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NINJ1 / Ninjurin-1 · IHC design guide
Design Immunohistochemistry for NINJ1
Plan paraffin section NINJ1 IHC using the catalog antibody's 5 μg/mL starting concentration (datasheet). Liver hepatocytes show medium staining, but tissue IHC reliability is uncertain (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NINJ1 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 | Cell membrane expected; tissue compartment unverified (UniProt) | |
| Staining pattern | Endothelial cells and hepatocytes; compartment unspecified (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Liver | |
| 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 | Tissue staining reliability is uncertain (HPA tissue IHC) | |
| Regulation | Expression regulation is not established (UniProt) | |
| Isoform / epitope | No isoforms; the secreted 1–56 chain lacks the cytoplasmic region (UniProt) |
Section 1
Recommended NINJ1 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by one published NINJ1 IHC protocol for neonatal intestinal tissue (PMC13237964).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A08377); 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-NINJ1, 5 μg/mL (datasheet A08377) |
| 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 | NINJ1-positive staining in hepatocytes of liver (HPA tissue IHC: Medium). HPA tissue profile: Expressed in endothelial cells and liver. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval); the published intestinal protocol uses Tris-EDTA pH 9.0 (PMC13237964).
Section 2
What Is the Expected NINJ1 Staining Pattern?
NINJ1 is a membrane protein with two transmembrane segments and extracellular regions; a secreted form is also annotated (UniProt Q92982 topology and processing). In paraffin-section IHC, look for staining in liver hepatocytes, reported at medium intensity, and assess endothelial cells identified in the tissue profile (HPA tissue IHC). Treat this as a provisional pattern: HPA rates tissue staining and the listed antibody’s IHC validation as Uncertain (HPA tissue IHC; HPA antibody HPA045063).
| Hepatocytes show a discernible cell-edge signal of moderate strength. | This fits the reported medium hepatocyte staining and the predicted membrane compartment (HPA tissue IHC: liver hepatocytes Medium; UniProt Q92982 topology). HPA does not assign a subcellular pattern to those hepatocytes, so judge the edge pattern against controls rather than treating it as independently validated. |
| Signal outlines endothelial cells in a vessel-associated pattern. | Endothelial expression is part of HPA’s tissue profile, but the supplied profile gives no endothelial intensity or antibody-specific confirmation (HPA tissue IHC: expressed in endothelial cells and liver; reliability Uncertain). Confirm the cell identity from section morphology before scoring it as NINJ1. |
| Strong staining is confined to nuclei, with no plausible cell-edge signal. | An exclusively nuclear pattern conflicts with the annotated membrane and secreted locations (UniProt Q92982 subcellular location). Consider it an artefact or off-target signal and compare it with the no-primary control; UniProt topology alone cannot validate the antibody’s staining pattern. |
| Prominent signal appears in cells reported as unstained by HPA, or blankets unrelated cell types. | For example, HPA reports adipocytes and bone-marrow hematopoietic cells as Not detected by tissue IHC (HPA tissue IHC). Check cross-reactivity and endogenous detection activity, while allowing for HPA’s Uncertain reliability and UniProt’s broad tissue-expression annotation (HPA tissue IHC; UniProt Q92982 tissue specificity). |
| The slide is diffusely colored, or the liver control has no discernible hepatocyte signal. | Diffuse color reduces confidence that staining marks a cell compartment; absent liver staining misses the supplied positive reference (HPA tissue IHC: hepatocytes Medium). Use control sections to separate background, detection failure, and a possible discrepancy with HPA’s uncertain tissue result (HPA tissue IHC: reliability Uncertain). |
💡Expected NINJ1 appearanceCall an IHC result provisionally positive when hepatocytes show distinguishable, approximately medium cell-associated staining with a plausible membrane emphasis (HPA tissue IHC: hepatocytes Medium; UniProt Q92982 topology); a uniform haze or exclusive nuclear stain fails that pattern check (UniProt Q92982 subcellular location).
| Antibody evidence | The listed rabbit polyclonal antibody HPA045063 has an Uncertain IHC assessment, and the tissue profile is also Uncertain (HPA antibody HPA045063; HPA tissue IHC). Use the pattern as a working expectation and interpret isolated strong staining cautiously. |
| Membrane topology and epitope position | NINJ1 has extracellular residues 1–78 and 139–152, separated by two transmembrane segments and a short cytoplasmic region (UniProt Q92982 topology). The supplied record does not locate the antibody epitope, so it cannot predict which region the IHC antibody detects. |
| Processing and extracellular signal | UniProt annotates a secreted NINJ1 chain spanning residues 1–56 alongside the full-length 1–152 chain (UniProt Q92982 processing). Extracellular staining is therefore biologically possible, but these annotations cannot establish that a particular extracellular deposit on a section is specific. |
| Tissue reference limits | HPA reports medium staining in liver hepatocytes and Not detected staining in several listed cell populations (HPA tissue IHC). Its Uncertain reliability and UniProt’s broad tissue-specificity statement limit how firmly any single negative population can exclude NINJ1 (HPA tissue IHC; UniProt Q92982 tissue specificity). |
| IF/ICC expectation? | A membrane pattern is plausible from UniProt topology and HPA’s subcellular summary (UniProt Q92982 topology; HPA subcellular: Membrane). The supplied HPA entry has no ICC-IF image cell lines and no ICC validation for HPA045063; it cannot provide an IF/ICC reference pattern or protocol (HPA subcellular; HPA antibody HPA045063). |
| Situation | Likely cause | Next action |
|---|---|---|
| Liver control shows no hepatocyte signal. | The run may lack detectable staining, or this specimen may differ from HPA’s medium hepatocyte result (HPA tissue IHC: hepatocytes Medium; reliability Uncertain). | Check that tissue and detection controls worked, then optimize general IHC conditions such as retrieval and antibody concentration. No NINJ1-specific retrieval condition or fixation sensitivity is supplied. |
| Color covers the section without clear cell boundaries. | Diffuse staining cannot be assigned confidently to the annotated membrane compartment (UniProt Q92982 subcellular location); general IHC background is another possibility. | Compare a no-primary control, inspect blocking and wash performance, and score only signal distinguishable from background. Do not interpret a uniform haze as the HPA hepatocyte pattern (HPA tissue IHC). |
| Signal appears mainly inside nuclei. | Nuclear confinement disagrees with UniProt’s membrane and secreted annotations (UniProt Q92982 subcellular location). | Review morphology and the no-primary control, then reassess antibody specificity before reporting nuclear NINJ1. The supplied HPA IHC assessment is Uncertain (HPA antibody HPA045063). |
| Adipocytes or bone-marrow hematopoietic cells stain strongly. | Those populations are reported as Not detected in the supplied HPA tissue IHC entries; cross-reactivity or endogenous detection activity merits investigation (HPA tissue IHC). | Inspect no-primary and detection controls and compare staining with cell morphology. Treat the discrepancy as unresolved until checked, because HPA calls its tissue IHC evidence Uncertain (HPA tissue IHC). |
| Staining seems extracellular rather than confined to intact cell edges. | A secreted residues 1–56 form is annotated, but the record does not show that extracellular section staining represents that form (UniProt Q92982 processing). | Record the location separately from membrane staining, check background controls, and avoid calling it specific secreted NINJ1 without further validation. |
| An IF/ICC image is being used to judge the IHC section. | HPA summarizes NINJ1 as membrane-associated but supplies no ICC-IF image cell lines or ICC validation for the listed antibody (HPA subcellular; HPA antibody HPA045063). | Judge the paraffin-section result against the IHC tissue observations and controls (HPA tissue IHC). Consult the separate IF/ICC guide for that application; the supplied record provides no IF/ICC protocol. |
Experiment evidence
IHC & IF images showing NINJ1 staining
A08377 · IHCImmunohistochemistry of NINJ1 in mouse brain tissue with NINJ1 antibody at 5 μg/ml.
A08377 · IFImmunofluorescence of NINJ1 in mouse brain tissue with NINJ1 antibody at 20 μg/ml.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000131669-NINJ1/tissue/cerebral+cortex#imid_12416837
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000131669-NINJ1/tissue/colon#imid_12416619
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000131669-NINJ1/tissue/liver#imid_12416620
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000131669-NINJ1/tissue/kidney#imid_12416629
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NINJ1 IHC & IF
🧪Run liver first and expect staining in hepatocytes (Medium; HPA: liver hepatocytes); run adipose tissue as the negative comparator and expect adipocytes to be unstained (HPA: adipose adipocytes, Not detected). On the liver slide, assess nonhepatocyte cells as internal negatives only if their NINJ1-negative status is independently established; the supplied HPA row reports hepatocytes alone (HPA: liver hepatocytes).
Positive control tissue: Liver (Hepatocytes, 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 NINJ1; derive a cell-line control from the positive tissue's cell type (Hepatocytes) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host and immunoglobulin class, and NINJ1-knockout tissue as a biological negative (standard IHC practice). For chromogenic liver IHC, block endogenous peroxidase and check background staining in the controls (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A08377 mouse-brain IHC caption does not state its fixative (selected-SKU caption: fixative not stated). Antigen-retrieval dependency is unreported; optimize retrieval on paraffin sections with the controls above (supplied target/application evidence; standard IHC practice). The supplied evidence does not establish that frozen sections or IF are easier, or identify a NINJ1-specific tissue artefact; interpret membrane staining in light of NINJ1 localisation (UniProt Q92982: cell membrane; HPA: membrane).
HPA tissue IHC evidence for NINJ1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Antibody staining in cells/structures not annotated, view images.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Liver | Hepatocytes | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NINJ1 IHC Tips
Troubleshoot NINJ1 staining in paraffin sections by checking retrieval, antibody specificity, cellular localisation, and the reliability of each comparison tissue.
What retrieval conditions should I start with for NINJ1 in paraffin sections?
Start with citrate pH 6.0 heat-induced antigen retrieval at 95–98 °C for 20 min (page retrieval rule). Allow sections to cool in buffer before rinsing, and compare a retrieved section with an otherwise matched section processed without retrieval (standard IHC practice). If staining remains weak, test a second retrieval condition on adjacent sections while keeping antibody concentration and detection time fixed (standard IHC practice). The catalog image shows NINJ1 staining in mouse brain at 5 μg/ml, but its caption does not report fixation or retrieval, so it cannot establish which retrieval condition produced that image (catalog antibody A08377 caption).
Could fixation explain weak or uneven NINJ1 staining?
Target-specific fixation sensitivity for NINJ1 is unknown from the supplied evidence; the catalog mouse brain IHC caption does not state its fixative (catalog antibody A08377 caption). Record fixative, fixation duration, tissue thickness, and processing history for each specimen before comparing staining intensity (standard IHC practice). Compare adjacent sections from similarly processed blocks using the same retrieval and detection conditions, then review whether weak areas coincide with visibly poor tissue preservation (standard IHC practice). Do not assign a NINJ1-specific fixation effect from the reported tissue staining pattern or from its membrane topology; neither provides a controlled fixation comparison (HPA tissue IHC; UniProt Q92982 topology).
Where should convincing chromogenic NINJ1 staining appear?
Prioritise staining at cell boundaries because NINJ1 is annotated at the cell membrane, including synaptic membranes (UniProt Q92982 subcellular; HPA subcellular: Membrane). Its 2 transmembrane segments span residues 79–103 and 114–138, so a predominantly diffuse nuclear signal warrants a specificity check (UniProt Q92982 topology). NINJ1 also has an annotated secreted chain comprising residues 1–56; extracellular staining therefore needs interpretation alongside membrane staining and antibody epitope information (UniProt Q92982 processing). Inspect cell outlines at high magnification and compare adjacent sections with a negative detection control before calling faint diffuse chromogen positive (standard IHC practice).
How does epitope position change the interpretation of NINJ1 staining?
Check the antibody’s documented immunogen or mapped epitope before interpreting the stained compartment; none is supplied for the catalog antibody here (catalog antibody A08377 caption). NINJ1 has 0 annotated isoforms, but the record lists a full-length 1–152 chain and a secreted 1–56 chain (UniProt Q92982 isoforms; UniProt Q92982 processing). An epitope within residues 1–56 could detect both chains, whereas one beyond residue 56 would exclude the listed secreted chain (UniProt Q92982 processing). Consider the glycosylation site at residue 60 when evaluating epitope accessibility, without assuming an effect on this antibody’s staining (UniProt Q92982 glycosylation; standard IHC practice).
How can IF help resolve an ambiguous NINJ1 IHC pattern?
Use IF on a comparable specimen to test whether NINJ1 signal outlines cells that also carry a validated marker for the cell type under study, such as an endothelial marker when assessing endothelial staining (HPA tissue IHC: expressed in endothelial cells; standard IF practice). Choose a far-red NINJ1 fluorophore when shorter wavelengths show substantial tissue autofluorescence, and inspect single-stain controls before judging overlap (standard IF practice). Set permeabilisation according to the antibody epitope: extracellular regions are residues 1–78 and 139–152, while residues 104–113 face the cytoplasm (UniProt Q92982 topology). The supplied caption documents tissue IHC, not IF validation for this antibody (catalog antibody A08377 caption).
How do I distinguish NINJ1 signal from chromogenic background?
Inspect a section processed without primary antibody to identify detection-system staining, and check whether chromogen accumulates at tissue edges, folds, or damaged areas (standard IHC practice). Apply a peroxidase block when using an enzyme-based chromogenic workflow, and compare matched sections with and without that step if endogenous enzyme activity is suspected (standard IHC practice). Titrate the primary antibody and shorten chromogen development if both expected cell outlines and unrelated tissue acquire diffuse colour (standard IHC practice). The catalog mouse brain image used 5 μg/ml antibody, but that concentration is image-specific evidence rather than a universal working concentration (catalog antibody A08377 caption).
What should I measure when comparing NINJ1 staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the compartment and cell population before scoring; membrane-associated staining is consistent with the NINJ1 localisation record (UniProt Q92982 subcellular; HPA subcellular: Membrane). For a defined population, report the percentage of positive cells and an intensity-weighted H-score, keeping the positivity threshold and chromogen exposure consistent across batches (standard IHC practice). If counting spatially distinct positive cells, report density per mm² of evaluable tissue and exclude folds, necrosis, and missing tissue by a prespecified rule (standard IHC practice). Normalise comparisons to the number of evaluable cells or tissue area in the same anatomical compartment, and record staining batch and section thickness (standard IHC practice).
When is a positive NINJ1 IHC result biologically credible?
A credible result has reproducible cell-associated staining in the expected compartment and survives comparison with negative detection controls and neighbouring sections (UniProt Q92982 subcellular; standard IHC practice). Interpret the cell identity carefully: HPA reports expression in endothelial cells and medium hepatocyte staining, while rating its tissue IHC reliability Uncertain (HPA tissue IHC). Diffuse nuclear colour, isolated edge staining, necrotic debris, or signal attributable to endogenous enzyme activity should prompt further checks before a positive call (UniProt Q92982 subcellular; standard IHC practice). NINJ1 participates in membrane rupture during programmed cell death, but staining alone cannot establish that rupture occurred in the scored cells (UniProt Q92982 function; standard IHC interpretation).
Boster reagents
Best NINJ1 / Ninjurin-1 IHC Antibodies
A08377 has IHC and IF images from mouse brain tissue (catalog image captions). Human, mouse, and rat are listed as reactive species (catalog: reactivity); the supplied images show mouse tissue only (catalog image captions).
Real IHC data
Anti-Ninjurin-1 NINJ1 Antibody
Cat # A08377
A08377 will render with an IHC-P image of mouse brain tissue stained at 5 μg/mL (A08377 IHC image caption; catalog: applications). Its IF image also shows mouse brain tissue, stained at 20 μg/mL (A08377 IF image caption).
Which to pick: For paraffin-section tissue IHC, choose A08377: IHC-P is listed and its own image shows mouse brain tissue at 5 μg/mL (catalog: applications; A08377 IHC image caption); the fixative is unreported (A08377 IHC image caption). For IF, A08377 has a mouse brain tissue image at 20 μg/mL (A08377 IF image caption); ICC validation is unreported (catalog: applications and image captions). For human or rat samples, A08377 lists both species as reactive, while the supplied IHC and IF images show mouse tissue only (catalog: reactivity; A08377 image captions); clonality is unreported (catalog: clone).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q92982 (NINJ1_HUMAN, Ninjurin-1).
- Human Protein Atlas. NINJ1 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. NINJ1 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. NINJ1 antibody validation summary (1 antibodies).
- NINJ1 as a novel biomarker of intestinal injury in necrotizing enterocolitis. BMC pediatrics 2026 — PMC13237964.
- Ninjurin1 drives lung tumor formation and progression by potentiating Wnt/β-Catenin signaling through Frizzled2-LRP6 assembly. Journal of experimental & clinical cancer research : CR 2022 — PMC8991582.
- Targeting NINJ1-Mediated Plasma Membrane Rupture in Tubular Epithelial Cell Prevents Inflammatory Response in Acute Kidney Injury. International journal of biological sciences 2025 — PMC12435343.
- Ninjurin1 Plays a Crucial Role in Pulmonary Fibrosis by Promoting Interaction between Macrophages and Alveolar Epithelial Cells. Scientific reports 2018 — PMC6277454.
- PubMed PMID:8780658 — UniProt-cited evidence.
- PubMed PMID:9465296 — UniProt-cited evidence.
- PubMed PMID:15164053 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NINJ1 (Ninjurin-1). Expected localisation Cell membrane expected; tissue compartment unverified (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A08377 at 5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Liver (Hepatocytes); 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 A08377); 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-NINJ1, 5 μg/mL (datasheet A08377); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Tissue staining reliability is uncertain (HPA tissue IHC); Isoform / epitope: No isoforms; the secreted 1–56 chain lacks the cytoplasmic region (UniProt); Regulation: Expression regulation is not established (UniProt). Evidence: UniProt Q92982, HPA tissue IHC · Uncertain, HPA ICC-IF · Membrane, Datasheet A08377, PMC13237964. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NINJ1/immunohistochemistry-ihc-guide.