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NLRP3 / NACHT, LRR and PYD domains-containing protein 3 · IHC design guide
Design Immunohistochemistry for NLRP3
Plan NLRP3 paraffin IHC around the reported cytoplasmic tissue pattern (HPA tissue IHC). Compare stained sections with appropriate controls, and interpret intensity cautiously because HPA rates the tissue evidence uncertain (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NLRP3 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 in tissue (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in several tissues, including bone marrow (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear 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 paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Tissue staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | LPS-treated granulocytes express NLRP3 (UniProt) | |
| Isoform / epitope | 6 isoforms; map the antibody epitope to each (UniProt) |
Section 1
Recommended NLRP3 IHC & IF Protocols
The catalog antibody’s IHC protocol is followed by 4 published NLRP3 staining protocols (PMC6842944; PMC4807043; PMC12720632; PMC5331985).
| Sample | Paraffin-embedded human lung carcinoma tissue; fixative not specified (datasheet M00034) |
| Fixation | Image fixative and duration unreported (datasheet M00034); 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 Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear 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 monoclonal (clone FBD-14) anti-NLRP3, 1:50-1:200 (datasheet M00034) |
| 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 | NLRP3-positive staining in glandular cells of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in several tissues including bone marrow. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 heat retrieval at 95–98 °C for 20 min (page retrieval rule).
Section 2
What Is the Expected NLRP3 Staining Pattern?
NLRP3 is mainly cytosolic in resting macrophages and can associate with organelle membranes (UniProt Q96P20). It has no transmembrane segment (UniProt Q96P20 topology). HPA reports cytoplasmic staining in several tissues, including bone marrow, but rates its tissue IHC pattern Uncertain because antibody staining and RNA expression have low consistency (HPA tissue IHC).
| Cytoplasmic staining in macrophages. | Fits the main resting-cell location and predominant cell type (UniProt Q96P20). |
| Strong, uniform nuclear staining in tissue cells. | Check for artefact; resting macrophage NLRP3 is mainly cytosolic (UniProt Q96P20). |
| Strong staining in adipocytes. | Check cross-reactivity or detection activity; HPA reports adipocytes Not detected (HPA tissue IHC). |
| Diffuse stain across cells and empty areas. | Suspect background; check a no-primary control (standard IHC practice). |
| No signal in bone marrow. | Check the run; HPA reports cytoplasmic expression there, with Uncertain reliability (HPA tissue IHC). |
💡Expected NLRP3 appearanceLook for cytoplasmic staining in macrophages (UniProt Q96P20); uniform nuclear stain warrants review (UniProt Q96P20).
| Activation state (UniProt Q96P20) | NLRP3 can move toward the MTOC and dispersed trans-Golgi vesicles (UniProt Q96P20). |
| Cell type (UniProt Q96P20) | Macrophages predominate; resting monocytes have very weak protein expression (UniProt Q96P20). |
| Tissue IHC confidence (HPA tissue IHC) | HPA rates the pattern Uncertain; antibody HPA012878 is also IHC Uncertain (HPA antibodies). |
| Situation | Likely cause | Next action |
|---|---|---|
| No cytoplasmic signal in bone marrow (HPA tissue IHC). | A failed run is possible; HPA tissue IHC reliability is Uncertain (HPA tissue IHC). | Check tissue preservation and a positive control (standard IHC practice). |
| Stain appears mainly nuclear in macrophages. | Unexpected for resting macrophages (UniProt Q96P20). | Review morphology and a no-primary control (standard IHC practice). |
| Adipocytes stain strongly. | HPA reports adipocytes Not detected (HPA tissue IHC). | Check cross-reactivity and endogenous detection activity (standard IHC practice). |
| Diffuse chromogen covers the section. | Nonspecific background or endogenous detection activity is possible (standard IHC practice). | Review blocking, washes and a no-primary control (standard IHC practice). |
| HPA tissue patterns disagree with this slide. | HPA rates tissue IHC reliability Uncertain (HPA tissue IHC). | Compare cell type and compartment before scoring (standard IHC practice). |
| Can IF/ICC confirm the punctate pattern? | HPA lists no ICC-IF images or main location (HPA subcellular). | Use the separate IF/ICC guide; this HPA record cannot confirm puncta (HPA subcellular). |
Experiment evidence
IHC & IF images showing NLRP3 staining
M00034 · IHCImmunohistochemical analysis of paraffin-embedded human lung carcinoma, using NALP3 Antibody.
M00034 · IFImmunofluorescent analysis of Raw264.7 cells, using NALP3 Antibody.
M00034-1 · IHCRat kidney tissue was stained with Anti-NLRP3 rabbit Antibody
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000162711-NLRP3/tissue/cerebral+cortex#imid_3263684
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000162711-NLRP3/tissue/colon#imid_3263610
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000162711-NLRP3/tissue/liver#imid_3263603
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000162711-NLRP3/tissue/kidney#imid_3263612
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NLRP3 IHC & IF
🧪Run bronchus first: respiratory epithelial cells should stain at the HPA Medium level (HPA: Bronchus, respiratory epithelial cells, Medium). Use heart muscle as the negative tissue, assessing cardiomyocytes for no detectable staining (HPA: Heart muscle, cardiomyocytes, Not detected); on the bronchus slide, morphologically identified cells that prove unstained should show only background signal, but do not assume all nonepithelial cells are negative because NLRP3 is expressed in macrophages (UniProt Q96P20 tissue specificity).
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 NLRP3; 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 section and an isotype control matched to the primary antibody’s host species and monoclonal subclass or polyclonal IgG class; use NLRP3 knockout material as a biological negative where available, or an immunizing-peptide block if the peptide is known (standard IHC practice). Block endogenous peroxidase for chromogenic detection and check inflammatory cells for residual signal, since macrophages can express NLRP3 (standard IHC practice; UniProt Q96P20 tissue specificity).
⚠️Feasibility: A target-specific fixation window and the effect of antigen retrieval are unreported in the supplied evidence; optimize retrieval empirically for paraffin sections (standard IHC practice). The selected M00034 paraffin-section lung carcinoma caption does not report a fixative, and the HPA bronchus staining does not establish specimen processing (M00034 tissue-IHC caption; HPA: Bronchus, respiratory epithelial cells, Medium). The evidence does not establish whether frozen sections or IF are easier; in bronchus, inflammatory-cell staining may complicate epithelial scoring because macrophages express NLRP3 (UniProt Q96P20 tissue specificity).
HPA tissue IHC evidence for NLRP3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low consistency between antibody staining and RNA expression data.). 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 → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NLRP3 IHC Tips
Troubleshoot NLRP3 staining in paraffin sections by checking retrieval, cell identity, compartment, and controls before interpreting chromogenic signal.
How should I adjust retrieval when NLRP3 staining is weak or uneven?
Start with Tris-EDTA at pH 9.0, heated to 95–98 °C for 20 min (page retrieval setting). If staining is weak, compare a shorter heating period on adjacent sections before trying a different buffer, keeping section thickness, antibody incubation, and detection constant (standard IHC practice). Record whether any gain appears in cells with plausible cytoplasmic staining, because NLRP3 is predominantly expressed in macrophages and is mainly cytosolic or associated with organelle membranes at rest (UniProt Q96P20). If stronger retrieval raises staining across unrelated cells or tissue edges, compare the change with a no-primary control before accepting it as improved detection (standard IHC practice).
Could fixation explain inconsistent NLRP3 staining across paraffin blocks?
Target-specific NLRP3 sensitivity to fixative type or fixation duration is unknown from the supplied evidence, so document each block’s processing history before attributing differences to fixation (supplied evidence). The selected M00034 image shows paraffin-embedded human lung carcinoma, but its caption does not state the fixative (M00034 caption). Compare matched blocks with similar tissue composition and processing, then apply the same retrieval, primary-antibody incubation, and chromogenic development to adjacent sections (standard IHC practice). If staining remains inconsistent, inspect morphology and include a no-primary control; avoid treating a different cellular mix as a fixation effect, because NLRP3 expression varies by cell type (UniProt Q96P20).
Which NLRP3 staining compartments should I accept in tissue sections?
Evaluate cytoplasmic signal first, while allowing staining near intracellular membranes: resting macrophage NLRP3 is mainly cytosolic or associated with endoplasmic reticulum, mitochondrial, and Golgi membranes (UniProt Q96P20). Activation can move NLRP3 through the microtubule organizing center to dispersed trans-Golgi vesicle membranes, but routine chromogenic sections may not resolve those structures (UniProt Q96P20; standard IHC practice). Record nuclear staining separately rather than automatically rejecting it, because nuclear localization is described for Th2 helper T cells by similarity, whereas Th1 localization is mainly cytoplasmic (UniProt Q96P20). Compare each pattern with cell identity, morphology, and a no-primary control before assigning a compartment-specific result (standard IHC practice).
How can isoforms and epitope masking complicate NLRP3 IHC?
NLRP3 has 6 listed isoforms and contains a pyrin domain at residues 1–93, FISNA at 140–210, and NACHT at 220–536 (UniProt Q96P20). Identify the antibody’s immunogen or mapped epitope before claiming that all isoforms are detected; neither the selected caption nor the supplied record establishes this antibody’s epitope (M00034 caption; supplied evidence). If signal differs after retrieval changes, compare adjacent sections under identical detection conditions, since accessibility in paraffin sections can change without proving an isoform difference (standard IHC practice). Document the antibody identifier, retrieval condition, and staining compartment, and reserve isoform-specific interpretation for an independently established epitope map (standard IHC practice).
How should I follow up an NLRP3 IHC pattern with multiplex IF?
On a separate IF workflow, pair NLRP3 with a validated marker identifying the suspected positive cell population; macrophages are a relevant starting population because NLRP3 is predominantly expressed there (UniProt Q96P20). Choose fluorophores after checking unstained tissue in each channel, shifting the NLRP3 readout away from strong tissue autofluorescence and using single-stain controls to assess bleed-through (standard IF practice). Because NLRP3 lacks a transmembrane segment and occupies cytosol and intracellular organelle membranes, optimize permeabilisation for access to the antibody’s mapped epitope (UniProt Q96P20; standard IF practice). Interpret apparent puncta with cell identity and appropriate controls; the supplied HPA subcellular record contains no ICC/IF images establishing this antibody’s staining pattern (HPA subcellular record).
What should I check when NLRP3 chromogenic staining appears widespread?
Inspect a no-primary section for endogenous enzyme activity, then check whether peroxidase blocking and the chromogenic detection reagents were applied consistently (standard IHC practice). Compare tissue edges, folds, necrotic areas, and intact regions at the same development time, since uneven reagent access and damaged morphology can mimic diffuse staining (standard IHC practice). Titrate the primary antibody as a serial dilution while holding retrieval and detection constant, and judge whether plausible cytoplasmic cellular signal remains distinct from background (standard IHC practice; UniProt Q96P20). Interpret broad staining cautiously: the HPA tissue profile is rated Uncertain because antibody staining has low consistency with RNA expression data (HPA tissue IHC).
How should I quantify NLRP3 staining across heterogeneous tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and intact tissue area before scoring, because NLRP3 is predominantly expressed in macrophages but is also reported in several other cell types (UniProt Q96P20). For cellular staining, report the percentage of positive cells or an H-score using intensity categories 0–3; for dispersed positive cells, report counts per mm² of evaluable tissue (standard IHC practice). Normalize cell-based results to the number of cells of the same identified population, and area-based results to the measured viable tissue area (standard IHC practice). Apply one positivity threshold and identical chromogen development across compared sections, recording excluded necrotic or folded regions before analysis (standard IHC practice).
How do I distinguish genuine NLRP3 signal from staining artefacts?
Favor staining in morphologically intact cells with a plausible cytoplasmic distribution, especially within a separately identified macrophage population, while recognizing that other cell types can express NLRP3 (UniProt Q96P20). Review isolated nuclear staining in its cellular context: nuclear localization is described for Th2 helper T cells by similarity, so compartment alone cannot establish identity (UniProt Q96P20). Treat edge-restricted color, necrotic deposits, and signal reproduced in a no-primary section as possible processing or endogenous-enzyme artefacts (standard IHC practice). Require concordance across matched sections and cell markers before a biological conclusion, particularly because HPA rates its tissue IHC profile Uncertain (HPA tissue IHC; standard IHC practice).
Boster reagents
Best NLRP3 / NACHT, LRR and PYD domains-containing protein 3 IHC Antibodies
Catalog IHC images show human lung carcinoma in paraffin sections and rat kidney tissue; an IF image shows Raw264.7 cells (M00034 and M00034-1 image captions).
Real IHC data
Anti-NALP3 NLRP3 Rabbit Monoclonal Antibody
Cat # M00034
Real IHC data
Anti-NLRP3 Rabbit Monoclonal Antibody
Cat # M00034-1
M00034 has an IHC image of paraffin-embedded human lung carcinoma and an IF image of Raw264.7 cells; its listed reactivity is human (M00034 image captions; catalog reactivity). M00034-1 has an IHC image of rat kidney tissue and lists IHC and IF applications with human, mouse and rat reactivity (M00034-1 IHC image caption; catalog applications and reactivity).
Which to pick: For human tissue IHC in paraffin sections, choose M00034: its own image documents that preparation, though the fixative is unreported (M00034 IHC image caption). For IF/ICC, choose M00034 when the listed human reactivity fits; it lists both applications and has an IF image, while its Raw264.7 image does not establish additional species reactivity (M00034 catalog applications and reactivity; IF image caption). For work requiring listed human, mouse and rat reactivity, choose M00034-1; its rat kidney IHC caption does not report section processing or fixative (M00034-1 catalog reactivity; IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q96P20 (NLRP3_HUMAN, NACHT, LRR and PYD domains-containing protein 3).
- Human Protein Atlas. NLRP3 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. NLRP3 subcellular location (ICC-IF): Highest expression in THP-1: 68.3 nTPM.
- Human Protein Atlas. NLRP3 antibody validation summary (1 antibodies).
- Expression and Contribution of NLRP3 Inflammasome During the Follicular Development Induced by PMSG. Frontiers in cell and developmental biology 2019 — PMC6842944.
- NLRP3 Inflammasome Plays an Important Role in the Pathogenesis of Collagen-Induced Arthritis. Mediators of inflammation 2016 — PMC4807043.
- Kaempferol activity on Cryptosporidium parvum infection in an experimentally infected immunocompromised mouse model: In silico and in vivo investigations. Pharmaceutical biology 2026 — PMC12720632.
- Microvesicles released from fat-laden cells promote activation of hepatocellular NLRP3 inflammasome: A pro-inflammatory link between lipotoxicity and non-alcoholic steatohepatitis. PloS one 2017 — PMC5331985.
- PubMed PMID:11687797 — UniProt-cited evidence.
- PubMed PMID:12355493 — UniProt-cited evidence.
- PubMed PMID:11786556 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NLRP3 (NACHT, LRR and PYD domains-containing protein 3). Expected localisation Cytoplasmic staining in tissue (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: M00034 at 1:50-1:200 (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: Paraffin-embedded human lung carcinoma tissue; fixative not specified (datasheet M00034); 2. Fixation: Image fixative and duration unreported (datasheet M00034); 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 Tris-EDTA buffer, pH 9.0 , 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 monoclonal (clone FBD-14) anti-NLRP3, 1:50-1:200 (datasheet M00034); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Tissue staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 6 isoforms; map the antibody epitope to each (UniProt); Regulation: LPS-treated granulocytes express NLRP3 (UniProt). Evidence: UniProt Q96P20, HPA tissue IHC · Uncertain, HPA cell-line RNA · Highest expression in THP-1: 68.3 nTPM, Datasheet M00034, PMC6842944, PMC4807043. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NLRP3/immunohistochemistry-ihc-guide.