This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
NREP / Neuronal regeneration-related protein · IHC design guide
Design Immunohistochemistry for NREP
Plan NREP chromogenic IHC in paraffin sections using cytoplasmic staining as the expected pattern (HPA tissue IHC). Compare high-staining bronchial respiratory epithelium with adipocytes where staining was not detected (HPA tissue IHC), and start the IHC-validated antibody at 1:100–1:300 (datasheet).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NREP 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 staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Appendix+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 conditions consistent. (standard IHC practice; not target-specific) | |
| Caveat | Staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Staining-linked regulation unreported (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage needs checking (UniProt) |
Section 1
Recommended NREP IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with published NREP staining of tissue sections and cartilage (PMC11066005; PMC10765488).
| Sample | Paraffin-embedded human heart tissue; fixative not specified (datasheet A09054-1) |
| Fixation | Image fixative and duration unreported (datasheet A09054-1); 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-NREP, 1:100 - 1:300 (datasheet A09054-1) |
| 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 | NREP-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min for cytoplasmic NREP (page retrieval rule; UniProt Q16612).
Section 2
What Is the Expected NREP Staining Pattern?
NREP is annotated as cytoplasmic, with no transmembrane segment (UniProt Q16612). In tissue IHC, expect cytoplasmic staining in many cell types, including strongly stained glandular cells in appendix and respiratory epithelial cells in bronchus (HPA tissue IHC). HPA rates the tissue pattern Approved but reports low consistency between antibody staining and RNA expression; treat the pattern as a guide for interpretation, with external verification still pending (HPA tissue IHC).
| Cytoplasmic signal in bronchial respiratory epithelium or appendix glands, with recognizable cell boundaries (HPA tissue IHC). | This fits the reported high staining in those cells and UniProt's cytoplasmic location (HPA tissue IHC; UniProt Q16612). Compare signal with adjacent cells and a no-primary control before scoring; HPA's tissue pattern has low antibody–RNA consistency (standard IHC practice; HPA tissue IHC). |
| Predominantly nuclear or sharply cell-surface staining, with little cytoplasmic signal. | Neither compartment is the expected tissue pattern; NREP has no annotated transmembrane segment (HPA tissue IHC; UniProt Q16612). Treat a compartment-restricted result as possible artefact until controls and an independent stain support it (standard IHC practice). |
| Strong staining in adipocytes, skeletal myocytes, or splenic red-pulp cells (HPA tissue IHC). | Those cells were reported as not detected (HPA tissue IHC). Check for antibody cross-reactivity or endogenous detection activity before calling them positive; the HPA assessment remains pending external verification (standard IHC practice; HPA tissue IHC). |
| Uniform color over tissue and empty spaces, obscuring cell outlines. | This is background rather than a scoreable cytoplasmic pattern (standard IHC practice; HPA tissue IHC). Review blocking, antibody concentration, washing, and chromogen development with appropriate controls (standard IHC practice). |
| No cytoplasmic signal in bronchial epithelium or appendix glands (HPA tissue IHC). | These are reported high-staining cells, so first assess the staining run and tissue preservation (HPA tissue IHC; standard IHC practice). A blank slide alone does not establish NREP absence, especially given HPA's low antibody–RNA consistency (standard IHC practice; HPA tissue IHC). |
💡Expected NREP appearanceCall a positive when discrete cytoplasmic staining is strong in the reported high-staining glandular or respiratory epithelial cells; isolated nuclear, surface-only, or cell-free color warrants investigation (HPA tissue IHC; UniProt Q16612; standard IHC practice).
| Cell and tissue choice | HPA reports high staining in appendix glands and bronchial epithelium, low staining in colon glands, and no detection in adipocytes (HPA tissue IHC). Choose a reported high-staining tissue for a run control, but do not treat an HPA-negative cell as a universal negative control because antibody–RNA consistency is low (HPA tissue IHC; standard IHC practice). |
| Compartment and protein features | NREP is cytoplasmic, has no transmembrane segment or signal peptide, and is annotated as one chain spanning residues 1–68 (UniProt Q16612). Those annotations support a cytoplasmic reading of tissue IHC; they do not establish a fixation or antigen-retrieval effect (UniProt Q16612). |
| Antibody evidence | HPA000545 is listed as IHC Approved, while the tissue profile has low antibody–RNA consistency and awaits external verification (HPA antibodies; HPA tissue IHC). Record the antibody used and judge unexpected staining with controls or independent evidence rather than treating Approved as definitive specificity proof (standard IHC practice). |
| IF/ICC Q&A: why might staining look punctate? | HPA reports vesicular localization in ICC-IF, while its tissue IHC profile is cytoplasmic (HPA subcellular; HPA tissue IHC). Cytoplasmic puncta can fit that IF observation, but the ICC-IF result does not require puncta in chromogenic paraffin IHC (HPA subcellular; HPA tissue IHC). |
| Isoforms and modification | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in a reported high-staining tissue (HPA tissue IHC). | A staining-run failure or poorly preserved section is possible; a blank result alone does not identify the cause (standard IHC practice). | Check the run control, reagent sequence, detection chemistry, and tissue morphology; review antigen retrieval against the antibody's validated IHC instructions if available (standard IHC practice). |
| Widespread weak brown haze masks cytoplasm. | Concentrated primary antibody, insufficient washing, or extended chromogen development can raise background (standard IHC practice). | Compare a no-primary control, then optimize primary concentration, washes, and development time one variable at a time (standard IHC practice). |
| Color persists without primary antibody. | Endogenous detection activity can contribute; tissue peroxidase is relevant when an HRP chromogen is used (standard IHC practice). | Confirm the detection system and assess its appropriate endogenous-activity block alongside the no-primary control (standard IHC practice). |
| Nuclear-only or surface-only signal dominates. | That distribution conflicts with the reported cytoplasmic tissue pattern and no-transmembrane annotation (HPA tissue IHC; UniProt Q16612). | Review morphology and controls, then seek independent support before scoring the compartment as NREP (standard IHC practice; HPA tissue IHC). |
| Adipocytes or skeletal myocytes stain strongly (HPA tissue IHC). | Those cells were reported as not detected; cross-reactivity or background is possible, though HPA's pattern awaits external verification (HPA tissue IHC; standard IHC practice). | Compare a reported high-staining tissue in the same run and use detection controls; avoid interpreting the unexpected cells from intensity alone (HPA tissue IHC; standard IHC practice). |
| IF/ICC shows puncta while tissue IHC looks broadly cytoplasmic. | HPA reports vesicles in ICC-IF and cytoplasmic expression in tissue IHC; the observations come from different assay contexts (HPA subcellular; HPA tissue IHC). | Score each assay against its reported compartment and controls without imposing an IF punctate pattern on paraffin IHC (HPA subcellular; HPA tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing NREP staining
A09054-1 · IHCImmunohistochemistry analysis of paraffin-embedded human heart tissue, using C5orf13 Antibody. The picture on the right is blocked with the
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000134986-NREP/tissue/cerebral+cortex#imid_182586
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000134986-NREP/tissue/colon#imid_182512
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000134986-NREP/tissue/liver#imid_182529
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000134986-NREP/tissue/kidney#imid_182522
HPA · ICC-IF Rh30Human Protein Atlas immunofluorescence, Rh30, antibody HPA000545; green = NREP, blue = nucleus, red = microtubules.
HPA · ICC-IF Rh30Human Protein Atlas immunofluorescence, Rh30, antibody HPA000545; green = NREP, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NREP IHC & IF
🧪Run appendix first: glandular cells should stain (HPA: High in appendix glandular cells). Use skeletal muscle myocytes as a biological negative (HPA: Not detected in skeletal muscle myocytes); on the appendix slide, assess adjacent nonglandular cells for background staining, without assuming they are NREP-negative.
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NREP in Rh30, SH-SY5Y, U2OS, with annotated localisation: Vesicles (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) slide and a concentration-matched isotype control appropriate to the primary antibody’s host species and clonality (standard IHC practice). A peptide-blocked slide provides an additional specificity check (A09054-1 tissue-IHC caption: signal blocked with synthesized peptide); quench endogenous peroxidase for chromogenic detection, especially when assessing inflammatory cells in appendix (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and the effect of antigen retrieval are unreported in the supplied evidence; compare retrieval conditions empirically (standard IHC practice). The A09054-1 paraffin-section caption does not state a fixative (selected-SKU caption: fixative not stated). There is no supplied comparison showing that frozen sections or IF are easier; in appendix, inflammatory-cell peroxidase can complicate chromogenic interpretation (standard IHC practice).
HPA tissue IHC evidence for NREP
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data. 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NREP IHC Tips
Troubleshoot NREP staining in paraffin sections by checking retrieval, cell type, compartment, controls and scoring before interpreting chromogenic signal.
What retrieval should I start with when NREP staining is weak?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 minutes for paraffin-section IHC (page retrieval setting). After cooling and rinsing, compare the same control section across runs so changes in staining can be attributed to retrieval rather than section choice (standard IHC practice). NREP is annotated as cytoplasmic, with no transmembrane segment, so assess intracellular staining rather than expecting a membrane rim (UniProt Q16612 localization and topology). If signal remains weak, test a different retrieval buffer on adjacent sections as a fallback, while holding antibody concentration, detection and development time constant (standard IHC practice).
Could fixation explain weak or inconsistent NREP staining?
The human heart image for the catalog antibody identifies paraffin-embedded tissue but does not report its fixative, and target-specific fixation sensitivity is unknown (A09054-1 tissue-IHC caption). Record the actual fixative and fixation duration for each specimen before comparing staining intensity across cases (standard IHC practice). If fixation varies, compare similarly processed sections first and use adjacent sections for controlled retrieval trials at citrate pH 6.0, 95–98 °C for 20 minutes (page retrieval setting; standard IHC practice). Treat a weak result as unresolved until tissue processing, detection and controls have been assessed; the available caption cannot establish a fixation-dependent NREP effect (A09054-1 tissue-IHC caption).
Where should convincing NREP staining appear in a paraffin section?
Prioritize cytoplasmic staining: UniProt assigns NREP to the cytoplasm, while HPA describes cytoplasmic expression in most tissues (UniProt Q16612 localization; HPA tissue IHC). A vesicular pattern may also fit the approved HPA subcellular location, but that location comes from cellular imaging and should be checked against the section’s morphology (HPA subcellular). NREP has no annotated transmembrane segment, so an isolated sharp membrane outline needs scrutiny rather than automatic acceptance (UniProt Q16612 topology). Compare candidate cells with nearby tissue and a no-primary control, and document whether the chromogenic deposit stays within intact cell boundaries (standard IHC practice).
How do isoforms and epitope accessibility affect NREP IHC?
NREP has two annotated isoforms, so an antibody signal cannot be assigned to one isoform without knowing the recognized epitope and its presence in each sequence (UniProt Q16612 isoforms). The record also lists phosphoserine at position 59; do not infer phosphorylation specificity unless the antibody’s epitope and validation establish it (UniProt Q16612 modified residues). The catalog image includes a synthesized-peptide blocking comparison in paraffin-embedded human heart, which supports evaluating peptide competition for that image but does not map the epitope (A09054-1 tissue-IHC caption). Review the antibody’s documented immunogen, then compare adjacent sections under identical retrieval and detection conditions before interpreting discordant staining (standard IHC practice).
How should I check an NREP pattern by multiplex IF?
For a parallel IF check, pair NREP with a marker of the expected cell type, such as pan-cytokeratin when examining respiratory epithelial cells reported as high by HPA (HPA tissue IHC; standard IF practice). Choose a spectrally separated, preferably far-red channel for the weaker signal and inspect unstained tissue for autofluorescence before assigning puncta to NREP (standard IF practice). Because NREP is cytoplasmic and has no transmembrane segment, permeabilize adequately to reach intracellular epitopes, then verify that the pattern remains within intact cells (UniProt Q16612 localization and topology; standard IF practice). HPA reports vesicles in cellular imaging, but that observation does not by itself validate the chromogenic section pattern (HPA subcellular).
How can I distinguish background from weak NREP signal?
Inspect a no-primary section and an unstained section alongside the test section; diffuse deposit, pigment and edge-heavy staining should be evaluated before calling cells positive (standard IHC practice). Include a peroxidase block before chromogenic detection and control DAB development time, because endogenous enzyme activity and overdevelopment can mimic weak cellular signal (standard IHC practice). Titrate the IHC-validated antibody against a consistent positive control, keeping citrate pH 6.0 retrieval at 95–98 °C for 20 minutes fixed during that comparison (page retrieval setting; standard IHC practice). Expect any accepted signal to fit NREP’s cytoplasmic annotation and the relevant cell morphology (UniProt Q16612 localization; standard IHC practice).
What should I measure when comparing NREP staining across cases? ⚠ ANSWER MARKED FOR VERIFICATION
Define the anatomical region and eligible intact cells before scoring, and exclude folds, necrosis and section edges using the same rules for every case (standard IHC practice). For cellular chromogenic staining, report the percentage of positive cells and an H-score based on intensity categories applied consistently across slides (standard IHC practice). Normalize each result to the number of eligible cells or tissue area, rather than comparing raw DAB-covered area from sections with different cellularity (standard IHC practice). Record cytoplasmic signal separately from other compartments, and compare only similarly processed batches because HPA flags low agreement between antibody staining and RNA data (UniProt Q16612 localization; HPA tissue-IHC reliability).
What makes a positive NREP IHC result credible?
A credible call combines intracellular staining in intact cells, appropriate tissue morphology, and a clean no-primary control; NREP is annotated as cytoplasmic rather than transmembrane (UniProt Q16612 localization and topology; standard IHC practice). HPA reports high staining in bronchial respiratory epithelial cells but no detection in skeletal-muscle myocytes, offering contextual comparators rather than absolute validation controls (HPA tissue IHC). Reject deposits confined to necrosis, section edges or endogenous-peroxidase-rich areas until those artefacts are excluded experimentally (standard IHC practice). Interpret intensity cautiously: the human heart peptide-blocking image concerns the catalog antibody, while HPA labels its tissue staining approved with low RNA agreement and pending external verification (A09054-1 tissue-IHC caption; HPA tissue-IHC reliability).
Boster reagents
Best NREP / Neuronal regeneration-related protein IHC Antibodies
The anti-NREP catalog antibody A09054-1 has an IHC image from paraffin-embedded human heart tissue (catalog IHC caption); IF/ICC and human, mouse, and rat reactivity are listed (catalog applications/reactivity).
Real IHC data
Anti-C5orf13 NREP Antibody
Cat # A09054-1
A09054-1 shows IHC staining of paraffin-embedded human heart tissue alongside a peptide-blocked comparison (catalog IHC caption). A09054-1 also lists IF/ICC applications and human, mouse, and rat reactivity; no IF image is supplied (catalog applications/reactivity; catalog IF image alts).
Which to pick: For tissue IHC, choose A09054-1 at the listed 1:100–1:300 dilution; its own image documents paraffin-embedded human heart tissue, but the fixative is unreported (datasheet: 1:100–1:300; catalog IHC caption). For IF/ICC, A09054-1 is the listed option, with an IF dilution of 1:50 and no supplied IF image (catalog applications; catalog IF dilution; catalog IF image alts). For cross-species work, A09054-1 is a rabbit polyclonal antibody listed as reactive with human, mouse, and rat; its IHC image documents human tissue only (catalog host/clonality/reactivity; catalog IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q16612 (NREP_HUMAN, Neuronal regeneration-related protein).
- Human Protein Atlas. NREP tissue IHC expression (reliability: Approved).
- Human Protein Atlas. NREP subcellular location (ICC-IF): Localized to vesicles..
- Human Protein Atlas. NREP antibody validation summary (1 antibodies).
- NREP, transcriptionally upregulated by HIF-1α, aggravates breast cancer cell growth and metastasis by promoting glycolysis. Cell death discovery 2024 — PMC11066005.
- YTHDF1-Mediated m6A Modification of NREP Promotes Corneal Fibrosis via TGF-β-Smad Signaling. Investigative ophthalmology & visual science 2026 — PMC13206844.
- The silencing of NREP aggravates OA cartilage damage through the TGF-β1/Smad2/3 pathway in chondrocytes. Journal of orthopaedic translation 2024 — PMC10765488.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:11358844 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NREP (Neuronal regeneration-related protein). Expected localisation Cytoplasmic staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A09054-1 at 1:100 - 1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human heart tissue; fixative not specified (datasheet A09054-1); 2. Fixation: Image fixative and duration unreported (datasheet A09054-1); 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-NREP, 1:100 - 1:300 (datasheet A09054-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation conditions consistent. (standard IHC practice; not target-specific); Caveat: Staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage needs checking (UniProt); Regulation: Staining-linked regulation unreported (UniProt). Evidence: UniProt Q16612, HPA tissue IHC · Approved, HPA ICC-IF · vesicles., Datasheet A09054-1, PMC11066005, PMC10765488. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NREP/immunohistochemistry-ihc-guide.