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NASP / Nuclear autoantigenic sperm protein · IHC design guide
Design Immunohistochemistry for NASP
Plan chromogenic NASP IHC in paraffin sections using the observed nuclear tissue pattern as a guide (HPA tissue IHC). Compare proliferating cell populations and include an adipocyte-rich area as a low-signal reference (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
NASP 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 staining in most tissues (HPA tissue IHC); cytoplasmic location also annotated (UniProt) | |
| Staining pattern | Nuclear staining, strongest in proliferating cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A05383-1) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A05383-1) | |
| Caveat | Adipocytes may lack signal despite broad tissue staining (HPA tissue IHC) | |
| Regulation | Most abundant in proliferating cells (HPA tissue IHC) | |
| Isoform / epitope | Four isoforms; isoform 1 is testis/sperm-specific; epitope impact unknown (UniProt) |
Section 1
Recommended NASP IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A05383-1) sits alongside three published NASP IHC protocols (PMC5294820; PMC11009454; PMC8524314).
| Sample | Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A05383-1) |
| Fixation | Image fixative and duration unreported (datasheet A05383-1); 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 A05383-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A05383-1) |
| Primary antibody | Rabbit anti-NASP, 2-5μg/ml (datasheet A05383-1) |
| Primary incubation | Overnight at 4 °C (datasheet A05383-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A05383-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | NASP-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Nuclear expression in most tissues, most abundant in proliferating cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet: A05383-1); the cited citrate protocols used other antibodies (PMC5294820; PMC11009454).
Section 2
What Is the Expected NASP Staining Pattern?
NASP should appear chiefly in nuclei across many tissues, with stronger staining in proliferating cells (HPA tissue IHC: Enhanced reliability; medium consistency with RNA). High staining is reported in colon glandular cells and esophageal squamous epithelial cells (HPA tissue IHC). UniProt also places NASP in the cytoplasm and reports no transmembrane segment (UniProt P49321: subcellular location; topology).
| Distinct nuclear staining in colon glandular cells, with nearby structures remaining interpretable. | This matches a reported high signal in colon glandular cells (HPA tissue IHC). Compare nuclei within the same section; HPA describes nuclear expression in most tissues, especially proliferating cells (HPA tissue IHC). |
| Strong membrane outlines or predominantly cytoplasmic staining, with little nuclear signal. | A membrane-dominant result conflicts with the absence of a transmembrane segment (UniProt P49321: topology). Predominantly cytoplasmic IHC conflicts with the reported nuclear tissue pattern, although UniProt also lists cytoplasm (HPA tissue IHC; UniProt P49321: subcellular location). Check specificity before scoring it as NASP. |
| Prominent chromogenic staining in adipocytes while expected positive cells remain faint. | Adipocytes are reported as not detected (HPA tissue IHC: adipose tissue). This mismatch raises concern for cross-reactivity or endogenous detection activity; the stain alone cannot distinguish those causes. |
| Diffuse color across tissue and spaces between cells, obscuring nuclear boundaries. | Treat this as background until control sections clarify it. General IHC causes include excess antibody or detection reagent and incomplete blocking or washing; diffuse color does not establish NASP localization. |
| No nuclear staining in an otherwise intact colon glandular region. | That conflicts with the reported high glandular signal (HPA tissue IHC: colon). Review run controls and section quality before interpreting the specimen as NASP negative; HPA tissue observations do not guarantee every section will stain. |
💡Expected NASP appearanceCall a positive result when nuclei of expected cells are clearly marked, such as high signal in colon glandular cells (HPA tissue IHC: colon); dominant membrane staining or color spread across cell-free spaces should prompt specificity or background checks (UniProt P49321: topology; general IHC practice).
| Tissue and cell population | HPA reports high staining in appendix, colon, duodenum and fallopian tube glandular cells, and in esophageal and oral squamous epithelium (HPA tissue IHC). Select a documented positive cell population for comparison. |
| Proliferation context | The atlas describes nuclear expression in most tissues, most abundant in proliferating cells (HPA tissue IHC). Interpret weaker areas against the cell population present, rather than assigning one intensity to the whole organ. |
| Isoform scope | UniProt lists 4 isoforms and describes isoform 1 as testis- and sperm-specific (UniProt P49321: isoforms; tissue specificity). Without an antibody epitope, these data cannot establish which isoforms the assay detects. |
| Antibody validation | HPA lists IHC as Enhanced for HPA028136, HPA030518 and HPA030520 (HPA antibodies). That supports the reported pattern; HPA still describes only medium consistency between antibody staining and RNA expression (HPA tissue IHC). |
| Chromogenic detection | Endogenous enzyme activity or nonspecific detection can produce color independently of the primary antibody (general IHC practice). A primary-antibody omission control helps assess detection background; it does not prove that every remaining signal is specific. |
| Situation | Likely cause | Next action |
|---|---|---|
| The positive-control section has no nuclear signal. | The run may have failed, or the selected field may lack the documented positive cell population; colon glandular cells are reported high (HPA tissue IHC: colon). | Confirm the glandular region and section integrity, then check reagent activity, antigen retrieval and antibody conditions using the assay's validated IHC protocol (general IHC practice). No NASP-specific retrieval requirement is supplied. |
| Color is strongest at membranes or throughout cytoplasm. | A membrane-dominant pattern conflicts with NASP topology, while a cytoplasm-dominant tissue pattern differs from HPA's nuclear profile (UniProt P49321: topology; HPA tissue IHC). | Compare nuclear and extranuclear signal in a documented positive tissue, inspect the omission control, and confirm the antibody's IHC validation. Do not infer that all cytoplasmic signal is artefactual: UniProt also lists cytoplasm (UniProt P49321: subcellular location). |
| Adipocytes stain conspicuously. | HPA reports NASP as not detected in adipocytes (HPA tissue IHC: adipose tissue); cross-reactivity or endogenous detection activity is possible. | Check a primary-antibody omission control and detection blocking, then compare with a documented positive tissue. Treat adipocyte color as unresolved unless controls support specificity (general IHC practice). |
| Diffuse color makes nuclear scoring unreliable. | Excess reagent, incomplete blocking or insufficient washing can raise chromogenic background (general IHC practice). | Review omission-control staining and the assay's blocking, dilution and wash conditions. Adjust one condition at a time, then assess whether distinct nuclei reappear (general IHC practice). |
| Different tissue regions have different staining intensities. | HPA reports a broad nuclear pattern enriched in proliferating cells and different levels across cell populations (HPA tissue IHC). | Score identified cell populations separately and compare like regions across sections. Preserve the distinction between weak staining and no detectable staining (general IHC practice). |
| IF/ICC images show nucleoplasmic signal plus puncta. | HPA reports nucleoplasm as the enhanced main location and vesicles as an additional location (HPA subcellular ICC-IF). | Interpret IF/ICC on its separate guide page; compare nuclear and punctate channels with appropriate controls. The ICC-IF pattern is contextual evidence, not an IHC-P protocol option (HPA subcellular ICC-IF). |
Experiment evidence
IHC & IF images showing NASP staining
A05383-1 · IHCIHC analysis of NASP using anti-NASP antibody (A05383-1). NASP was detected in a paraffin-embedded section of human liver cancer tissue. Hea
A05383-1 · IHCIHC analysis of NASP using anti-NASP antibody (A05383-1). NASP was detected in a paraffin-embedded section of human stomach cancer tissue. H
A05383-1 · IFIF analysis of NASP using anti-NASP antibody (A05383-1) and anti-Tubulin Alpha antibody (M03989-3). NASP was detected in immunocytochemical
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000132780-NASP/tissue/cerebral+cortex#imid_13831489
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000132780-NASP/tissue/colon#imid_13831423
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000132780-NASP/tissue/liver#imid_13831424
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000132780-NASP/tissue/kidney#imid_13831409
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA028136; green = NASP, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA028136; green = NASP, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for NASP IHC & IF
🧪Run colon first: glandular cells should stain (HPA: High in colon glandular cells). Use adipose tissue as the negative tissue; adipocytes should lack specific staining (HPA: Not detected in adipocytes), while cells outside the glands on the colon slide should be assessed for background rather than assumed to be NASP-negative (HPA: colon result identifies glandular cells only).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show NASP in A-431, U-251MG, U2OS, HEK293, KOLF2.1J, U2OS, siRNA 1 (10x), U2OS, siRNA 1 (40x), U2OS, scrambled (10x), U2OS, scrambled (40x), NIH 3T3, with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and an isotype control using rabbit IgG matched to the primary antibody’s clonality once confirmed (selected A05383-1 caption: rabbit primary and HRP-conjugated goat anti-rabbit secondary). Use NASP knockout material as a biological specificity control, and block endogenous peroxidase before DAB detection in the positive tissue (selected A05383-1 caption: HRP/DAB; standard IHC practice).
⚠️Feasibility: The selected A05383-1 paraffin-section caption does not report a fixative, and the supplied evidence reports no NASP-specific fixation window or fixation effect (selected A05383-1 caption). That caption documents heat-mediated EDTA retrieval at pH 8.0 and a 2 μg/ml primary incubation overnight at 4°C; whether retrieval is essential for NASP is unreported (selected A05383-1 caption). Frozen-section ease is unreported, while IF/ICC images show nucleoplasmic localisation without establishing an easier workflow (HPA: subcellular); assess colon glandular staining against luminal background (standard IHC practice).
HPA tissue IHC evidence for NASP
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Medium | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced NASP IHC Tips
Use the catalog antibody’s paraffin-section evidence alongside NASP localisation and tissue patterns when troubleshooting chromogenic IHC.
Which retrieval conditions should I start with for NASP paraffin-section IHC?
Start with heat-mediated retrieval in EDTA pH 8.0 for paraffin sections (datasheet A05383-1). This condition produced staining in a human liver cancer section using 2 μg/ml primary antibody overnight at 4°C (datasheet A05383-1). If nuclear staining is weak, compare retrieval heating and cooling consistency across sections before changing conditions (standard IHC practice). If staining remains weak, test an alternative buffer or pH only as a controlled fallback, with the same antibody concentration and detection steps (standard IHC practice). Judge any improvement by nuclear signal in viable cells and by background in a no-primary control (HPA: nuclear expression in most tissues; standard IHC practice).
How should I troubleshoot fixation-related variation in NASP IHC?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (datasheet A05383-1). Record each block’s fixative and fixation duration, then compare sections processed with the same EDTA pH 8.0 retrieval and detection conditions (standard IHC practice; datasheet A05383-1). Keep the reported 2 μg/ml primary incubation overnight at 4°C as a reference while assessing fixation variation (datasheet A05383-1). Evaluate nuclear signal in viable cells alongside tissue morphology, since damaged morphology makes compartment scoring unreliable (HPA: nuclear expression in most tissues; standard IHC practice). Do not assign a NASP-specific fixation effect without a direct comparison of fixation conditions (standard IHC practice).
What staining compartment should I expect, and how should I assess cytoplasmic signal?
Expect predominantly nuclear staining in tissue IHC, particularly among proliferating cells (HPA: nuclear expression in most tissues, most abundant in proliferating cells). Nucleoplasmic localisation is supported by cellular imaging, while both nucleus and cytoplasm are annotated for NASP (HPA: nucleoplasm, enhanced; UniProt P49321: subcellular location). Score nuclear and cytoplasmic staining separately rather than merging them into one positive fraction (standard IHC practice). A cytoplasmic component can be biologically plausible because NASP participates in a cytoplasmic HSP90–linker-histone complex (UniProt P49321: function). If staining is exclusively diffuse cytoplasmic, compare it with nuclear-positive tissue and a no-primary control before interpreting it as NASP (HPA: tissue profile; standard IHC practice).
Can this IHC assay distinguish NASP isoforms or epitope masking?
NASP has 4 annotated isoforms, and isoform 1 is testis- and sperm-specific (UniProt P49321: isoforms and tissue specificity). The supplied caption establishes paraffin-section staining for A05383-1 but does not identify its epitope or demonstrate isoform discrimination (datasheet A05383-1). Therefore, report positive staining as NASP immunoreactivity rather than assigning it to one isoform (UniProt P49321: isoforms; standard IHC interpretation). NASP has annotated acetylation and phosphorylation sites, but their effects on this antibody’s tissue staining are unreported (UniProt P49321: modified residues; datasheet A05383-1). To investigate possible epitope masking, compare retrieval conditions on adjacent sections while keeping antibody concentration, development and scoring consistent (standard IHC practice).
How should I relate multiplex IF findings to this chromogenic NASP IHC assay?
Use IF as a separate readout: nucleoplasmic localisation has cellular imaging support, while the selected antibody caption documents chromogenic paraffin-section IHC (HPA: subcellular location; datasheet A05383-1). For a 2-color IF comparison, pair NASP with a validated marker of the cell population being assessed, such as the glandular cells highlighted in colon tissue data (HPA: high in colon glandular cells; standard IF practice). Choose fluorophores after checking tissue autofluorescence and include single-channel controls to assess spectral bleed-through (standard IF practice). Because NASP has no transmembrane segment and has nuclear and cytoplasmic locations, assess permeabilisation for access to intracellular epitopes (UniProt P49321: topology and subcellular location; standard IF practice). Score IF compartments separately before comparing them with the chromogenic nuclear pattern (HPA: tissue profile; standard IHC/IF practice).
What should I check when NASP DAB staining appears widespread?
First compare stained sections with a no-primary control, especially where DAB appears outside viable cell nuclei (standard IHC practice; HPA: nuclear tissue profile). The reported workflow used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and HRP/DAB detection (datasheet A05383-1). Block endogenous peroxidase before HRP detection, and keep DAB development comparable across sections (standard chromogenic IHC practice). If background persists, reassess blocking, washing and primary concentration while changing one condition at a time (standard IHC practice). Treat edge staining, necrotic areas and staining reproduced without primary antibody as potential artefacts rather than evidence of broad NASP expression (standard IHC practice).
How should I quantify NASP staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue compartment and viable cell population before scoring, because NASP staining is predominantly nuclear and enriched in proliferating cells (HPA: tissue profile). Report the percentage of positive nuclei and, where intensity is reproducible, an H-score from 0–300 using intensity grades 0–3 (standard IHC scoring practice). Alternatively, report positive nuclei per mm², normalised to viable tissue area, and include total viable nuclei when cell density differs (standard image-analysis practice). Hold retrieval, antibody incubation, DAB development, imaging and positivity thresholds constant across the comparison (standard IHC practice). Analyse nuclear and cytoplasmic signal separately, and document excluded folds, edges and necrosis so that artefacts do not shift the score (UniProt P49321: subcellular location; standard IHC practice).
How can I distinguish convincing NASP positivity from tissue artefacts?
Convincing staining should include viable-cell nuclear signal consistent with the tissue profile, particularly in proliferating cell populations (HPA: nuclear expression in most tissues, most abundant in proliferating cells). High staining in colon glandular cells provides a documented tissue comparison, whereas adipocytes are reported as undetected (HPA: colon glandular cells high; adipose adipocytes not detected). Examine unexpected exclusively cytoplasmic staining separately, since NASP also has a cytoplasmic annotation but tissue IHC is mainly nuclear (UniProt P49321: subcellular location; HPA: tissue profile). Discount edge effects, necrotic regions and deposits that do not follow intact cell morphology (standard IHC practice). A no-primary control helps identify endogenous peroxidase or nonspecific DAB signal before assigning an unusual pattern to NASP (standard chromogenic IHC practice).
Boster reagents
Best NASP / Nuclear autoantigenic sperm protein IHC Antibodies
A05383-1 is an IHC/IF-tested anti-NASP antibody with images from human cancer sections, mouse testis sections, and U2OS cells (catalog applications and image captions).
Real IHC data
Anti-NASP antibody ®
Cat # A05383-1
The A05383-1 card shows IHC in paraffin-embedded human liver cancer, human stomach cancer, and mouse testis sections (A05383-1 IHC image captions). Its IF images show U2OS cells and paraffin-embedded human stomach cancer and mouse testis sections (A05383-1 IF image captions).
Which to pick: For tissue IHC, choose A05383-1 at 2–5 μg/ml; its IHC captions document heat retrieval in EDTA at pH 8.0 (catalog dilution; A05383-1 IHC image captions). For IF/ICC, the same SKU lists 5 μg/ml and has IF images; clonality is unreported (catalog applications and dilution; A05383-1 IF image captions; catalog clone field). For cross-species work, A05383-1 lists Human, Mouse, and Rat reactivity, while its IHC images cover human and mouse only; the paraffin-section captions do not report a fixative (catalog reactivity; A05383-1 IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P49321 (NASP_HUMAN, Nuclear autoantigenic sperm protein).
- Human Protein Atlas. NASP tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. NASP subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. NASP antibody validation summary (3 antibodies).
- microRNA-29c inhibits cell proliferation by targeting NASP in human gastric cancer. BMC cancer 2017 — PMC5294820.
- NASP Promotes Triple-negative Breast Cancer Progression and Metastasis by Stabilizing YAP in a USP15-Dependent Way. International journal of biological sciences 2025 — PMC12223775.
- Nuclear autoantigenic sperm protein facilitates glioblastoma progression and radioresistance by regulating the ANXA2/STAT3 axis. CNS neuroscience & therapeutics 2024 — PMC11009454.
- Knockdown of long noncoding RNA colorectal neoplasia differentially expressed inhibits hepatocellular carcinoma progression by mediating the expression of nuclear autoantigenic sperm protein. Oncology reports 2021 — PMC8524314.
- PubMed PMID:1426632 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16710414 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for NASP (Nuclear autoantigenic sperm protein). Expected localisation Nuclear staining in most tissues (HPA tissue IHC); cytoplasmic location also annotated (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A05383-1). Validated IHC antibody: A05383-1 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A05383-1); 2. Fixation: Image fixative and duration unreported (datasheet A05383-1); 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 A05383-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A05383-1); 8. Primary antibody: Rabbit anti-NASP, 2-5μg/ml (datasheet A05383-1); 9. Primary incubation: Overnight at 4 °C (datasheet A05383-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A05383-1); Caveat: Adipocytes may lack signal despite broad tissue staining (HPA tissue IHC); Isoform / epitope: Four isoforms; isoform 1 is testis/sperm-specific; epitope impact unknown (UniProt); Regulation: Most abundant in proliferating cells (HPA tissue IHC). Evidence: UniProt P49321, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoplasm., Datasheet A05383-1, PMC5294820, PMC11009454. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/NASP/immunohistochemistry-ihc-guide.