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NFAT5 / Nuclear factor of activated T-cells 5 · IHC design guide
Design Immunohistochemistry for NFAT5
Plan paraffin-section NFAT5 IHC with the catalog antibody at 2–5 μg/mL (datasheet A01815-3). Assess nuclear and cytoplasmic staining by cell type, using the reported high goblet-cell signal as a reference (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NFAT5 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 | Nuclear and cytoplasmic staining (HPA tissue IHC) | |
| Staining pattern | High goblet-cell signal; general nuclear and cytoplasmic pattern (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A01815-3) | |
| Positive control | Bronchus+4 more · see all | |
| Negative control | Lymph node+3 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across samples (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Hypertonic nuclear shift, by similarity (UniProt) | |
| Isoform / epitope | 5 isoforms (A–E); check epitope coverage (UniProt) |
Section 1
Recommended NFAT5 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A01815-3) is followed by four published NFAT5 IHC protocols (PMC7758185; PMC5355657; PMC5613209; PMC7304660).
| Sample | Paraffin-embedded human bladder cancer tissue; fixative not specified (datasheet A01815-3) |
| Fixation | Image fixative and duration unreported (datasheet A01815-3); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A01815-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01815-3) |
| Primary antibody | Rabbit anti-NFAT5, 2-5μg/ml (datasheet A01815-3) |
| Primary incubation | Overnight at 4 °C (datasheet A01815-3) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A01815-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NFAT5-positive staining in goblet cells of bronchus (HPA tissue IHC: High). HPA tissue profile: General nuclear and cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA at pH 8.0 for the catalog antibody (datasheet: A01815-3). Citrate at pH 6.0 is a published alternative (PMC7758185).
Section 2
What Is the Expected NFAT5 Staining Pattern?
NFAT5 is found in the nucleus and cytoplasm and has no transmembrane segment (UniProt O94916). In paraffin sections, expect nuclear and cytoplasmic staining, with the strongest HPA tissue observations in bronchial and nasopharyngeal goblet cells (HPA: High). Interpret intensity by cell type: HPA rates tissue IHC as Approved but reports only medium consistency between antibody staining and RNA expression (HPA: Approved; medium consistency).
| Goblet cells show clear nuclear staining, with or without cytoplasmic staining, in bronchus or nasopharynx. | This fits the reported High staining in those cell types (HPA: High in bronchial and nasopharyngeal goblet cells). Nuclear signal is consistent with NFAT5 localization; cytoplasmic signal can also be genuine (UniProt O94916; HPA: general nuclear and cytoplasmic tissue expression). Compare cells within the same section before judging intensity. |
| Signal is confined to cell borders, extracellular material, or a uniform surface rim. | A membrane-only pattern is inconsistent with the reported nuclear and cytoplasmic distribution (UniProt O94916; HPA: tissue IHC profile). NFAT5 has no transmembrane segment (UniProt O94916 topology). Check morphology and the no-primary control before treating such staining as NFAT5; cytoplasmic staining alone is not automatically erroneous. |
| Strong staining appears in lymph-node germinal-center cells while an expected positive area is weak. | HPA reports germinal-center cells as Not detected and bronchial goblet cells as High (HPA: tissue IHC). Review the cell identification and compare controls processed together. Staining in an unexpected cell type can reflect cross-reactivity or endogenous detection activity (general IHC practice), but one slide cannot establish either cause. |
| Color spreads through stroma, lumens, or most cells without a discernible nuclear or cytoplasmic pattern. | This is hard to reconcile with a cell-associated NFAT5 readout (UniProt O94916 localization; HPA: tissue IHC profile). Diffuse color can arise from nonspecific binding or detection background (general IHC practice). Inspect a no-primary control and avoid scoring the haze as positive cells. |
| Bronchial or nasopharyngeal goblet cells show no detectable signal. | These are useful positive-control cell types because HPA reports High staining there (HPA: tissue IHC); their absence prompts a run check, not an immediate biological conclusion. Confirm that goblet cells are present, then inspect the control, detection reagents, and staining conditions (general IHC practice). |
💡Expected NFAT5 appearanceCall a section positive when identifiable bronchial or nasopharyngeal goblet cells show clear nuclear staining, possibly with cytoplasmic staining (HPA: High; UniProt O94916); diffuse acellular color is background (general IHC practice).
| Cell type and reference tissue | HPA reports High staining in bronchial and nasopharyngeal goblet cells, Medium in several listed cell types, and Not detected in lymph-node germinal-center cells (HPA: tissue IHC). Use those cell-level observations when selecting comparisons; do not assign one expected intensity to an entire organ. |
| Nuclear versus cytoplasmic distribution | Both compartments are reported (UniProt O94916; HPA: tissue IHC profile). UniProt notes increased nuclear distribution under hypertonic conditions by similarity (UniProt O94916); this is a biological qualifier, not a predicted intensity change for a particular paraffin section. |
| Antibody and isoform interpretation | HPA069711 has Approved IHC status, while the tissue profile has medium staining–RNA consistency (HPA: antibody validation; tissue IHC). UniProt lists five NFAT5 isoforms (UniProt O94916). Without an epitope map or isoform-specific validation, do not infer which isoforms contribute to a positive stain. |
| IF/ICC: where should signal appear? | Mainly in the nucleoplasm, with additional cytosolic signal (HPA: enhanced ICC-IF localization). This supports a compartment comparison only; the IF/ICC workflow belongs in its own guide, and these images do not establish an IHC dilution or retrieval condition. |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in the positive-control area | Goblet cells may be absent from the examined field, or the staining run may have failed (general IHC practice; HPA: High in bronchial and nasopharyngeal goblet cells). | Locate intact goblet cells, compare a concurrently processed positive control, then verify reagent delivery and detection steps (general IHC practice). |
| Weak signal in otherwise suitable goblet cells | Staining conditions may need adjustment (general IHC practice); the supplied sources do not establish NFAT5-specific fixation sensitivity. | Optimize the IHC-validated antibody's dilution and retrieval conditions against controls, following its validated IHC procedure (general IHC practice). |
| Nuclear signal is absent but cytoplasmic signal is distinct | Cytoplasmic localization is reported, so this observation alone does not prove an artefact (UniProt O94916; HPA: tissue IHC profile). | Check nuclear counterstain and cell boundaries, then compare the positive control and staining controls before assigning a localization change (general IHC practice). |
| Unexpected cells stain strongly | Possible nonspecific binding or endogenous detection activity (general IHC practice); HPA's Not detected calls apply to specified cell types, not every cell in those tissues (HPA: tissue IHC). | Confirm cell identity and inspect no-primary and detection controls; adjust blocking or detection as indicated by those controls (general IHC practice). |
| Diffuse brown background obscures cells | Nonspecific binding, residual endogenous enzyme activity, or excessive detection can obscure chromogenic IHC (general IHC practice). | Review the no-primary control; optimize blocking, antibody dilution, washing, and chromogen development one variable at a time (general IHC practice). |
| Results differ between tissues | HPA reports cell-specific levels from High to Not detected and only medium staining–RNA consistency (HPA: tissue IHC). | Score identified cell types and compartments separately, with controls from the same run; avoid calling an entire tissue positive or negative from a single field (general IHC practice). |
Experiment evidence
IHC & IF images showing NFAT5 staining
A01815-3 · IHCIHC analysis of NFAT5 using anti-NFAT5 antibody (A01815-3). NFAT5 was detected in a paraffin-embedded section of human bladder cancer tissue
A01815-3 · IFIF analysis of NFAT5 using anti-NFAT5 antibody (A01815-3) and anti-Tubulin Alpha antibody (M03989-3). NFAT5 was detected in immunocytochemic
P01815 · IHCImmunohistochemical analysis of paraffin-embedded human liver cancer. 1, Antibody was diluted at 1:200(4° overnight). 2, Tris-EDTA,pH9.0 was
P01815 · IFImmunofluorescence analysis of COS7 cells, using NFAT5 (Phospho-Ser1197) Antibody. The picture on the right is blocked with the phospho pept
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000102908-NFAT5/tissue/cerebral+cortex#imid_19043603
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000102908-NFAT5/tissue/colon#imid_19043519
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000102908-NFAT5/tissue/liver#imid_19043520
HPA · GallbladderHuman Protein Atlas IHC, Gallbladder. Source: https://www.proteinatlas.org/ENSG00000102908-NFAT5/tissue/gallbladder#imid_19043496
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA051528; green = NFAT5, blue = nucleus, red = microtubules.
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA051528; green = NFAT5, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NFAT5 IHC & IF
🧪Run bronchus first and expect staining in goblet cells (HPA: High in bronchial goblet cells); use lymph node germinal center cells as the negative tissue comparison (HPA: Not detected). On the bronchus slide, use unstained areas outside the goblet cell population to judge background, without assuming those cells are biologically NFAT5-negative (HPA: High in bronchial goblet cells).
Positive control tissue: Bronchus (Goblet cells, HPA High)
Negative control tissue: Lymph node (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NFAT5 in Hep-G2, U2OS, A-549, RT-4, SiHa, NIH 3T3, with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a concentration-matched nonimmune rabbit IgG isotype control; confirm specificity with NFAT5 knockout material or an immunizing-peptide competition control if available (caption: rabbit primary antibody; standard IHC controls). For chromogenic bronchus sections, block endogenous peroxidase and check whether goblet-cell mucus retains background stain (HPA: bronchial goblet cells High; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A01815-3 tissue-IHC caption does not state the fixative (caption: fixative not stated). The documented paraffin-section procedure uses heat retrieval in EDTA at pH 8.0, but the evidence does not establish whether NFAT5 staining depends on that retrieval method (caption: EDTA retrieval). HPA shows nucleoplasmic and cytosolic ICC-IF signal, but provides no basis to call frozen sections or IF easier; in bronchus, assess mucus-associated background before scoring goblet-cell staining (HPA: subcellular ICC-IF; HPA: bronchial goblet cells High; standard IHC practice).
HPA tissue IHC evidence for NFAT5
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium 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 |
|---|---|---|---|---|
| Bronchus | Goblet cells | High | Protein (IHC) | HPA → |
| Nasopharynx | Goblet cells | High | Protein (IHC) | HPA → |
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Seminal vesicle | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | Chondrocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NFAT5 IHC Tips
Troubleshoot NFAT5 staining in paraffin sections by checking retrieval, background, and nuclear versus cytoplasmic signal against the documented tissue patterns.
What retrieval should I try first if NFAT5 staining is weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet A01815-3). The catalog antibody produced NFAT5 staining in a paraffin section of human bladder cancer after that retrieval, followed by 2 μg/ml primary antibody overnight at 4°C (datasheet A01815-3). If signal is weak, first check that sections stayed covered during heating and that the retrieval solution reached the intended temperature throughout the run (standard IHC practice). Compare retrieved and unretrieved serial sections using the same detection settings, and judge improvement by cellular staining rather than diffuse DAB deposition (standard IHC practice).
Could fixation explain weak or patchy NFAT5 staining?
Target-specific fixation sensitivity is unknown: the catalog image describes a paraffin section but does not state its fixative (datasheet A01815-3). Record the fixative, fixation duration, section thickness, and storage history for each specimen before changing the antibody conditions (standard IHC practice). When comparing specimens, hold EDTA retrieval at pH 8.0 and the primary concentration at 2 μg/ml constant initially (datasheet A01815-3). Uneven staining across a section warrants checking tissue processing and section adhesion alongside a matched control; it does not, by itself, establish NFAT5-specific fixation damage (standard IHC practice).
Should NFAT5 appear in nuclei, cytoplasm, or both?
Expect a mixture of nuclear and cytoplasmic staining rather than requiring one compartment in every cell (HPA: general nuclear and cytoplasmic expression; UniProt O94916: nucleus and cytoplasm). HPA subcellular imaging places NFAT5 mainly in the nucleoplasm, with additional cytosolic signal (HPA: subcellular). Nuclear distribution can increase under hypertonic conditions, although that annotation is based on similarity and does not establish the conditions in a tissue section (UniProt O94916: subcellular location). Score nuclear and cytoplasmic DAB separately against the counterstain, using the same thresholds across specimens; a change in compartment alone needs biological context and controls (standard IHC practice).
Could NFAT5 isoforms or epitope masking cause discordant staining?
NFAT5 has 5 listed isoforms, C, A, B, D, and E, so an antibody's epitope determines which forms it can detect (UniProt O94916: isoforms). The supplied catalog caption gives the primary concentration but no epitope map; its tissue stain therefore cannot establish isoform coverage (datasheet A01815-3). NFAT5 has no transmembrane segment, while documented modified residues include phosphorylation and acetylation sites; neither fact establishes whether this antibody's epitope is affected (UniProt O94916: topology and modified residues). If a second antibody is available, compare antibodies with documented, distinct epitopes on serial sections and keep retrieval and DAB development matched (standard IHC practice).
How can IF help assess an ambiguous chromogenic NFAT5 pattern?
Use IF on a separate section to compare NFAT5 with a marker for the cell population being assessed, then return to matched compartments in the chromogenic IHC section (standard IF/IHC practice). HPA reports high NFAT5 staining in bronchial goblet cells, providing one documented cell population for a matched tissue comparison (HPA: High in bronchus goblet cells). Choose spectrally separated fluorophores, favoring red or far-red detection when tissue autofluorescence obscures shorter wavelengths, and include single-channel controls (standard IF practice). NFAT5 has no transmembrane segment and is reported in nucleoplasm and cytosol, so intracellular IF detection requires permeabilisation; optimize it because this antibody's epitope is unspecified (UniProt O94916: topology; HPA: subcellular; standard IF practice).
How do I reduce diffuse brown staining without losing NFAT5 signal?
First compare the test section with a no-primary control to locate background from secondary reagents, endogenous peroxidase, or DAB deposition (standard IHC practice). Block endogenous peroxidase before detection and inspect tissue edges and damaged regions separately from intact cells (standard chromogenic IHC practice). The catalog image used 10% goat serum, peroxidase-conjugated anti-rabbit secondary, and DAB, giving starting conditions for the same antibody (datasheet A01815-3). If background remains high, titrate primary concentration around the documented 2 μg/ml while holding retrieval at EDTA pH 8.0 and development time constant, then compare cellular contrast (datasheet A01815-3; standard IHC practice).
How should I quantify NFAT5 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Choose a cell-based measure before scoring: nuclear H-score from 0–300, percentage of positive cells, or positive-cell density per mm² are standard IHC options (standard IHC practice). Record cytoplasmic staining separately because both nuclear and cytoplasmic expression are reported for NFAT5 (HPA: tissue profile; UniProt O94916: subcellular location). Normalize positive counts to the number of evaluable cells, or density to viable tissue area, within the same defined cell population and sampling scheme (standard IHC practice). Apply one staining threshold and imaging setup across batches, and interpret modest differences cautiously because HPA describes only medium consistency between staining and RNA data (HPA: Approved, medium consistency).
What distinguishes a true NFAT5-positive cell from staining artefact?
Look for staining within intact cells and compare its nuclear and cytoplasmic distribution with the counterstain; both compartments are plausible for NFAT5 (HPA: general nuclear and cytoplasmic expression). HPA reports high staining in bronchial goblet cells but no detected signal in lymph-node germinal center cells, so cell identity and tissue context matter when judging a pattern (HPA: tissue IHC). Discount staining confined to section edges, necrotic areas, or the no-primary control, and check whether endogenous peroxidase accounts for brown deposits (standard chromogenic IHC practice). A positive DAB reaction supports protein detection, while nuclear enrichment by itself does not prove NFAT5 transcriptional activity (UniProt O94916: function and subcellular location; standard IHC interpretation).
Boster reagents
Best NFAT5 / Nuclear factor of activated T-cells 5 IHC Antibodies
Validated NFAT5 antibodies have IHC images from human paraffin-embedded bladder and liver cancer sections (catalog IHC captions); A01815-3 also has IF images from human bladder sections and A549 cells (catalog IF captions).
Real IHC data
Anti-NFAT5 Antibody ®
Cat # A01815-3
Real IHC data
Anti-Phospho NFAT5 (Ser1197) Antibody
Cat # P01815
A01815-3 will render with IHC data from a human paraffin-embedded bladder cancer section; it lists human, mouse and rat reactivity and IF/ICC applications (A01815-3 caption; catalog applications/reactivity). P01815 will render with IHC data from a human paraffin-embedded liver cancer section; it lists human and mouse reactivity (P01815 caption; catalog reactivity).
Which to pick: For tissue IHC, choose A01815-3 for total NFAT5 in paraffin sections or P01815 when the target is phospho-NFAT5 Ser1197; each has its own paraffin-section IHC image (catalog titles; A01815-3 and P01815 IHC captions). For IF/ICC or rat reactivity, choose A01815-3 because those applications and rat reactivity are listed for it (catalog applications/reactivity). The IHC captions report paraffin embedding but do not report the fixative for either SKU (A01815-3 and P01815 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O94916 (NFAT5_HUMAN, Nuclear factor of activated T-cells 5).
- Human Protein Atlas. NFAT5 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. NFAT5 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol..
- Human Protein Atlas. NFAT5 antibody validation summary (4 antibodies).
- NFAT5 promotes oral squamous cell carcinoma progression in a hyperosmotic environment. Laboratory investigation; a journal of technical methods and pathology 2021 — PMC7758185.
- NFAT5 participates in seawater inhalation‑induced acute lung injury via modulation of NF-κB activity. Molecular medicine reports 2016 — PMC5355657.
- Transcription Factor NFAT5 Promotes Glioblastoma Cell-driven Angiogenesis via SBF2-AS1/miR-338-3p-Mediated EGFL7 Expression Change. Frontiers in molecular neuroscience 2017 — PMC5613209.
- Recurrent Amplification of the Osmotic Stress Transcription Factor NFAT5 in Adrenocortical Carcinoma. Journal of the Endocrine Society 2020 — PMC7304660.
- PubMed PMID:10051678 — UniProt-cited evidence.
- PubMed PMID:10377394 — UniProt-cited evidence.
- PubMed PMID:11528118 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NFAT5 (Nuclear factor of activated T-cells 5). Expected localisation Nuclear and cytoplasmic staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A01815-3). Validated IHC antibody: A01815-3 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bronchus (Goblet cells); negative Lymph node. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human bladder cancer tissue; fixative not specified (datasheet A01815-3); 2. Fixation: Image fixative and duration unreported (datasheet A01815-3); 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 retrieval: EDTA pH 8.0 (datasheet A01815-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01815-3); 8. Primary antibody: Rabbit anti-NFAT5, 2-5μg/ml (datasheet A01815-3); 9. Primary incubation: Overnight at 4 °C (datasheet A01815-3). Watch for: Fixation: Keep fixation consistent across samples (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 5 isoforms (A–E); check epitope coverage (UniProt); Regulation: Hypertonic nuclear shift, by similarity (UniProt). Evidence: UniProt O94916, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A01815-3, PMC7758185, PMC5355657. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NFAT5/immunohistochemistry-ihc-guide.