear tissue staining, including high staining in pancreatic exocrine glandular cells and adipocytes (HPA tissue IHC). Interpret these patterns cautiously: HPA rates its tissue IHC reliability Uncertain because antibody staining and RNA expression have low consistency, with a possible splice or transcript discrepancy (HPA tissue IHC).

What am I looking at on my slide?
Cytoplasmic staining, with possible membranous or nuclear staining, in identifiable cells.	This fits the reported tissue pattern (HPA tissue IHC) and NEDD9 localization (UniProt Q14511). Judge signal within the relevant cell type; staining across several compartments alone does not establish specificity because the tissue IHC assessment is Uncertain (HPA tissue IHC).
Signal is confined to an extracellular deposit or an empty tissue space rather than cells.	That location is unsupported by the reported cellular localizations (UniProt Q14511; HPA tissue IHC). Treat it as possible staining artefact and inspect the section and detection controls (general IHC practice). Do not classify nuclear or membranous signal alone as misplaced: both occur in the supplied localization evidence (HPA tissue IHC).
Strong signal appears in unexpected structures while the intended cell population is unstained.	Review cell identification and compare the distribution with a negative detection control; cross-reactivity or endogenous detection activity is possible (general IHC practice). A tissue name alone is insufficient to call the pattern wrong: HPA reports low tissue specificity and widely distributed NEDD9 staining (HPA tissue IHC).
Chromogen forms a diffuse haze across cells and surrounding tissue.	This cannot be scored confidently as cellular NEDD9. Check the negative detection control, blocking, washing and chromogen development (general IHC practice). HPA's reported cytoplasmic, membranous and nuclear pattern describes staining within cells, not an undifferentiated background (HPA tissue IHC).
There is no signal in pancreatic exocrine glandular cells or adipocytes.	Both are reported as High in HPA tissue IHC, so first check section integrity, the detection control and staining run (HPA tissue IHC; general IHC practice). A negative result is not conclusive evidence of absent NEDD9: the HPA tissue IHC reliability is Uncertain (HPA tissue IHC).

💡Expected NEDD9 appearanceA plausible positive shows discernible cytoplasmic staining, potentially with membranous or nuclear signal, in identifiable pancreatic exocrine glandular cells or adipocytes reported as High; diffuse extracellular chromogen is suspect (HPA tissue IHC; general IHC practice).

How each factor affects the staining
Tissue and cell selection	HPA reports High staining in pancreatic exocrine glandular cells and adipocytes, but Low staining in bronchial respiratory epithelial cells (HPA tissue IHC). UniProt reports NEDD9 protein in respiratory epithelium (UniProt Q14511); use the specific cell and assay observation when choosing a comparison section.
Compartment and topology	NEDD9 is reported in the cytoplasm, nucleus, cell cortex and focal adhesions, and lacks a transmembrane segment (UniProt Q14511). HPA describes cytoplasmic, membranous and nuclear tissue staining (HPA tissue IHC). Membranous staining can therefore be plausible without implying that NEDD9 spans the membrane.
Antibody evidence	HPA rates the tissue IHC pattern Uncertain and lists IHC as Uncertain for HPA038768, HPA072955 and CAB009720 (HPA tissue IHC; HPA antibodies). Concordance with the expected cell type and compartment helps interpretation, but does not upgrade that validation rating.
Isoforms and antigen recognition	UniProt lists three NEDD9 isoforms and phosphorylated residues (UniProt Q14511). The supplied record does not map an IHC antibody epitope or show whether isoforms or phosphorylation alter staining; do not infer which form produced a positive cell.
IF/ICC Q&A	What should IF/ICC show? Mainly nucleoplasm and cytosol; both are supported HPA locations (HPA subcellular ICC-IF). This is localization evidence for cells, not a validated paraffin IHC protocol or a guarantee that every tissue cell shows both compartments (HPA subcellular ICC-IF; HPA tissue IHC).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Expected positive cells are unstained.	The staining run or detection may have failed; the chosen cells may also differ from the reported HPA pattern (general IHC practice; HPA tissue IHC).	Check a run control and compare identifiable pancreatic exocrine glandular cells or adipocytes, both reported High (general IHC practice; HPA tissue IHC). If adjusting antigen retrieval, treat it as general IHC optimization; NEDD9-specific fixation sensitivity is unreported here.
Only bronchial respiratory epithelial cells look weak.	HPA records Low tissue IHC staining there, although UniProt reports NEDD9 protein in respiratory epithelium (HPA tissue IHC; UniProt Q14511).	Use a reported High cell population to assess the run, and record the bronchial result without forcing agreement between the two sources (HPA tissue IHC; UniProt Q14511).
Membranous or nuclear signal seems unexpected.	Either compartment can occur in the supplied localization evidence (HPA tissue IHC; UniProt Q14511).	Confirm that signal lies within identifiable cells, then compare its distribution with a negative detection control (general IHC practice). Do not reject a section solely for membranous or nuclear staining (HPA tissue IHC).
Brown signal appears in the negative detection control.	Endogenous detection activity or nonspecific detection chemistry may contribute (general IHC practice).	Address the relevant endogenous activity and review blocking and detection reagents before assigning the same pattern to NEDD9 (general IHC practice).
The whole section has diffuse background.	Nonspecific reagent binding, inadequate washing or excessive chromogen development may obscure cell boundaries (general IHC practice).	Review blocking, washes and development against the negative detection control; score only resolved cellular staining (general IHC practice).
Two antibodies give different tissue patterns.	HPA reports low consistency between antibody staining and RNA expression, a possible splice or transcript discrepancy, and Uncertain IHC reliability (HPA tissue IHC; HPA antibodies).	Document each antibody and its validation status, compare the same identifiable cell types, and report unresolved disagreement rather than assigning specificity from intensity alone (HPA antibodies; general IHC practice).

Source: UniProt Q14511 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing NEDD9 staining

IHC analysis of HEF1 using anti-HEF1 antibody (PB9289). HEF1 was detected in a paraffin-embedded section of human breast cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-HEF1 Antibody (PB9289) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

PB9289 · IHCIHC analysis of HEF1 using anti-HEF1 antibody (PB9289). HEF1 was detected in a paraffin-embedded section of human breast cancer tissue. Heat

IF analysis of HEF1 using anti-HEF1 antibody (PB9289). HEF1 was detected in an immunocytochemical section of Hela cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 5 μg/mL rabbit anti-HEF1 Antibody (PB9289) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

PB9289 · IFIF analysis of HEF1 using anti-HEF1 antibody (PB9289). HEF1 was detected in an immunocytochemical section of Hela cells. Enzyme antigen retr

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000111859-NEDD9/tissue/cerebral+cortex#imid_14635163

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000111859-NEDD9/tissue/colon#imid_14630718

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000111859-NEDD9/tissue/liver#imid_14630735

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000111859-NEDD9/tissue/kidney#imid_14630720

HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA039270; green = NEDD9, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for NEDD9 IHC & IF

🧪Start with adipose tissue and expect adipocytes to stain (HPA: High in adipocytes). HPA detects NEDD9 in all 45 scored tissues, so there is no validated negative tissue; use no-primary and isotype controls, and treat unstained cells within the positive section as a background reference rather than confirmed NEDD9-negative cells (HPA: no negative rows).

Positive control tissue: Adipose tissue (Adipocytes, HPA High)

Negative control tissue: None in HPA: NEDD9 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NEDD9 in Hep-G2, U-251MG, U2OS, EFO-21, SK-MEL-30, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Nucleoplasm (supported), Cytosol (supported) (HPA subcellular).

Technical controls: Run a secondary-only section without primary antibody and a concentration-matched rabbit IgG isotype control alongside the catalog rabbit antibody (PB9289 caption: rabbit primary antibody); a matched NEDD9-knockout specimen would provide a biological negative. Quench endogenous peroxidase and assess background in the adipose section before interpreting HRP/DAB staining (PB9289 caption: HRP/DAB detection).

⚠️Feasibility: Paraffin-section IHC has been demonstrated with heat retrieval in EDTA at pH 8.0, but that example does not establish that retrieval is required (PB9289 caption: breast cancer section, EDTA pH 8.0). The caption does not report a fixative, and the supplied evidence reports no NEDD9-specific fixation window or fixation effect (PB9289 caption: fixative unreported). Frozen sections and IF cannot be judged easier from these data; in adipose sections, lipid extraction can leave empty vacuoles that complicate interpretation (HPA: High in adipocytes; standard histology practice).

HPA tissue IHC evidence for NEDD9

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low consistency between antibody staining and RNA expression data. 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
Adipose tissue	Adipocytes	High	Protein (IHC)	HPA →
Caudate	Glial cells	High	Protein (IHC)	HPA →
Cerebellum	Cells in molecular layer	High	Protein (IHC)	HPA →
Cerebral cortex	Glial cells	High	Protein (IHC)	HPA →
Gallbladder	Glandular cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
None in HPA: NEDD9 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

Section 3

Advanced NEDD9 IHC Tips

Troubleshoot NEDD9 staining in paraffin sections by checking retrieval, compartment, cell type and controls before comparing chromogenic scores (UniProt Q14511; standard IHC practice).

What retrieval should I try first when NEDD9 staining is weak?

Use heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections stained with PB9289 (datasheet PB9289). The selected tissue image used this retrieval before incubation with 2 μg/ml primary antibody overnight at 4°C (datasheet PB9289). If staining remains weak, compare a carefully controlled alternative retrieval condition on adjacent sections while keeping antibody concentration and detection constant (standard IHC practice). Include a positive control alongside the test section, because HPA reports high staining in adipocytes and several glandular and glial cell populations (HPA tissue IHC). Excessively harsh retrieval can damage morphology or increase nonspecific signal, so judge both staining and tissue preservation (standard IHC practice).

Could fixation explain weak or uneven NEDD9 staining?

The PB9289 tissue caption identifies a paraffin section but does not report its fixative, so NEDD9-specific fixation sensitivity is unknown (datasheet PB9289). Record the fixative, fixation duration and processing history for every section, then compare matched tissue processed under the same conditions (standard IHC practice). Keep EDTA retrieval at pH 8.0 and the antibody at the caption's 2 μg/ml during that comparison (datasheet PB9289). Uneven fixation or section handling can create patchy staining and compromised morphology, which should be assessed alongside the DAB signal (standard IHC practice). Neither NEDD9 localisation nor its phosphorylation sites establish a fixation-dependent staining effect in these sections (UniProt Q14511).

Is nuclear staining plausible when I expected NEDD9 at focal adhesions?

Nuclear and cytoplasmic staining are plausible for NEDD9: UniProt lists the nucleus, cytoplasm, cortex and focal adhesions, while HPA supports nucleoplasm and cytosol (UniProt Q14511; HPA subcellular). Assess nuclear and cytoplasmic DAB separately, using morphology and a counterstain to distinguish nuclei from overlapping cytoplasm (standard IHC practice). Focal adhesion signal may appear peripheral or punctate, but its absence in a tissue section does not by itself exclude NEDD9 (UniProt Q14511; standard IHC practice). Review the same compartment in a positive control and an adjacent negative control before assigning faint staining (standard IHC practice). Avoid interpreting membrane staining as proof of an integral membrane protein, because NEDD9 has no transmembrane segment (UniProt Q14511 topology).

Could NEDD9 isoforms or phosphorylation change what this antibody detects?

NEDD9 has 3 reported isoforms and multiple modified residues, including ABL1-linked phosphotyrosines, so epitope identity matters when comparing staining patterns (UniProt Q14511). The supplied PB9289 caption gives an IHC condition but no epitope sequence or isoform coverage; do not infer either from a positive image (datasheet PB9289). Obtain the antibody's epitope information before claiming that a negative section lacks all NEDD9 forms, or that signal reports phosphorylation (standard IHC practice). Compare staining with an independent antibody directed to a documented distinct epitope if isoform coverage is central to the study (standard IHC practice). Keep retrieval and detection matched when comparing such reagents, since these steps affect signal independently of epitope recognition (standard IHC practice).

How should I check a NEDD9 pattern by multiplex IF?

Use IF as a spatial follow-up to chromogenic IHC, and validate the IF antibody and conditions independently of PB9289's paraffin-section image (datasheet PB9289; standard IF practice). Multiplex NEDD9 with a validated marker for the cell population being scored; HPA reports high staining in adipocytes, glial cells and some glandular cells (HPA tissue IHC). Choose fluorophores after inspecting unstained tissue for autofluorescence, and include single-label controls to evaluate spectral bleed-through (standard IF practice). For an intracellular NEDD9 epitope, optimise permeabilisation because supported locations include nucleoplasm and cytosol, and NEDD9 lacks a transmembrane segment (HPA subcellular; UniProt Q14511 topology). Evaluate peripheral signal separately from nuclear and cytosolic signal, with background controls for each channel (UniProt Q14511 localisation; standard IF practice).

How can I reduce diffuse brown staining without losing NEDD9 signal?

First compare stained sections with a primary-antibody omission control to assess detection-system background, and inspect tissue edges for disproportionate DAB deposition (standard IHC practice). The PB9289 image used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and peroxidase-based DAB detection (datasheet PB9289). Include a peroxidase-blocking step when using HRP, and verify that washing and detection times are consistent across sections (standard IHC practice). If background persists, titrate the primary antibody below the caption condition while retaining a positive control and unchanged retrieval (standard IHC practice). Score cellular signal only where morphology supports an interpretable compartment; HPA describes NEDD9 staining across cytoplasmic, membranous and nuclear locations (HPA tissue IHC).

What is a defensible way to quantify NEDD9 IHC across sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring, because reported NEDD9 staining spans cytoplasmic, membranous and nuclear patterns (HPA tissue IHC). For each compartment, record the percentage of positive cells and an intensity-based H-score from 0–300, or measure positive-cell density per mm² when cell counts are the endpoint (standard IHC practice). Normalise counts to the number of eligible cells or viable tissue area, and apply identical thresholds, exposure and section-selection rules across samples (standard IHC practice). Keep the PB9289 retrieval and detection conditions consistent across a comparison set (datasheet PB9289; standard IHC practice). Report compartments separately and annotate excluded folds, necrosis and edge regions so artefacts do not inflate the score (standard IHC practice).

When is an apparent NEDD9-positive region likely to be artefact?

Treat reproducible cellular staining in plausible NEDD9 compartments as more credible than isolated edge staining or signal over necrotic tissue (UniProt Q14511 localisation; standard IHC practice). Nucleoplasm, cytosol and focal adhesion-associated regions are plausible, whereas an exclusively extracellular pattern needs additional validation because NEDD9 has no signal peptide or transmembrane segment (HPA subcellular; UniProt Q14511 topology). Check cell identity: HPA reports high staining in adipocytes, glial cells and some glandular cells, but rates its tissue IHC reliability uncertain (HPA tissue IHC). Compare a primary-omission control and a peroxidase-blocked section when DAB appears diffuse or associated with endogenous enzyme activity (standard IHC practice). Resolve a discordant pattern with an independent antibody or orthogonal expression evidence before making a biological claim (standard IHC practice).

Boster reagents

Best NEDD9 / Enhancer of filamentation 1 IHC Antibodies

PB9289 has IHC data from human paraffin-embedded breast cancer tissue and IF/ICC data from HeLa cells (catalog image captions); listed reactivity includes human, monkey, mouse and rat (catalog).

Real IHC data

Anti-HEF1/NEDD9 Antibody ®

Cat # PB9289

View product →

PB9289 is listed for IHC and IF/ICC (catalog applications). Its figures show IHC on a human paraffin-embedded breast cancer section and IF/ICC on HeLa cells (PB9289 image captions).

Which to pick: Choose PB9289 for paraffin-section IHC: its own caption documents heat retrieval in EDTA, pH 8.0, and 2 μg/ml primary antibody; the fixative is unreported (PB9289 IHC caption). The same rabbit antibody is listed for IF/ICC at 5 μg/ml and shown on HeLa cells; clonality is unreported (catalog; PB9289 IF caption). For other species, PB9289 lists monkey, mouse and rat reactivity, but the supplied IHC and IF/ICC figures document human samples only (catalog reactivity; PB9289 image captions).

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

References

UniProt Consortium. UniProt entry Q14511 (CASL_HUMAN, Enhancer of filamentation 1).
Human Protein Atlas. NEDD9 tissue IHC expression (reliability: Uncertain).
Human Protein Atlas. NEDD9 subcellular location (ICC-IF): Mainly localized to the nucleoplasm and cytosol..
Human Protein Atlas. NEDD9 antibody validation summary (5 antibodies).
Role of NEDD9 in invasion and metastasis of lung adenocarcinoma. Experimental and therapeutic medicine 2012 — PMC3493698.
POSITIVE EXPRESSION OF NEDD9 IN HEAD AND NECK CANCER IS RELATED TO BETTER SURVIVAL PERIOD. Acta clinica Croatica 2019 — PMC7314307.
The overexpression of scaffolding protein NEDD9 promotes migration and invasion in cervical cancer via tyrosine phosphorylated FAK and SRC. PloS one 2013 — PMC3776827.
Elevated expression of NEDD9 is associated with metastatic activity in gastric cancer. OncoTargets and therapy 2015 — PMC4360801.
PubMed PMID:8668148 — UniProt-cited evidence.
PubMed PMID:8879209 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.