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ATXN3 / Ataxin-3 · IHC design guide
Design Immunohistochemistry for ATXN3
Plan ATXN3 paraffin IHC around cytoplasmic and nuclear staining in most tissues (HPA tissue IHC). Start with the catalog antibody at 0.5–1 μg/mL (datasheet PB9423), and include specificity controls because the tissue IHC evidence flags possible cross-reactivity (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ATXN3 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 and nuclear in most tissues (HPA tissue IHC) | |
| Staining pattern | Most tissues show cytoplasmic and nuclear staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet PB9423) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | Liver+1 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep formalin fixation consistent across sections (standard IHC practice; not target-specific); assess its effects empirically. | |
| Caveat | Possible cross-reactivity with another gene product (HPA tissue IHC) | |
| Regulation | Amino acid loss recruits ATXN3 to lysosomes (UniProt) | |
| Isoform / epitope | 5 isoforms; map the antibody epitope across them (UniProt) |
Section 1
Recommended ATXN3 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet: PB9423) with three published ATXN3 chromogenic IHC protocols (PMC11254720; PMC4642276; PMC4624233).
| Sample | Paraffin-embedded Human Lung Cancer tissue; fixative not specified (datasheet PB9423) |
| Fixation | Image fixative and duration unreported (datasheet PB9423); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet PB9423) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9423) |
| Primary antibody | Rabbit anti-ATXN3, 0.5-1μg/ml (datasheet PB9423) |
| Primary incubation | Overnight at 4 °C (datasheet PB9423) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9423) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ATXN3-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nuclear expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate pH 6 retrieval for the catalog antibody (datasheet: PB9423); the published protocols specify either citrate without pH or heat retrieval without buffer (PMC4624233; PMC11254720).
Section 2
What Is the Expected ATXN3 Staining Pattern?
ATXN3 is predominantly nuclear but also shows cytoplasmic staining in most tissues (UniProt P54252 subcellular location; HPA tissue IHC profile). Expect staining in adipocytes and several glandular or epithelial cell populations (HPA tissue IHC). ATXN3 has no transmembrane segment (UniProt P54252 topology). Interpret patterns cautiously: HPA rates its tissue IHC reliability Uncertain and notes that the staining may detect protein from more than one gene (HPA tissue IHC reliability).
| Nuclear staining with some cytoplasmic signal in adipocytes or adrenal glandular cells. | This fits the reported compartments and High staining in those cells (UniProt P54252 subcellular location; HPA tissue IHC). Judge each cell population separately; a positive cell beside a negative one is plausible because HPA reports cell-specific differences (HPA tissue IHC). |
| Predominantly extracellular staining, or membrane-only staining without nuclear or cytoplasmic signal. | This does not match the reported tissue IHC pattern (HPA tissue IHC profile). Check morphology and controls before calling it ATXN3; a membrane signal alone is inconclusive, especially given the Uncertain tissue IHC reliability (HPA tissue IHC reliability). |
| Strong signal in liver cholangiocytes or smooth muscle cells. | HPA reports ATXN3 as Not detected in these cell populations (HPA tissue IHC). Review cell identity, antibody specificity and chromogen-only controls: staining in an unexpected population can reflect cross-reactivity or endogenous detection activity (general IHC practice). |
| Diffuse color across nuclei, cytoplasm and tissue spaces with little cell-to-cell distinction. | This is difficult to score as specific staining (general IHC practice). Review the negative control and background from the detection system, then adjust blocking, antibody concentration or washes as appropriate (general IHC practice). |
| No detectable signal in adipocytes or adrenal glandular cells. | These are reported High-staining populations, so an entirely blank result warrants a workflow check (HPA tissue IHC). Inspect tissue preservation, retrieval, primary-antibody conditions and detection controls before treating the blank slide as evidence of ATXN3 absence (general IHC practice). |
💡Expected ATXN3 appearanceA convincing positive result shows discernible nuclear staining, potentially with cytoplasmic staining, in a reported High-staining population such as adipocytes or adrenal glandular cells (UniProt P54252 subcellular location; HPA tissue IHC); uniform extracellular color or signal confined to an unexpected cell population calls for control review (general IHC practice; HPA tissue IHC).
| Tissue and cell selection | Use an HPA High population, such as adipocytes, as a positive reference (HPA tissue IHC). Cholangiocytes and smooth muscle cells are reported Not detected, while cerebellar granular-layer cells, glia in hippocampus or caudate, and skeletal myocytes are Low (HPA tissue IHC); these are different expectations, not interchangeable controls. |
| Antibody validation | HPA rates tissue IHC reliability Uncertain, cites only medium agreement with RNA data, and warns that staining may detect protein from more than one gene (HPA tissue IHC reliability). Both listed IHC-tested antibodies, HPA024123 and CAB021976, have Uncertain IHC status (HPA antibodies); compartment agreement alone cannot establish specificity. |
| Subcellular interpretation | UniProt describes ATXN3 as predominantly nuclear, including the inner nuclear matrix, but not exclusively nuclear (UniProt P54252 subcellular location). HPA reports cytoplasmic and nuclear tissue staining (HPA tissue IHC profile); scoring only nuclear color could miss a reported component. |
| Isoforms and epitope coverage | UniProt lists five ATXN3 isoforms (UniProt P54252 isoforms). No antibody epitope or isoform coverage is supplied, so a difference between antibodies or tissues cannot be attributed to a particular isoform from these records alone. |
| IF/ICC Q: Should plasma-membrane signal count as an IHC-P positive? | A: HPA approves nucleoplasm and nucleoli as main ICC-IF locations and plasma membrane as an additional ICC-IF location (HPA subcellular). That separate cell-imaging observation does not establish membrane-only staining as a tissue IHC-P criterion (HPA tissue IHC profile). |
| Situation | Likely cause | Next action |
|---|---|---|
| High-staining reference tissue appears blank. | An IHC workflow failure is possible; adipocytes and adrenal glandular cells are reported High (HPA tissue IHC). | Check tissue morphology, retrieval and primary-antibody conditions, then verify detection with appropriate controls (general IHC practice). Avoid interpreting a blank reference as true absence until the workflow works. |
| Nuclear signal is absent, but broad cytoplasmic color remains. | The pattern is incomplete relative to predominant nuclear localization and the reported nuclear-plus-cytoplasmic tissue profile (UniProt P54252 subcellular location; HPA tissue IHC profile). | Check the nuclear counterstain and compare primary-omission and positive-tissue controls; review antibody concentration and background before assigning subcellular localization (general IHC practice). |
| Unexpectedly strong staining appears in cholangiocytes or smooth muscle cells. | These populations are reported Not detected (HPA tissue IHC); cross-reactivity or endogenous detection activity is possible (general IHC practice). | Confirm cell identity on the section and inspect primary-omission and detection controls (general IHC practice). Treat any residual signal cautiously because HPA flags possible detection of more than one gene (HPA tissue IHC reliability). |
| Color spreads across the section and obscures cell boundaries. | Excess primary antibody, inadequate blocking or incomplete washing can raise nonspecific background (general IHC practice). | Compare with a primary-omission control, then optimize blocking, primary dilution and washes while retaining a High-staining reference population (general IHC practice; HPA tissue IHC). |
| The nuclear pattern varies between tissues or antibodies. | ATXN3 has five reported isoforms, but their contribution to these slides and the antibodies’ epitope coverage are unspecified (UniProt P54252 isoforms). HPA tissue IHC reliability is Uncertain (HPA tissue IHC reliability). | Score each tissue and cell population against its reported HPA level, document the antibody used, and seek independent specificity evidence before treating a pattern difference as biological (HPA tissue IHC; general IHC practice). |
| Only membrane-associated color is visible in a paraffin section. | UniProt reports no transmembrane segment; HPA’s additional plasma-membrane location comes from ICC-IF, while its tissue IHC profile is nuclear and cytoplasmic (UniProt P54252 topology; HPA subcellular; HPA tissue IHC). | Recheck morphology and background controls, and look for the reported nuclear or cytoplasmic pattern in a High-staining population before calling the section positive (general IHC practice; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing ATXN3 staining
PB9423 · IHCIHC analysis of Ataxin 3 using anti-Ataxin 3 antibody (PB9423). Ataxin 3 was detected in paraffin-embedded section of Human Lung Cancer Tiss
PB9423 · IFIF analysis of ATXN3 using anti-ATXN3 antibody (PB9423). ATXN3 was detected in immunocytochemical section of MCF-7 cell. Enzyme antigen retr
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000066427-ATXN3/tissue/cerebral+cortex#imid_5632116
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000066427-ATXN3/tissue/colon#imid_5632050
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000066427-ATXN3/tissue/liver#imid_5632051
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000066427-ATXN3/tissue/kidney#imid_5632052
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA069338; green = ATXN3, blue = nucleus, red = microtubules.
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA069338; green = ATXN3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ATXN3 IHC & IF
🧪Run adipose tissue first; its adipocytes should stain (HPA: High in adipocytes). Use smooth muscle as the negative tissue (HPA: Not detected in smooth muscle cells); extracellular matrix and adipocyte lipid vacuoles on the positive slide should remain unstained as internal background checks (standard IHC practice).
Positive control tissue: Adipose tissue (Adipocytes, HPA High)
Negative control tissue: Liver (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ATXN3 in MCF-7, Rh30, U2OS, with annotated localisation: Nucleoplasm (approved), Nucleoli (approved) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and concentration-matched rabbit IgG isotype controls, matched to the primary antibody’s clonality where applicable (caption: rabbit anti-ATXN3; standard IHC practice); use ATXN3 knockout tissue or a cognate-peptide block, if available, to test specificity (standard IHC practice). Quench endogenous peroxidase and check endogenous biotin background in the adipose section because the selected IHC detection uses a biotin–streptavidin complex with DAB (caption: SABC with DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected PB9423 tissue-IHC caption does not state its fixative (caption: fixative unreported). The paraffin-section example uses heat retrieval in citrate buffer at pH 6 for 20 minutes, but whether ATXN3 staining requires retrieval is unreported (caption: citrate retrieval). There is no supplied basis to favor frozen sections or IF over paraffin IHC; adipose lipid vacuoles can complicate interpretation of chromogenic staining (standard IHC practice).
HPA tissue IHC evidence for ATXN3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Medium consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene. Pending external verification.). 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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Adipocytes | High | Protein (IHC) | HPA → |
Section 3
Advanced ATXN3 IHC Tips
Troubleshoot ATXN3 staining in paraffin-section chromogenic IHC by checking retrieval, cellular localisation, antibody specificity and scoring against matched controls.
Where should I start if ATXN3 staining is weak after antigen retrieval?
Start with heat-mediated citrate retrieval at pH 6 for 20 minutes (datasheet PB9423). The selected paraffin-section example used that retrieval before 1 µg/mL primary antibody overnight at 4°C, so compare staining against those documented conditions (datasheet PB9423). If staining remains weak, vary heating time on adjacent sections while keeping antibody concentration, detection and imaging conditions fixed; excessive heating can damage tissue morphology (standard IHC practice). Include a no-primary control and a section processed with the documented retrieval in each run so increased background is not mistaken for improved ATXN3 detection (standard IHC practice).
How should I troubleshoot weak staining when fixation history is uncertain?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not report a fixative (datasheet PB9423). Record the available fixation and processing history, then compare sections processed together so differences in preservation are easier to separate from staining variation (standard IHC practice). Check tissue morphology and nuclear detail before changing retrieval, because poorly preserved sections can make cellular localisation difficult to score (standard IHC practice). If the history is unavailable, describe it as unreported and interpret weak staining alongside a concurrently stained positive-control section, rather than attributing it to a particular fixative effect (standard IHC practice).
Is cytoplasmic ATXN3 staining credible when I expect a nuclear signal?
ATXN3 is predominantly nuclear but is not exclusively nuclear, and the inner nuclear matrix is a reported location (UniProt P54252 subcellular location). Tissue IHC describes cytoplasmic and nuclear expression in most tissues, although that antibody profile has uncertain reliability (HPA tissue IHC). Score nuclear and cytoplasmic staining separately, checking intact cellular boundaries and a nuclear counterstain before assigning compartments (standard IHC practice). Amino acid deprivation can recruit ATXN3 to the lysosomal membrane, but a punctate chromogenic pattern alone cannot establish that mechanism or identify a lysosome (UniProt P54252 subcellular location; standard IHC interpretation).
Could isoforms or epitope position explain inconsistent ATXN3 staining?
ATXN3 has five listed isoforms, so establish which sequence region the antibody recognizes before interpreting a negative section as loss of all ATXN3 forms (UniProt P54252 isoforms; standard IHC interpretation). The record places the Josephin domain at residues 1–180 and ubiquitin-interacting motifs at 224–243, 244–263 and 331–349 (UniProt P54252 domains). Phosphoserines are listed at 219, 265, 272 and 328, but epitope overlap or staining effects cannot be inferred without antibody mapping (UniProt P54252 modified residues; standard IHC interpretation). Compare the stated immunogen with isoform sequences when available, and use an independent epitope or orthogonal assay to investigate discordant staining (standard IHC practice).
How can IF help check the cellular source of an ATXN3 IHC signal?
For a parallel IF investigation, multiplex ATXN3 with a marker for the expected cell type and a nuclear stain; bronchial respiratory epithelial cells are one documented high-staining population for comparison (HPA tissue IHC; standard IF practice). Choose fluorophores and imaging channels after checking tissue autofluorescence and single-label controls, particularly when a weak signal overlaps intrinsic fluorescence (standard IF practice). ATXN3 has no transmembrane segment, while nuclear and lysosomal-membrane locations are reported, so intracellular epitope access should guide permeabilisation; the record does not establish a luminal epitope orientation (UniProt P54252 topology and subcellular location; standard IF practice). Keep IF localisation evidence distinct from the paraffin-section chromogenic result when interpreting agreement (standard IHC/IF practice).
What should I check when DAB staining is diffuse or widespread?
The selected example used a biotinylated secondary, streptavidin–biotin detection and DAB, so inspect background in a no-primary section run through the same detection steps (datasheet PB9423; standard IHC practice). Apply a peroxidase block and assess endogenous biotin interference as general controls for this detection chemistry, rather than assuming diffuse colour represents ATXN3 (standard IHC practice). The example used 10% goat serum blocking and 1 µg/mL primary antibody; compare a lower primary concentration only after checking wash quality and secondary-only staining (datasheet PB9423; standard IHC practice). Evaluate whether colour follows intact cells or damaged tissue and section edges before scoring it (standard IHC practice).
How should I quantify ATXN3 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because tissue IHC reports both nuclear and cytoplasmic expression (HPA tissue IHC). For intact cells, report the percentage positive and an H-score from 0–300 using consistent intensity categories; score nuclear and cytoplasmic signals separately (standard IHC scoring). If discrete positive cells are the endpoint, report density per mm² of viable tissue and state how the analysed area was selected (standard IHC scoring). Normalise cell counts to the same eligible cell population or viable area across sections, and keep retrieval, detection and image thresholds consistent between comparisons (standard IHC practice).
How can I distinguish genuine ATXN3 staining from tissue artefact?
Look for reproducible staining within intact cells, with compartment assignment supported by the counterstain; nuclear signal is plausible, but cytoplasmic signal also occurs in the tissue IHC profile (UniProt P54252 subcellular location; HPA tissue IHC; standard IHC practice). Treat colour concentrated at section edges, necrotic areas or damaged cells cautiously, and compare it with a no-primary control for endogenous enzyme or detection background (standard IHC practice). A signal confined to an unexpected cell population warrants review against matched morphology and controls, rather than assignment from colour alone (standard IHC interpretation). The HPA tissue antibody profile is rated uncertain and may detect products of more than one gene, so confirm consequential findings independently (HPA tissue IHC).
Boster reagents
Best ATXN3 / Ataxin-3 IHC Antibodies
PB9423 has IHC data from a human paraffin-embedded lung cancer section and IF/ICC data from MCF-7 cells (catalog image captions); its listed reactivity is human and rat (catalog).
Real IHC data
Anti-Ataxin 3/ATXN3 Antibody ®
Cat # PB9423
PB9423 will render with its own IHC figure showing staining of a human paraffin-embedded lung cancer section (PB9423 IHC image caption). The catalog also lists IF/ICC and shows ATXN3 staining in MCF-7 cells; human and rat are listed as reactive species (PB9423 applications, IF image caption, reactivity).
Which to pick: Choose PB9423 for paraffin-section IHC: its own caption documents citrate pH 6 retrieval and staining of human lung cancer tissue; the fixative is unreported (PB9423 IHC image caption). PB9423 is also the IF/ICC choice based on its listed applications and MCF-7 IF image, while rat is listed as reactive without a rat IHC or IF example in the supplied captions (PB9423 applications, reactivity, image captions). Clonality is unreported (PB9423 catalog payload).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P54252 (ATX3_HUMAN, Ataxin-3).
- Human Protein Atlas. ATXN3 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. ATXN3 subcellular location (ICC-IF): Mainly localized to the nucleoplasm and nucleoli. In addition localized to the plasma membrane..
- Human Protein Atlas. ATXN3 antibody validation summary (3 antibodies).
- ATXN3 functions as a tumor suppressor through potentiating galectin-9-mediated apoptosis in human colon adenocarcinoma. The Journal of biological chemistry 2024 — PMC11254720.
- Rad23b exacerbates pathological aggregates through disrupting proteasome functions in Spinocerebellar ataxia type 3. Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics 2026 — PMC13224077.
- Differential recruitment of UBQLN2 to nuclear inclusions in the polyglutamine diseases HD and SCA3. Neurobiology of disease 2015 — PMC4642276.
- Limited Effect of Chronic Valproic Acid Treatment in a Mouse Model of Machado-Joseph Disease. PloS one 2015 — PMC4624233.
- PubMed PMID:7874163 — UniProt-cited evidence.
- PubMed PMID:9274833 — UniProt-cited evidence.
- PubMed PMID:11450850 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ATXN3 (Ataxin-3). Expected localisation Cytoplasmic and nuclear in most tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet PB9423). Validated IHC antibody: PB9423 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative Liver. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded Human Lung Cancer tissue; fixative not specified (datasheet PB9423); 2. Fixation: Image fixative and duration unreported (datasheet PB9423); 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: Citrate pH 6 , 20 min (datasheet PB9423); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9423); 8. Primary antibody: Rabbit anti-ATXN3, 0.5-1μg/ml (datasheet PB9423); 9. Primary incubation: Overnight at 4 °C (datasheet PB9423). Watch for: Fixation: Keep formalin fixation consistent across sections (standard IHC practice; not target-specific); assess its effects empirically.; Caveat: Possible cross-reactivity with another gene product (HPA tissue IHC); Isoform / epitope: 5 isoforms; map the antibody epitope across them (UniProt); Regulation: Amino acid loss recruits ATXN3 to lysosomes (UniProt). Evidence: UniProt P54252, HPA tissue IHC · Uncertain, HPA ICC-IF · Mainly localized to the nucleoplasm and nucleoli. In addition…, Datasheet PB9423, PMC11254720, PMC4642276. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ATXN3/immunohistochemistry-ihc-guide.