This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
NLRP9 / NACHT, LRR and PYD domains-containing protein 9 · IHC design guide
Design Immunohistochemistry for NLRP9
Plan NLRP9 chromogenic IHC in paraffin sections using intestinal glandular staining as a reference (HPA tissue IHC). This guide covers fixation, controls and interpretation, including the reported mismatch between staining and RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NLRP9 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 | Membranous intestinal staining (HPA tissue IHC); cytoplasmic location (UniProt) | |
| Staining pattern | Membranous staining in intestinal glandular cells (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 | Bone marrow+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show very low consistency (HPA tissue IHC) | |
| Regulation | Expression regulation is not established (UniProt) | |
| Isoform / epitope | 2 isoforms; assess epitope coverage (UniProt) |
Section 1
Recommended NLRP9 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet) is accompanied by published protocols for gastric and colorectal cancers (PMC8262223) and MS brain lesions (PMC6138928).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A14119); 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-NLRP9, 10 μg/mL (datasheet A14119) |
| 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 | NLRP9-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Membranous expression in intestines. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval guidance); the MS brain protocol uses pH 9 microwave retrieval (PMC6138928).
Section 2
What Is the Expected NLRP9 Staining Pattern?
NLRP9 is annotated as cytoplasmic, with no transmembrane segment (UniProt Q7RTR0), while tissue IHC reports membranous staining in intestines (HPA tissue IHC). Look first in intestinal glandular cells, including appendix and small intestine, and in pancreatic endocrine cells, all reported High (HPA tissue IHC). HPA rates the IHC evidence Approved but reports very low agreement between antibody staining and RNA expression (HPA tissue IHC).
| Intestinal glandular cells show a membrane-associated chromogenic signal. | This matches the reported intestinal IHC profile (HPA tissue IHC: membranous expression in intestines). Record its cellular distribution as observed; UniProt places NLRP9 in the cytoplasm and lists no transmembrane segment (UniProt Q7RTR0). The two location descriptions should remain visible when interpreting the slide. |
| Appendix or small-intestine glandular cells, or pancreatic endocrine cells, stain more strongly than surrounding cells. | These are useful positive comparators because each cell population is reported High by tissue IHC (HPA tissue IHC). High is a reported staining category, not a promised intensity for every section; assess the signal alongside tissue morphology and controls (general IHC practice). |
| A strong nuclear-only signal dominates the expected positive cells. | Nuclear-only staining does not match the cytoplasmic UniProt annotation or HPA's intestinal membrane-associated profile (UniProt Q7RTR0; HPA tissue IHC). Treat it as suspect and inspect background and detection controls before assigning it to NLRP9 (general IHC practice). |
| Cells reported as unstained show widespread chromogenic signal. | For example, HPA reports bone-marrow hematopoietic cells and lymph-node germinal-center cells as Not detected (HPA tissue IHC). Signal there calls for control review; cross-reactivity or endogenous detection activity are possible explanations, not conclusions from tissue identity alone (general IHC practice). |
| The whole section looks hazy, or a known-positive cell population has no signal. | Haze without cellular definition is difficult to score (general IHC practice). Absence of staining in appendix or small-intestine glandular cells needs a run-level check because HPA reports those cells High (HPA tissue IHC); a negative slide by itself does not establish absent NLRP9. |
💡Expected NLRP9 appearanceA credible positive IHC result shows defined signal in appendix or small-intestine glandular cells, or pancreatic endocrine cells reported High, with intestinal membrane-associated staining documented by HPA; strong nuclear-only or uniform background signal is suspect (HPA tissue IHC; UniProt Q7RTR0; general IHC practice).
| Compartment interpretation | UniProt lists cytoplasm and inflammasome and no transmembrane segment, whereas HPA describes membranous intestinal IHC (UniProt Q7RTR0; HPA tissue IHC). Do not infer a membrane-spanning protein or use either annotation alone to dismiss a well-controlled slide. |
| Choice of tissue and cell population | Appendix and small-intestine glandular cells and pancreatic endocrine cells are reported High; colon and duodenal glandular cells are Medium (HPA tissue IHC). Score the named cells rather than averaging signal across an entire organ. |
| Strength of antibody evidence | The tissue IHC result is Approved, with very low antibody-staining versus RNA consistency; HPA042623 is Approved for IHC (HPA tissue IHC; HPA antibodies). These labels support cautious comparison with controls, not an independently confirmed compartment assignment. |
| Isoforms and epitope scope | UniProt lists two isoforms and pyrin and NACHT domains but supplies no antibody epitope in this record (UniProt Q7RTR0). An isoform-specific staining prediction cannot be made from the supplied evidence. |
| Processing and retrieval limits | UniProt lists no signal peptide, propeptide, glycosylation sites, or processed chain beyond residues 1–991 (UniProt Q7RTR0). Target-specific effects of fixation or antigen retrieval are unreported here; choose and evaluate retrieval using the antibody's validated IHC method (general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain appears in appendix or small-intestine glandular cells. | These cells are reported High, so a blank result may reflect a failed staining run or sample issue; tissue identity alone cannot distinguish them (HPA tissue IHC; general IHC practice). | Check morphology and a positive control processed in the same run, then verify the catalog antibody's IHC-P conditions and detection reagents (general IHC practice). |
| A strong nuclear-only signal appears. | That location conflicts with UniProt's cytoplasmic annotation and HPA's intestinal membrane-associated profile (UniProt Q7RTR0; HPA tissue IHC). | Compare with a detection-only control and review whether the signal follows nuclei, tissue edges, or cellular morphology before scoring it (general IHC practice). |
| Staining is widespread in cells expected to be unstained. | HPA reports bone-marrow hematopoietic cells and lymph-node germinal-center cells as Not detected; cross-reactivity or endogenous detection activity may contribute (HPA tissue IHC; general IHC practice). | Check primary-omission and detection controls, then assess whether staining remains restricted to defined cells in a reported positive tissue (general IHC practice; HPA tissue IHC). |
| Diffuse brown background obscures cell boundaries. | Nonspecific reagent binding or endogenous chromogenic detection activity can obscure cellular staining (general IHC practice). | Inspect primary-omission controls; review blocking, antibody dilution, washing, and endogenous-activity control for the detection system (general IHC practice). |
| Intestinal membrane-associated signal seems inconsistent with a cytoplasmic annotation. | The descriptions differ: HPA reports membranous intestinal IHC, while UniProt annotates cytoplasm and no transmembrane segment (HPA tissue IHC; UniProt Q7RTR0). | Document the actual distribution and compare named positive cells and controls; avoid treating membrane-associated chromogen as proof of membrane topology (general IHC practice; UniProt Q7RTR0). |
| Can IF/ICC establish the same subcellular pattern? | HPA provides no main ICC-IF location and lists no cell lines with ICC-IF images for NLRP9 (HPA subcellular). | Use the separate IF/ICC guide for that application; this IHC result cannot validate an IF compartment pattern, though cytoplasm is UniProt's annotated location (HPA subcellular; UniProt Q7RTR0). |
Experiment evidence
IHC & IF images showing NLRP9 staining
A14119 · IHCImmunohistochemistry of NOD6 in human testis tissue with NOD6 antibody at 10 μg/mL.
A14119 · IFImmunofluorescence of NOD6 in human testis tissue with NOD6 antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000185792-NLRP9/tissue/cerebral+cortex#imid_11684694
HPA · Small intestineHuman Protein Atlas IHC, Small intestine. Source: https://www.proteinatlas.org/ENSG00000185792-NLRP9/tissue/small+intestine#imid_11684603
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000185792-NLRP9/tissue/colon#imid_11684620
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000185792-NLRP9/tissue/liver#imid_11684629
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NLRP9 IHC & IF
🧪Run appendix first and expect staining in glandular cells (HPA: Appendix glandular cells, High); use bone marrow hematopoietic cells as the negative tissue (HPA: Bone marrow hematopoietic cells, Not detected). On the appendix slide, use adjacent nonglandular cells as a background check, but do not assume they are a validated internal negative population (HPA: Appendix glandular cells, High).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for NLRP9; 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; a host-species- and clonality-matched isotype control where applicable; and knockout tissue as a biological negative, or a peptide-block control if the matching immunizing peptide is available. For chromogenic appendix IHC, quench endogenous peroxidase and check background in glandular structures; block endogenous biotin if using biotin-based detection (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A14119 tissue-IHC caption does not state a fixative (A14119 caption: fixative not stated). Retrieval dependence is unreported, so compare retrieved and unretrieved paraffin sections during optimization (supplied target/application evidence: no retrieval condition). Neither frozen sections nor IF can be judged easier from the supplied evidence; assess glandular background carefully in appendix, and note that no ICC-IF image-bearing cell lines are listed (HPA subcellular: no ICC-IF images).
HPA tissue IHC evidence for NLRP9
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Very low consistency between antibody staining and RNA expression data. External characterization data supports antibody staining.). 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 → |
| Pancreas | Pancreatic endocrine cells | High | Protein (IHC) | HPA → |
| Small intestine | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | Medium | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NLRP9 IHC Tips
Troubleshoot NLRP9 staining in paraffin sections by checking retrieval, compartment, cell identity, controls and scoring before interpreting signal (UniProt Q7RTR0; HPA tissue IHC).
How should I adjust retrieval when NLRP9 staining is weak?
Start with citrate pH 6.0 heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval setting). If staining is weak, compare a cautiously adjusted retrieval condition on serial sections while keeping antibody concentration, detection and development time constant (standard IHC practice). Include a known staining compartment, such as small-intestinal glandular cells, and a matched no-primary control in the comparison (HPA: High in small-intestinal glandular cells; standard IHC practice). Score signal and tissue preservation together: stronger diffuse staining with damaged morphology does not establish improved NLRP9 detection (standard IHC practice).
Could fixation explain inconsistent NLRP9 staining between paraffin blocks?
Target-specific NLRP9 sensitivity to fixation is unknown from the supplied evidence; the testis IHC caption gives an antibody concentration of 10 µg/mL but does not state the fixative (A14119 tissue-IHC caption). Compare blocks with documented fixation and processing histories, and stain matched sections in the same run to reduce procedural variation (standard IHC practice). Use the page's citrate pH 6.0, 95–98 °C, 20 min retrieval setting consistently before changing other variables (page retrieval setting). If morphology and signal vary together, record that association without assigning a specific fixation effect to NLRP9 (standard IHC practice).
Should NLRP9 appear cytoplasmic or membranous in chromogenic sections?
Expect cytoplasmic NLRP9 based on its annotated location and lack of a transmembrane segment (UniProt Q7RTR0: Cytoplasm; no transmembrane segment). The tissue atlas nevertheless describes membranous staining in intestines, with High staining in small-intestinal glandular cells (HPA tissue IHC). Examine serial sections for reproducible intracellular distribution and compare it with cell boundaries and the no-primary control before calling a crisp membrane rim target-specific (standard IHC practice; UniProt Q7RTR0 topology). Record cytoplasmic and apparent membranous staining separately, since the atlas reports very low consistency between staining and RNA expression despite its Approved antibody-staining assessment (HPA reliability description).
How can isoforms and epitope location affect NLRP9 IHC interpretation?
NLRP9 has 2 annotated isoforms, while the supplied material does not identify this antibody's epitope or establish isoform coverage (UniProt Q7RTR0: isoforms 1 and 2; A14119 tissue-IHC caption). Map a documented immunogen to both isoform sequences before interpreting discordant staining as an isoform difference (standard antibody-validation practice; UniProt Q7RTR0 isoforms). The annotated pyrin domain spans residues 1–94 and the NACHT domain 146–465, so epitope position could affect which molecular forms an antibody recognizes (UniProt Q7RTR0 domains; standard antibody-validation practice). Treat any epitope-dependent explanation as provisional until supported by antibody-specific mapping or an independent validation method (standard antibody-validation practice).
How can IF help assess ambiguous NLRP9 staining in tissue?
On matched tissue sections, multiplex NLRP9 with a marker identifying the expected glandular epithelial cells, then check whether signal falls within the marked cells (HPA: High in small-intestinal glandular cells; standard IF practice). Select a fluorophore in a relatively low-autofluorescence channel for the specimen and include unstained and secondary-only controls (standard IF practice). Because NLRP9 is annotated as cytoplasmic and has no transmembrane segment, permeabilise sufficiently to expose an intracellular epitope, while checking that cell morphology remains intact (UniProt Q7RTR0: Cytoplasm; no transmembrane segment; standard IF practice). Interpret IF as a separate assay requiring its own antibody validation; the A14119 caption documents tissue IHC, not IF (A14119 tissue-IHC caption).
What should I check when NLRP9 DAB staining is diffuse?
Compare the stained section with no-primary and detection-reagent controls to locate background from the chromogenic workflow (standard IHC practice). For peroxidase-based detection, apply an endogenous-peroxidase block before DAB and check whether residual colour appears in controls (standard IHC practice). Titrate the primary antibody and shorten DAB development in matched sections if nonspecific colour obscures cells; the supplied testis caption reports 10 µg/mL for A14119 but does not establish a universal working concentration (A14119 tissue-IHC caption; standard IHC practice). Reassess any diffuse extracellular or uniform edge staining against NLRP9's cytoplasmic annotation and section morphology (UniProt Q7RTR0: Cytoplasm; standard IHC practice).
How should I quantify NLRP9 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and scoring region before reading slides; small-intestinal glandular cells are a documented stained population (HPA: High in small-intestinal glandular cells; standard IHC practice). Report the percentage of positive eligible cells and, where intensity is reproducible, an H-score from 0–300 using percentages in intensity categories 0–3 (standard IHC scoring practice). Normalise counts to the number of evaluable cells, or report positive-cell density per mm² of viable annotated tissue when cell counts are impractical (standard IHC scoring practice). Exclude folds, necrotic areas and damaged edges using the same prespecified rules across sections; record cytoplasmic and apparent membranous patterns separately (standard IHC practice; UniProt Q7RTR0: Cytoplasm; HPA tissue IHC).
When is an apparent NLRP9-positive cell convincing?
A convincing call has reproducible staining in an anatomically identified cell population, acceptable morphology and little corresponding colour in the no-primary control (standard IHC practice). Small-intestinal glandular staining has atlas support, but its reported membranous pattern warrants caution because NLRP9 is annotated as cytoplasmic without a transmembrane segment (HPA tissue IHC; UniProt Q7RTR0 topology and location). Question signal limited to tissue edges, necrosis or endogenous-enzyme-rich areas, and compare it with a peroxidase-blocked control (standard IHC practice). Interpret biological differences cautiously: the atlas reports very low agreement between antibody staining and RNA expression, even though external characterization supports the staining (HPA reliability description).
Boster reagents
Best NLRP9 / NACHT, LRR and PYD domains-containing protein 9 IHC Antibodies
A14119 has human testis IHC and IF images (catalog image captions) and lists Human and Mouse reactivity (catalog reactivity).
Real IHC data
Anti-NOD6 NLRP9 Antibody
Cat # A14119
A14119 is listed for IHC-P and shows human testis IHC at 10 μg/mL (catalog applications; IHC image caption). The same SKU is listed for IF and shows human testis IF at 20 μg/mL (catalog applications; IF image caption).
Which to pick: Choose A14119 for paraffin-section tissue IHC: IHC-P is listed, and its own image shows human testis staining; the fixative is unreported (catalog applications; IHC image caption). For IF, choose A14119 on the strength of its human testis image; ICC validation is unreported (catalog applications; IF image caption). A14119 also lists Mouse reactivity, but the supplied IHC and IF images show Human tissue only; it is rabbit-hosted, with clonality unreported (catalog reactivity, image captions and host/clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q7RTR0 (NLRP9_HUMAN, NACHT, LRR and PYD domains-containing protein 9).
- Human Protein Atlas. NLRP9 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. NLRP9 subcellular location (ICC-IF): Highest expression in SK-MEL-30: 0.1 nTPM.
- Human Protein Atlas. NLRP9 antibody validation summary (1 antibodies).
- Somatic Mutation of NLRP Genes in Gastric and Colonic Cancers. Pathology oncology research : POR 2021 — PMC8262223.
- Exome sequencing study in patients with multiple sclerosis reveals variants associated with disease course. Journal of neuroinflammation 2018 — PMC6138928.
- PubMed PMID:12563287 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:12766759 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NLRP9 (NACHT, LRR and PYD domains-containing protein 9). Expected localisation Membranous intestinal staining (HPA tissue IHC); cytoplasmic location (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A14119 at 10 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Bone marrow. 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 A14119); 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-NLRP9, 10 μg/mL (datasheet A14119); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: Staining and RNA show very low consistency (HPA tissue IHC); Isoform / epitope: 2 isoforms; assess epitope coverage (UniProt); Regulation: Expression regulation is not established (UniProt). Evidence: UniProt Q7RTR0, HPA tissue IHC · Approved, HPA cell-line RNA · Highest expression in SK-MEL-30: 0.1 nTPM, Datasheet A14119, PMC8262223, PMC6138928. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NLRP9/immunohistochemistry-ihc-guide.