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NFAT5 / Nuclear factor of activated T-cells 5 · IHC design guide
Design Immunohistochemistry for NFAT5
Plan chromogenic NFAT5 IHC in paraffin sections using the catalog antibody’s validated conditions. Compare nuclear and cytoplasmic staining with the tissue pattern, and account for the possible shift toward nuclear staining under hypertonic conditions (HPA tissue IHC; UniProt).
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 | Goblet cells: nuclear and cytoplasmic staining (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 conditions consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Hypertonicity may raise nuclear signal (UniProt) | |
| Isoform / epitope | 5 isoforms; verify epitope coverage (UniProt) |
Section 1
Recommended NFAT5 IHC & IF Protocols
The catalog antibody protocol is accompanied by four published NFAT5 IHC procedures, each drawn from a target-verified PMC article.
| 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 pH 8.0 retrieval for the catalog antibody (datasheet A01815-3). Citrate pH 6.0 is a published alternative (PMC7758185).
Section 2
What Is the Expected NFAT5 Staining Pattern?
NFAT5 should show nuclear and cytoplasmic staining, with the strongest reported tissue IHC signal in bronchial and nasopharyngeal goblet cells (HPA: general nuclear and cytoplasmic expression; High in goblet cells). NFAT5 is a nuclear, cytoplasmic and chromosome-associated transcription factor without a transmembrane segment (UniProt O94916: subcellular location and topology). Treat tissue comparisons as guides because HPA rates its IHC profile Approved, with medium consistency between staining and RNA expression (HPA: reliability).
| Goblet cells show clear nuclear staining with possible cytoplasmic staining in bronchus or nasopharynx. | This fits the reported High goblet-cell IHC signal and general nuclear/cytoplasmic profile (HPA: bronchus, nasopharynx and tissue profile). Score the nuclear and cytoplasmic compartments separately; a cytoplasmic component alone does not establish mislocalisation (HPA: tissue profile). |
| Strong staining outlines cell membranes while nuclei and cytoplasm remain largely unstained. | An isolated membrane pattern conflicts with the reported nuclear/cytoplasmic distribution and lack of a transmembrane segment (HPA: tissue profile; UniProt O94916: topology). Review morphology, detection and controls before assigning that signal to NFAT5 (general IHC practice). |
| Strong signal appears in cells reported as undetected, such as lymph-node germinal-center cells. | HPA reports NFAT5 as Not detected in those cells (HPA: lymph node). Consider antibody cross-reactivity or endogenous detection activity, and compare with a no-primary control (general IHC practice). HPA's medium consistency means this comparison is a warning rather than proof of nonspecificity (HPA: reliability). |
| Brown precipitate spreads across extracellular space, tissue folds and multiple cell types without clear boundaries. | Diffuse staining is difficult to assign to a compartment or cell type (general IHC practice). Check the no-primary control and detection reagents, then reassess blocking and washes (general IHC practice); this appearance alone cannot identify its cause. |
| A bronchus or nasopharynx section shows no goblet-cell signal. | The missing signal conflicts with HPA's High goblet-cell observations (HPA: bronchus and nasopharynx). Confirm that goblet cells are present on the section, then check antibody and detection controls (general IHC practice). One negative section does not establish absent NFAT5 expression. |
💡Expected NFAT5 appearanceA convincing positive shows readily identifiable nuclear staining, with possible cytoplasmic staining, in goblet cells of bronchus or nasopharynx, where HPA reports High signal; isolated membrane staining or widespread cell-free precipitate should prompt control review (HPA: tissue profile, bronchus and nasopharynx; UniProt O94916: topology; general IHC practice).
| Cell-type reference | Bronchial and nasopharyngeal goblet cells are reported High; adipocytes, adrenal glandular cells and several neural populations are Medium (HPA: tissue IHC). Use the named cell type, rather than the whole organ, when choosing and scoring a reference section (general IHC practice). |
| Physiological localisation | Nuclear distribution increases under hypertonic conditions, according to a by-similarity annotation (UniProt O94916: subcellular location). Record treatment conditions when comparing nuclear with cytoplasmic staining; the annotation does not supply an expected IHC intensity or a required nuclear-only pattern. |
| Antibody validation | The HPA tissue profile is Approved with medium staining–RNA consistency (HPA: reliability). Among listed HPA antibodies, HPA069711 has Approved IHC status, while the others have no listed IHC status (HPA: antibodies). Validation supports interpretation but does not make every unexpected positive specific (general IHC practice). |
| Protein form and epitope | UniProt lists five NFAT5 isoforms, an RHD at residues 264–443, no signal peptide and one annotated chain spanning residues 1–1531 (UniProt O94916: isoforms, domains and processing). These annotations do not identify the catalog antibody's epitope or establish equal recognition of every isoform. |
| IF/ICC Q: Where should NFAT5 appear? | A: Mainly in the nucleoplasm, with additional cytosolic signal (HPA: subcellular ICC-IF). This is a localisation comparison only; the HPA ICC-IF record does not provide an IF protocol or set the chromogenic IHC dilution. |
| Situation | Likely cause | Next action |
|---|---|---|
| Goblet cells in a reference section are unstained. | The section may lack identifiable target cells, or the IHC detection run may have failed (general IHC practice). | Confirm goblet-cell morphology in bronchus or nasopharynx, where HPA reports High signal (HPA: tissue IHC). Check the run's positive control and the IHC-validated antibody's instructions before adjusting general retrieval or detection steps (general IHC practice). |
| Signal is confined to cell membranes. | That distribution is inconsistent with reported nuclear/cytoplasmic localisation and no transmembrane segment (HPA: tissue profile; UniProt O94916: topology). | Compare with the no-primary control, inspect tissue edges and reassess whether staining follows cells or deposits (general IHC practice). Do not score an isolated membrane rim as the expected NFAT5 pattern. |
| Strong staining appears in HPA Not detected cells. | Cross-reactivity or endogenous detection activity is possible (general IHC practice); HPA lists lymph-node germinal-center cells as Not detected (HPA: lymph node). | Run a no-primary control and review blocking appropriate to the chromogenic detection system (general IHC practice). Interpret disagreement cautiously because the HPA tissue profile has medium staining–RNA consistency (HPA: reliability). |
| Background obscures nuclear boundaries. | Diffuse detection signal can prevent cell-level and compartment-level scoring (general IHC practice). | Check the no-primary control, reagent exposure, blocking and washes; adjust those general IHC steps only in response to the controls (general IHC practice). Score NFAT5 only where cellular boundaries remain interpretable. |
| Nuclear and cytoplasmic proportions differ between specimens. | NFAT5 occupies both compartments, and hypertonicity can increase nuclear distribution by similarity (HPA: tissue profile; UniProt O94916: subcellular location). | Document specimen conditions and score each compartment separately. Avoid calling cytoplasmic signal alone an artefact; HPA reports a general nuclear and cytoplasmic IHC pattern (HPA: tissue profile). |
| A new antibody gives a pattern unlike the HPA reference. | The HPA list does not assign IHC status to every antibody, and the record does not specify the new antibody's epitope or isoform coverage (HPA: antibodies; UniProt O94916: isoforms). | Check that the antibody is IHC validated, compare reference cell types and compartments, and include appropriate positive and no-primary controls (general IHC practice; HPA: tissue IHC). Avoid attributing the discrepancy to fixation sensitivity without target-specific evidence. |
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 require staining in goblet cells (High; HPA: bronchus goblet cells). Use lymph node germinal center cells as the negative comparator (HPA: Not detected); on the bronchus slide, assess adjacent nongoblet cells for background without assuming they lack NFAT5.
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) slide, a concentration-matched rabbit IgG isotype control (selected A01815-3 IHC caption: rabbit primary antibody), and NFAT5-knockout tissue or cells as a biological specificity control. Quench endogenous peroxidase and inspect goblet-cell mucus for nonspecific chromogen retention (selected A01815-3 IHC caption: HRP/DAB detection; HPA: bronchus goblet cells High).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the fixative for the selected paraffin-section image is unreported (selected A01815-3 IHC caption). Heat retrieval in EDTA at pH 8.0 produced the catalog image, but the evidence does not establish that retrieval is required (selected A01815-3 IHC caption). Frozen sections or IF/ICC cannot be judged easier from the supplied evidence; bronchial goblet-cell mucus warrants a background check during chromogenic scoring (HPA: bronchus goblet cells High; selected A01815-3 IHC caption: HRP/DAB detection).
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, compartment patterns, controls and scoring before interpreting biological differences.
How should I retrieve NFAT5 when nuclear staining is weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 for this catalog antibody (datasheet A01815-3). The selected paraffin-section example used that retrieval before applying 2 μg/ml primary antibody overnight at 4°C (caption A01815-3). If staining is weak, compare a longer heat exposure with the original method on adjacent sections, while monitoring tissue damage (standard IHC practice). Keep antibody concentration, incubation and DAB development constant during that comparison so a signal change can be attributed to retrieval (standard IHC practice). Assess nuclear and cytoplasmic staining separately because both compartments are reported for NFAT5 (HPA tissue IHC; UniProt O94916 subcellular location).
Can fixation explain inconsistent NFAT5 staining across paraffin blocks?
The selected example describes a paraffin section but does not state its fixative, so NFAT5-specific sensitivity to fixation is unknown (caption A01815-3). Record each block’s fixative and fixation duration, then compare sections processed with the same EDTA pH 8.0 retrieval (datasheet A01815-3; standard IHC practice). Use a shared control section and keep the 2 μg/ml primary incubation overnight at 4°C constant during the comparison (caption A01815-3; standard IHC practice). Variable preservation or accessibility can change immunostaining in general, but staining differences alone cannot establish a fixation effect specific to NFAT5 (standard IHC practice). Do not infer fixation sensitivity from tissue expression patterns or protein modifications (HPA tissue IHC; UniProt O94916 modified residues).
Should NFAT5 appear in nuclei, cytoplasm, or both?
Evaluate both compartments: tissue staining is described as generally nuclear and cytoplasmic, while enhanced subcellular imaging places NFAT5 mainly in the nucleoplasm with additional cytosolic signal (HPA tissue IHC; HPA subcellular). UniProt also lists nucleus, cytoplasm and chromosome, with increased nuclear distribution under hypertonic conditions reported by similarity (UniProt O94916 subcellular location). On adjacent paraffin sections, keep EDTA retrieval at pH 8.0 and the 2 μg/ml primary concentration fixed before comparing compartments (datasheet A01815-3; caption A01815-3). Score nuclear and cytoplasmic intensity separately within the same cell population, using the counterstain to define nuclei (standard IHC practice). A shift toward nuclear staining warrants biological follow-up; chromogenic localisation alone does not establish NFAT5 transcriptional activity (UniProt O94916 function; standard IHC practice).
Could isoforms or epitope accessibility cause discordant NFAT5 staining?
NFAT5 has 5 listed isoforms, C, A, B, D and E, so epitope coverage matters when comparing antibodies (UniProt O94916 isoforms). Check the catalog antibody’s documented immunogen or epitope against those isoforms before interpreting a negative compartment or cell population (standard IHC practice). The protein has an RHD at residues 264–443 and several documented modified residues, but these features do not establish that this antibody recognizes or misses a particular form (UniProt O94916 domains; UniProt O94916 modified residues). Compare serial paraffin sections under the same EDTA pH 8.0 retrieval and 2 μg/ml primary conditions when testing an independently documented antibody (datasheet A01815-3; caption A01815-3; standard IHC practice). Treat discordance as an epitope question until antibody specificity and tissue preservation have been assessed (standard IHC practice).
How can IF help resolve ambiguous NFAT5 IHC localisation?
Use IF as a separate localisation check, pairing NFAT5 with a marker for the expected cell type and a nuclear counterstain (standard IF practice). NFAT5 is mainly nucleoplasmic with additional cytosolic localisation, so inspect both compartments within marker-positive cells (HPA subcellular). Choose spectrally separated fluorophores and a detection channel with low tissue autofluorescence, then include single-label and no-primary controls (standard IF practice). Because NFAT5 has no transmembrane segment and is reported inside cells, use permeabilisation sufficient to expose intracellular epitopes and verify that it preserves cell boundaries (UniProt O94916 topology; HPA subcellular; standard IF practice). The paraffin IHC example used EDTA pH 8.0 retrieval and overnight primary incubation at 4°C; establish IF conditions independently rather than assuming those tissue conditions transfer (caption A01815-3; standard IF practice).
What should I change when NFAT5 IHC has diffuse brown background?
First compare a no-primary section with the stained section to identify signal from detection chemistry or tissue rather than primary binding (standard IHC practice). The selected example used 10% goat serum blocking, 2 μg/ml primary overnight at 4°C, and DAB detection (caption A01815-3). For diffuse background, check serum blocking and washing, then titrate the primary below that example concentration while holding retrieval at EDTA pH 8.0 (caption A01815-3; datasheet A01815-3; standard IHC practice). Include an endogenous peroxidase block and inspect whether brown signal persists without primary antibody (standard chromogenic IHC practice). Preserve detectable nuclear or cytoplasmic signal in the expected cells while reducing background; both compartments are plausible for NFAT5 (HPA tissue IHC).
How should I score NFAT5 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, then report nuclear and cytoplasmic NFAT5 separately (HPA tissue IHC; standard IHC practice). For comparable regions, record the percentage of positive cells and an H-score based on intensity categories, documenting the threshold used for positivity (standard IHC practice). If spatial abundance matters, report positive cells per mm² of viable tissue and normalise counts to the measured area or relevant cell population (standard IHC practice). Keep retrieval at EDTA pH 8.0, primary concentration at 2 μg/ml, and DAB development consistent within a comparison (datasheet A01815-3; caption A01815-3; standard IHC practice). Exclude necrosis and damaged edges from the analysed area, and compare like cell types across sections (standard IHC practice).
How do I distinguish real NFAT5 staining from artefact?
A credible result follows cellular boundaries and can include nuclear and cytoplasmic staining, with nucleoplasmic enrichment supported by subcellular imaging (HPA tissue IHC; HPA subcellular). Compare the observed cell type with reference patterns: goblet cells in bronchus and nasopharynx are reported high, while lymph-node germinal center cells are reported undetected (HPA tissue IHC). Treat staining confined to section edges, necrotic areas or structures positive on a no-primary control as suspect (standard IHC practice). Check endogenous peroxidase activity before interpreting DAB signal, and compare sections processed with the same EDTA pH 8.0 retrieval (datasheet A01815-3; standard chromogenic IHC practice). A nuclear increase can be consistent with a hypertonic response, but staining alone cannot demonstrate the cause or NFAT5 activity (UniProt O94916 subcellular location; UniProt O94916 function).
Boster reagents
Best NFAT5 / Nuclear factor of activated T-cells 5 IHC Antibodies
Two antibodies have IHC images from paraffin-embedded human bladder and liver cancer sections (catalog IHC captions); A01815-3 also has IF images from A549 cells and human bladder cancer tissue (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 its human bladder cancer paraffin-section IHC image; its catalog also lists IF/ICC and human, mouse and rat reactivity (A01815-3 catalog). P01815 will render with its human liver cancer paraffin-section IHC image and targets phospho-NFAT5 Ser1197; its catalog lists human and mouse reactivity (P01815 catalog).
Which to pick: For total NFAT5 tissue IHC, choose A01815-3, whose IHC caption shows a human bladder cancer paraffin section with EDTA pH 8 retrieval and 2 μg/ml primary antibody; the fixative is unreported (A01815-3 IHC caption). For IF/ICC, choose A01815-3 because those applications are listed and IF images are supplied; P01815 has an IF image, but IF is absent from its listed applications (catalog applications and IF captions). For the broadest listed species reactivity, choose A01815-3 (human, mouse and rat); choose the polyclonal P01815 when phospho-NFAT5 Ser1197 is the target, noting that its IHC caption shows a human liver cancer paraffin section with Tris-EDTA pH 9 retrieval and leaves the fixative unreported (catalog reactivity; P01815 dilution data and IHC caption).
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 conditions consistent across sections (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 5 isoforms; verify epitope coverage (UniProt); Regulation: Hypertonicity may raise nuclear signal (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.