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NENF / Neudesin · IHC design guide
Design Immunohistochemistry for NENF
Plan chromogenic NENF IHC on paraffin sections with the catalog antibody starting at 10 μg/mL (datasheet: IHC-P). Use the cytoplasmic and membranous tissue pattern as a provisional reference because HPA rates its tissue staining reliability as uncertain (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NENF 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 tissue staining (HPA tissue IHC) | |
| Staining pattern | Glandular cells show cytoplasmic and membranous staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Secreted NENF may stain away from its RNA source (HPA tissue IHC) | |
| Regulation | Tumor overexpression reported (UniProt) | |
| Isoform / epitope | No isoforms; the mature chain begins at residue 32 (UniProt) |
Section 1
Recommended NENF IHC & IF Protocols
The catalog antibody has an IHC-P protocol (datasheet); the published options below cover hepatocellular carcinoma and colorectal tissue staining (PMC12964953; PMC10605590).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A11147); 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-NENF, 10 μg/mL (datasheet A11147) |
| 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 | NENF-positive staining in glandular cells of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: General cytoplasmic and membranous expression. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen-retrieval setting); optimize with tissue controls.
Section 2
What Is the Expected NENF Staining Pattern?
NENF is secreted and is also reported in the endoplasmic reticulum and mitochondria, with no transmembrane segment (UniProt Q9UMX5 localization and topology). In paraffin sections, expect mainly cytoplasmic and membranous staining in glandular cells and selected neurons (HPA tissue IHC). Treat this as a provisional pattern: HPA rates tissue staining reliability Uncertain because antibody staining and RNA expression show low consistency, and secreted protein can appear away from its site of production (HPA tissue IHC).
| Moderate cytoplasmic or membranous staining in colon glandular cells, caudate neurons, or cerebellar Purkinje cells. | This matches reported positive cell types and the general tissue profile (HPA tissue IHC). Compare stained cells with tissue structure and a counterstain; intensity alone cannot establish specificity because the tissue IHC assessment is Uncertain (HPA tissue IHC). |
| Strong staining confined to nuclei, with little cytoplasmic or membranous signal. | An exclusively nuclear pattern is inconsistent with the reported tissue profile and listed subcellular locations (HPA tissue IHC; UniProt Q9UMX5 localization). Consider an artefact or off-target signal, and check control slides before interpreting it as NENF. |
| Prominent staining in adipocytes or lymph-node germinal center cells while expected glandular cells are weak. | Those cell types were reported as Not detected, whereas several glandular populations were Medium (HPA tissue IHC). The contrast raises concern for cross-reactivity or endogenous detection activity; an isolated positive cell does not resolve the uncertain antibody validation (HPA antibodies). |
| Diffuse color over cells, stroma, and empty spaces, obscuring cell boundaries. | A widespread deposit that does not follow the reported cytoplasmic and membranous cell pattern is difficult to score as specific staining (HPA tissue IHC). General IHC practice: inspect a negative control and assess blocking, washes, and chromogen development for background. |
| No signal in an adequately preserved colon section, including its glandular cells. | Colon glandular cells are reported at Medium intensity, so their absence is a useful warning sign, not proof that the specimen lacks NENF (HPA tissue IHC). The HPA assessment is Uncertain; assess tissue integrity and controls before calling the result negative (HPA tissue IHC). |
💡Expected NENF appearanceCall a section provisionally positive when glandular cells or selected neurons show predominantly cytoplasmic and membranous signal near the reported Medium level; isolated nuclear staining or widespread deposit is suspect (HPA tissue IHC; UniProt Q9UMX5 localization).
| Secretory processing and topology | NENF has a signal peptide at residues 1–31, a mature chain at 32–172, and no transmembrane segment (UniProt Q9UMX5 processing and topology). Interpret membranous staining alongside cytoplasmic staining and tissue architecture; topology alone does not identify a membrane-bound pool. |
| Intracellular localization | UniProt also lists endoplasmic reticulum and mitochondria, with localization to these compartments attributed to PINK1 and PARK7 (UniProt Q9UMX5 localization). This supports considering intracellular signal, but does not make a fine organelle pattern mandatory in chromogenic tissue IHC. |
| Tissue distribution and secretion | HPA reports Medium staining in several glandular populations and selected neurons, alongside Not detected calls in specific other cell types (HPA tissue IHC). Because NENF is secreted, tissue protein location need not match tissue RNA location (HPA tissue IHC; UniProt Q9UMX5 localization). |
| Strength of antibody evidence | Both listed antibodies, HPA026763 and HPA028488, have Uncertain IHC status (HPA antibodies). HPA also flags low agreement between antibody staining and RNA expression (HPA tissue IHC). Use the reported pattern to guide comparison, while keeping any single positive result provisional. |
| Situation | Likely cause | Next action |
|---|---|---|
| The expected glandular cells are unstained. | A weak assay signal or specimen issue is possible; the reported Medium glandular staining is itself uncertain (HPA tissue IHC). | General IHC practice: review section integrity, antibody and detection controls, and the antigen retrieval conditions used. Compare with a tissue containing a reported positive cell population (HPA tissue IHC); do not infer NENF-specific fixation sensitivity. |
| The whole section has diffuse brown background. | Nonspecific reagent binding, endogenous detection activity, or excess chromogen development can obscure a cell-associated pattern (general IHC practice). | General IHC practice: inspect a negative control, confirm appropriate blocking for the detection chemistry, and review washes and development time. Score only a distinguishable cellular pattern consistent with the HPA profile (HPA tissue IHC). |
| Signal is predominantly nuclear. | This differs from the reported cytoplasmic and membranous tissue profile and listed secretory or organelle locations (HPA tissue IHC; UniProt Q9UMX5 localization). | Check the counterstain and negative control, then repeat with reviewed detection conditions if needed (general IHC practice). Do not count an exclusively nuclear result as the expected NENF pattern. |
| Cells reported as Not detected stain strongly. | Off-target binding or endogenous detection activity is possible; HPA's IHC validation is Uncertain (HPA tissue IHC; HPA antibodies). | Compare the suspect cells with a reported Medium population in the same run, and examine negative controls (HPA tissue IHC; general IHC practice). Treat the unexpected signal as unresolved rather than confirming expression from color alone. |
| Two tissues show different apparent intensities. | HPA reports Medium, Low, and Not detected calls across cell types, while warning that its tissue IHC reliability is Uncertain (HPA tissue IHC). | Compare the same cell type and compartment under matched staining and scoring conditions (general IHC practice). Record differences as observed staining; avoid equating them directly with RNA abundance for a secreted protein (HPA tissue IHC). |
| Q: What pattern should IF/ICC show? | HPA summarizes NENF as Secreted but provides no main subcellular location or ICC-IF cell-line images (HPA subcellular). | A: Do not prescribe an IF/ICC organelle pattern from this IHC section. Use the separate IF/ICC guide for assay design; consider the secreted, endoplasmic reticulum, and mitochondrial annotations as context, not an image-validated IF result (UniProt Q9UMX5 localization; HPA subcellular). |
Experiment evidence
IHC & IF images showing NENF staining
A11147 · IHCImmunohistochemistry of NENF in rat kidney tissue with NENF antibody at 10 μg/mL.
A11147 · IFImmunofluorescence of NENF in rat kidney tissue with NENF antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000117691-NENF/tissue/cerebral+cortex#imid_6578296
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000117691-NENF/tissue/colon#imid_6578070
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000117691-NENF/tissue/liver#imid_6578063
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000117691-NENF/tissue/kidney#imid_6578072
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NENF IHC & IF
🧪Run breast first: glandular cells should stain at a medium level (HPA: Medium in breast glandular cells). Use adipose tissue as the negative tissue; adipocytes on the breast slide, if present, should provide an internal background comparator (HPA: Not detected in adipose tissue adipocytes).
Positive control tissue: Adrenal gland (Glandular cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for NENF; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) control, an isotype-matched control for the primary antibody’s host and clonality, and a biological specificity control such as knockout tissue or peptide blocking where available (standard IHC practice). Block endogenous peroxidase and check for endogenous biotin if using biotin-based detection (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported in the supplied evidence; the selected A11147 rat kidney IHC caption also leaves the fixative unreported (A11147 tissue-IHC caption). Retrieval dependency is unreported, so optimize antigen retrieval for the catalog antibody’s paraffin-section assay (standard IHC practice). The evidence does not establish whether frozen sections or IF are easier for NENF; account for possible extracellular staining from its secreted form and distinguish it from tissue background (UniProt Q9UMX5: Secreted, extracellular space; standard IHC practice).
HPA tissue IHC evidence for NENF
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low consistency between antibody staining and RNA expression data. Secreted protein, tissue location of RNA and protein is expected to differ. Pending external verification.). 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 | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
| Ovary | Follicle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NENF IHC Tips
Troubleshoot NENF chromogenic IHC by checking retrieval, signal location, controls, and scoring before interpreting staining intensity.
What retrieval should I try when NENF staining is weak in paraffin sections?
Start with citrate buffer at pH 6.0 for heat induced retrieval at 95–98 °C for 20 min (page IHC protocol). If staining remains weak, compare a longer exposure to the same buffer on matched sections, then consider another retrieval buffer as a fallback (standard IHC practice). Keep section thickness, antibody concentration, and chromogen development constant during that comparison so retrieval is the variable being assessed (standard IHC practice). Score signal in cells alongside tissue background, because stronger retrieval can also expose sites that increase nonspecific staining (standard IHC practice).
Could fixation be masking NENF in my paraffin sections?
NENF specific fixation sensitivity is unknown from the supplied evidence; the A11147 rat kidney IHC caption reports 10 µg/mL antibody but does not state a fixative (A11147 tissue IHC caption). Record fixative, fixation duration, and processing history for each section before comparing staining, since these variables can change antigen accessibility in paraffin IHC (standard IHC practice). On matched material, compare the page retrieval condition and one controlled adjustment while holding antibody concentration and detection time steady (page IHC protocol; standard IHC practice). Do not interpret a negative section as absent NENF until a documented positive control and tissue morphology are satisfactory (standard IHC practice).
Should NENF appear at the membrane, in cytoplasm, or outside cells?
NENF is annotated as secreted into extracellular space and as present in mitochondria and endoplasmic reticulum, with intracellular localisation linked to PINK1 and PARK7 (UniProt Q9UMX5 localisation). It has a 1–31 signal peptide, a 32–172 mature chain, and no transmembrane segment, so membrane adjacent staining alone does not establish membrane insertion (UniProt Q9UMX5 processing and topology). HPA reports a general cytoplasmic and membranous tissue pattern but rates its NENF IHC reliability uncertain (HPA tissue IHC). Compare cellular and extracellular signal with morphology and controls, and describe the observed compartment rather than assigning a mechanism from chromogenic staining alone (standard IHC interpretation).
How can I assess whether processing or epitope position explains discrepant staining?
The supplied record lists 0 isoforms, a 1–31 signal peptide, and a 32–172 mature NENF chain (UniProt Q9UMX5 processing and isoforms). An antibody recognizing the removed signal peptide could behave differently from one recognizing the mature chain, so check the catalog antibody’s stated immunogen or epitope before interpreting tissue staining (UniProt Q9UMX5 processing; standard IHC practice). NENF also has a heme binding domain at residues 44–129 and an annotated acetyllysine at residue 136, but the payload does not map this antibody’s epitope (UniProt Q9UMX5 domains and modified residues). Compare independent epitope antibodies only when their validation and matched controls are documented (standard IHC practice).
How should I compare NENF IHC with multiplex IF staining?
Treat IF as a separate assay and multiplex NENF with a validated marker for the expected cell type; HPA reports medium NENF staining in cerebellar Purkinje cells and colon glandular cells (HPA tissue IHC). Choose spectrally separated fluorophores after checking unstained tissue autofluorescence, and use single stain controls to assess bleed through (standard IF practice). If the antibody epitope is intracellular, test gentle permeabilisation; if it is extracellular and accessible, compare staining without permeabilisation, because this antibody’s epitope side is unspecified (UniProt Q9UMX5 localisation; standard IF practice). Do not treat agreement between fluorescence and chromogenic signal as proof of specificity without matched controls (standard IHC and IF practice).
What should I check when NENF DAB staining is diffuse or patchy?
First compare the stained section with a no primary control to identify signal arising from the chromogenic detection workflow (standard IHC practice). Block endogenous peroxidase before DAB development, then assess whether longer chromogen exposure or concentrated primary antibody increases diffuse colour on matched sections (standard IHC practice). The A11147 rat kidney image caption states 10 µg/mL antibody but does not specify fixation or a complete detection protocol, so its concentration is a comparison point rather than a universal setting (A11147 tissue IHC caption). Evaluate staining at section edges, damaged areas, and extracellular spaces separately, since NENF is annotated as secreted (UniProt Q9UMX5 localisation; standard IHC interpretation).
How should I score NENF staining across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored compartment and cell population before reading slides, because NENF may be secreted or intracellular and HPA describes cytoplasmic and membranous tissue staining (UniProt Q9UMX5 localisation; HPA tissue IHC). For cellular chromogenic signal, report the percentage of positive cells and an H-score of 0–300 using intensity categories 0–3; score extracellular deposits separately if relevant (standard IHC scoring practice). Normalise positive cell counts to the number of evaluable cells, or positive area to evaluable tissue area, and exclude folds and necrotic regions consistently (standard IHC scoring practice). Keep retrieval, development, thresholds, and region selection consistent across samples (standard IHC practice).
How can I distinguish credible NENF staining from artefact?
A credible result follows tissue morphology, survives comparison with a no primary control, and has a plausible cellular or extracellular location for NENF (UniProt Q9UMX5 localisation; standard IHC interpretation). Treat staining restricted to cut edges, necrotic regions, or sites with residual endogenous peroxidase as suspect until it passes control checks (standard IHC practice). Compare the stained cell type with an independently supported expectation: HPA reports medium staining in colon glandular cells but no detection in adipocytes, while rating its NENF tissue IHC reliability uncertain (HPA tissue IHC). Because NENF is secreted, protein location need not match RNA producing cells; avoid declaring a mismatch false staining on that basis alone (HPA tissue IHC).
Boster reagents
Best NENF / Neudesin IHC Antibodies
One anti-NENF antibody has IHC and IF images from rat kidney tissue (catalog image captions), with human, mouse and rat reactivity listed (catalog reactivity).
Real IHC data
Anti-Neudesin NENF Antibody
Cat # A11147
A11147 is listed for IHC-P, with an IHC image from rat kidney tissue at 10 μg/mL (catalog applications; IHC image caption). A11147 is also listed for IF, with an IF image from rat kidney tissue at 20 μg/mL (catalog applications; IF image caption).
Which to pick: Choose A11147 for paraffin-section tissue IHC because IHC-P is listed and its own IHC image shows rat kidney tissue; the fixative is unreported (catalog applications; IHC image caption). Choose A11147 for IF; ICC is not separately listed, so confirm performance for an ICC workflow (catalog applications). For work across species, A11147 lists human, mouse and rat reactivity, while the supplied images show rat kidney tissue only; clonality is unreported (catalog reactivity; catalog image captions; catalog clone field).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UMX5 (NENF_HUMAN, Neudesin).
- Human Protein Atlas. NENF tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. NENF subcellular location (ICC-IF): Secreted.
- Human Protein Atlas. NENF antibody validation summary (2 antibodies).
- Identification of novel circulating protein biomarkers for hepatocellular carcinoma superior to alpha-fetoprotein through a stemness index and secretome analysis. World journal of surgical oncology 2026 — PMC12964953.
- Neudesin is involved in anxiety behavior: structural and neurochemical correlates. Frontiers in behavioral neuroscience 2013 — PMC3766795.
- New Insights on the Progesterone (P4) and PGRMC1/NENF Complex Interactions in Colorectal Cancer Progression. Cancers 2023 — PMC10605590.
- Adult Hippocampal Neurogenesis Modulation by the Membrane-Associated Progesterone Receptor Family Member Neudesin. Frontiers in cellular neuroscience 2018 — PMC6275434.
- PubMed PMID:15605373 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:9771976 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NENF (Neudesin). Expected localisation Cytoplasmic and membranous tissue staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A11147 at 10 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular 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 A11147); 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-NENF, 10 μg/mL (datasheet A11147); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Secreted NENF may stain away from its RNA source (HPA tissue IHC); Isoform / epitope: No isoforms; the mature chain begins at residue 32 (UniProt); Regulation: Tumor overexpression reported (UniProt). Evidence: UniProt Q9UMX5, HPA tissue IHC · Uncertain, HPA ICC-IF · Secreted, Datasheet A11147, PMC12964953, PMC10605590. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NENF/immunohistochemistry-ihc-guide.