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HASPIN / Serine/threonine-protein kinase haspin · IHC design guide
Design Immunohistochemistry for HASPIN
Plan HASPIN IHC-P using the nuclear and cytoplasmic tissue pattern (HPA tissue IHC). Testis Leydig cells provide a high-staining reference, but staining has low concordance with RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HASPIN 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 tissue staining (HPA tissue IHC) | |
| Staining pattern | Leydig cells stain strongly; several tissues show nuclear and cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Testis+4 more · see all | |
| Negative control | Duodenum+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Use formalin-fixed paraffin sections (selected-SKU IHC image A31862-1); keep fixation consistent. (standard IHC practice; not target-specific) | |
| Caveat | Staining has low concordance with RNA expression (HPA tissue IHC) | |
| Regulation | Detected in proliferating cell lines (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unspecified (UniProt) |
Section 1
Recommended HASPIN IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by one published ovarian tissue protocol (PMC7826093).
| Sample | FFPE human cancer tissue (datasheet A31862-1) |
| Fixation | Image formalin-fixed; duration unreported (datasheet A31862-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-HASPIN, 1:50-1:100 (datasheet A31862-1) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | HASPIN-positive staining in leydig cells of testis (HPA tissue IHC: High). HPA tissue profile: Nuclear and cytoplasmic expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min (page retrieval rule); compare the published citrate method (PMC7826093).
Section 2
What Is the Expected HASPIN Staining Pattern?
HASPIN is nuclear during interphase and associates with chromosomes and the spindle during mitosis (UniProt Q8TF76); HPA reports nuclear and cytoplasmic tissue staining, with high staining in testis Leydig cells (HPA: tissue IHC). It has no transmembrane segment (UniProt Q8TF76 topology). Treat the pattern as provisional: HPA rates tissue staining Approved but reports low agreement with RNA data and pending external verification (HPA: tissue IHC).
| Distinct nuclear staining, with some cytoplasmic staining, in testis Leydig cells. | This fits HPA's high Leydig-cell staining and its broader nuclear/cytoplasmic tissue profile (HPA: tissue IHC). Nuclear localization also fits interphase HASPIN (UniProt Q8TF76). Score the stained cell type and compartment separately; HPA's Approved rating carries a low RNA-agreement caveat (HPA: tissue IHC). |
| Staining appears concentrated on chromosomes or the spindle in identifiable mitotic cells. | This can fit HASPIN's reported mitotic localization (UniProt Q8TF76). Its role in histone H3 Thr-3 phosphorylation supports a mitotic context, but staining alone does not measure kinase activity or H3T3ph (UniProt Q8TF76; general IHC interpretation). Check the mitotic morphology before assigning this pattern (general IHC practice). |
| Signal outlines cell membranes or fills extracellular spaces without a convincing intracellular pattern. | Treat this as suspect: UniProt places HASPIN in the nucleus, chromosomes and spindle and reports no transmembrane segment (UniProt Q8TF76). Review morphology and controls before calling it HASPIN; deposited chromogen or nonspecific binding can create misleading outlines (general chromogenic IHC practice). |
| Strong staining dominates a cell type that HPA reports as not detected, such as duodenal glandular cells. | That result conflicts with the reported pattern for that specific cell type (HPA: duodenal glandular cells, Not detected). Check staining controls and cell identification for cross-reactivity or endogenous detection activity (general IHC practice). HPA's pending external verification means the discrepancy needs investigation, not an automatic biological ruling (HPA: tissue IHC). |
| Broad, diffuse staining obscures nuclei and tissue boundaries, or testis Leydig cells show no signal. | Diffuse staining is difficult to score by compartment and may reflect background (general IHC practice). Absent Leydig-cell signal conflicts with HPA's high staining report (HPA: testis Leydig cells, High); check section quality, retrieval and detection controls before treating the specimen as HASPIN-negative (general IHC practice). |
💡Expected HASPIN appearanceA convincing positive shows distinct intracellular nuclear staining, potentially with cytoplasmic signal, and high staining in testis Leydig cells (HPA: tissue IHC); isolated membrane rims or extracellular color are suspect given HASPIN's intracellular localization and lack of a transmembrane segment (UniProt Q8TF76).
| Cell-cycle state | Interphase nuclear localization and mitotic chromosome/spindle association can produce different intracellular appearances (UniProt Q8TF76); compare like cells and recognizable mitotic figures when scoring (general IHC practice). |
| Tissue and cell-type choice | HPA reports high staining in testis Leydig cells, but no detected staining in duodenal glandular cells (HPA: tissue IHC). UniProt reports strong testis expression (UniProt Q8TF76). HPA cautions that antibody staining has low agreement with RNA data (HPA: tissue IHC). |
| Isoforms and antibody recognition | UniProt lists two HASPIN isoforms and a kinase domain at residues 484–798 (UniProt Q8TF76). The supplied sources do not map the IHC antibody's epitope, so isoform recognition cannot be assigned from these records (HPA: antibody validation; UniProt Q8TF76). |
| Fixation and antigen retrieval | Target-specific fixation sensitivity and retrieval conditions are unreported in the supplied UniProt and HPA records. Retrieval can be evaluated as a general paraffin-IHC workflow variable; do not infer a HASPIN-specific effect from tissue staining or topology (general IHC practice; HPA: tissue IHC; UniProt Q8TF76). |
| IF/ICC Q: where should signal appear? | A: HPA supports nucleoplasmic localization in ICC-IF images from A-431 and U-251MG, with no additional location listed (HPA: subcellular). This is IF/ICC evidence; tissue IHC has a broader nuclear/cytoplasmic profile (HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Leydig cells lack visible chromogen in a testis section. | The result conflicts with HPA's high Leydig-cell staining; the supplied sources do not identify a HASPIN-specific fixation cause (HPA: tissue IHC). | Confirm Leydig-cell identification and section quality, then check positive-control staining and the established paraffin-IHC retrieval and detection steps (general IHC practice). |
| Every compartment appears diffusely colored. | Background can hide the nuclear/cytoplasmic pattern HPA reports (HPA: tissue IHC; general IHC practice). | Inspect the no-primary control; review blocking, antibody dilution, washes and chromogen development as general chromogenic-IHC variables (general IHC practice). |
| Signal appears mainly at membranes or outside cells. | This distribution conflicts with HASPIN's intracellular locations and absent transmembrane segment (UniProt Q8TF76). | Compare with a no-primary control, inspect tissue edges and deposits, and score only signal tied to identifiable cells and compartments (general IHC practice). |
| Unexpected cell types stain strongly while the intended pattern is weak. | Cross-reactivity or endogenous detection activity is possible (general IHC practice); HPA also reports low antibody-staining/RNA agreement (HPA: tissue IHC). | Identify the stained cells, compare HPA's cell-type entries, and check no-primary and detection controls before assigning biological expression (HPA: tissue IHC; general IHC practice). |
| Nuclear signal seems absent, but cytoplasmic signal remains. | HPA allows cytoplasmic tissue staining, while UniProt places HASPIN in interphase nuclei; one compartment alone does not settle specificity (HPA: tissue IHC; UniProt Q8TF76). | Inspect nuclear counterstain and cell morphology; compare a documented positive cell type and control sections under the same scoring rules (general IHC practice). |
| A mitotic figure shows little obvious chromosome-associated signal. | UniProt reports chromosome and spindle association during mitosis, but the supplied sources do not establish how often an IHC section should capture a detectable mitotic pattern (UniProt Q8TF76). | Confirm that the figure is mitotic and technically interpretable; use the broader cellular and tissue pattern when assessing the section (general IHC practice; HPA: tissue IHC). |
Experiment evidence
IHC & IF images showing HASPIN staining
A31862-1 · IHCFormalin-fixed and paraffin-embedded human cancer tissue reacted with the primary antibody, which was peroxidase-conjugated to the secondary
A31862-2 · IFIF analysis of GSG2/HASPIN using anti-GSG2/HASPIN antibody (A31862-2). GSG2/HASPIN was detected in an immunocytochemical section of MG63 cel
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000177602-HASPIN/tissue/cerebral+cortex#imid_7634496
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000177602-HASPIN/tissue/colon#imid_7634430
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000177602-HASPIN/tissue/liver#imid_7634423
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000177602-HASPIN/tissue/kidney#imid_7634432
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA030698; green = HASPIN, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA030698; green = HASPIN, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HASPIN IHC & IF
🧪Run testis first and require staining in Leydig cells (HPA: High in testis Leydig cells). Use duodenum glandular cells as the negative tissue (HPA: Not detected in duodenum glandular cells); compare other cells on the testis slide with the no-primary control for background, without assuming they are HASPIN-negative.
Positive control tissue: Testis (Leydig cells, HPA High)
Negative control tissue: Duodenum (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show HASPIN in A-431, U-251MG, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host-species- and immunoglobulin-class-matched isotype controls appropriate to the catalog antibody, plus HASPIN knockout tissue or a peptide-block control if a suitable immunizing peptide is available. For chromogenic testis IHC, block endogenous peroxidase and inspect the control slide for residual staining (standard IHC practice).
⚠️Feasibility: The selected-SKU caption explicitly describes formalin-fixed, paraffin-embedded tissue and peroxidase/AEC detection (A31862-1 tissue-IHC caption), but no matched source reports a HASPIN-specific fixation window or fixation effect. Antigen-retrieval dependence and any advantage of frozen sections or IF are unreported in the supplied evidence; assess retrieval conditions empirically. Testis tissue should be scored by cell type because the HPA positive call is specifically for Leydig cells (HPA: High in testis Leydig cells); no testis-specific staining artefact is established by the supplied evidence.
HPA tissue IHC evidence for HASPIN
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data. 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 |
|---|---|---|---|---|
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Adipocytes | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Duodenum | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
| Pancreas | Exocrine glandular cells | Not detected | Protein (IHC) | HPA → |
| Small intestine | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced HASPIN IHC Tips
Troubleshoot HASPIN staining in paraffin sections by checking retrieval, compartment, cell type, and controls before interpreting chromogenic signal.
How should I retrieve HASPIN in paraffin sections when nuclear staining is weak?
Use Tris-EDTA pH 9.0 heat-induced retrieval at 95–98 °C for 20 min (page retrieval setting). If nuclear signal remains weak, compare a longer heating interval on adjacent sections while keeping the antibody concentration and detection conditions constant (standard FFPE IHC practice). Check whether tissue morphology and nuclear counterstaining remain intact, because excessive heating can make chromogenic signal difficult to assign to individual nuclei (standard FFPE IHC practice). Evaluate both interphase nuclei and mitotic figures: HASPIN is nuclear during interphase and associates with chromosomes and the spindle during mitosis (UniProt Q8TF76 localisation).
Could fixation explain weak or patchy HASPIN staining in my paraffin sections?
HASPIN-specific sensitivity to fixation is unknown from the supplied evidence, so treat fixation as a variable to test rather than an established cause of signal loss (supplied evidence). Compare sections with documented fixation and processing histories using the same retrieval, antibody incubation, and chromogen development conditions (standard FFPE IHC practice). Include a tissue control with an expected HASPIN-positive cell population, such as testis Leydig cells, on each staining run (HPA: High in testis Leydig cells). If only poorly preserved regions stain weakly, inspect morphology and processing records before changing the antibody dilution; that comparison cannot establish a HASPIN-specific fixation effect (standard FFPE IHC practice).
Should HASPIN stain nuclei, cytoplasm, or mitotic structures in chromogenic IHC?
Expect predominantly nuclear staining in interphase cells, with chromosome-associated signal possible in mitotic cells (UniProt Q8TF76 localisation; HPA subcellular: supported nucleoplasm). HASPIN can also associate with the spindle during mitosis, so assess apparent extranuclear staining against the cell’s mitotic morphology and counterstain (UniProt Q8TF76 localisation). HPA reports nuclear and cytoplasmic staining across several tissues, but describes low consistency between antibody staining and RNA expression data (HPA tissue IHC: profile and reliability description). Score nuclear and cytoplasmic compartments separately, and compare each with the no-primary control before treating diffuse cytoplasmic chromogen as specific signal (standard IHC control practice).
How can epitope uncertainty affect interpretation of HASPIN staining across cell populations?
HASPIN has 2 annotated isoforms, but the supplied evidence does not identify the catalog antibody’s epitope or establish which isoforms it detects (UniProt Q8TF76 isoforms; supplied evidence). Its kinase domain occupies residues 484–798, while listed phosphorylation sites include residues 58, 93, 97, 143, and 147 (UniProt Q8TF76 domains and modified residues). Do not attribute a difference in chromogenic staining to an isoform or phosphorylation state without epitope mapping or orthogonal validation (standard antibody validation practice). Compare the same cellular compartment across sections processed together, and document the antibody lot and retrieval conditions when assessing reproducibility (standard IHC practice).
How should I adapt HASPIN localisation checks for multiplex IF on adjacent material?
For an IF comparison, pair HASPIN with a marker that identifies the cell population being evaluated, and retain a nuclear counterstain to assess compartment assignment (standard multiplex IF practice). Choose fluorophores in channels with low background for the specimen, checking unstained tissue for autofluorescence before assigning a dim HASPIN signal (standard IF practice). HASPIN has no transmembrane segment and is reported in the nucleoplasm, chromosomes, and mitotic spindle; permeabilise cells sufficiently to access these intracellular epitopes (UniProt Q8TF76 topology and localisation; standard IF practice). Titrate permeabilisation against preserved nuclear and mitotic morphology, and use single-stain controls to check channel bleed-through (standard IF practice).
What should I check when HASPIN chromogen appears throughout the section?
Run no-primary and detection-only controls to identify signal from the detection system before changing the HASPIN antibody concentration (standard chromogenic IHC practice). Apply a peroxidase block when using enzyme-based detection, and compare any remaining deposit with tissue edges, folds, and damaged areas (standard chromogenic IHC practice). The selected antibody’s documented tissue image used a peroxidase-conjugated secondary antibody and AEC staining on formalin-fixed paraffin-embedded human cancer tissue (caption: SKU A31862-1). If diffuse cytoplasmic color persists, compare it with expected nuclear or mitotic localisation and with the HPA-reported cytoplasmic pattern, whose RNA agreement is low (UniProt Q8TF76 localisation; HPA tissue IHC: profile and reliability description).
How should I quantify HASPIN staining when only some cells are positive? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and score nuclear and cytoplasmic staining separately before viewing the experimental groups (standard IHC scoring practice; UniProt Q8TF76 localisation). Record the percentage of positive cells and staining intensity, or calculate an H-score from intensity categories and their corresponding cell percentages (standard IHC scoring practice). For sparse mitotic signal, report positive mitotic figures per mm² of evaluable tissue or per counted mitotic figures, stating the denominator (standard histologic quantification practice). Normalise comparisons to the same eligible cell type and tissue area, and exclude folds, necrosis, and sections with failed controls from the scored region (standard IHC scoring practice).
How can I distinguish true HASPIN staining from an artefact in a paraffin section?
Give greatest weight to signal in the expected compartment and a plausible cell population, such as nuclear staining in testis Leydig cells or chromosome-associated staining in mitotic cells (HPA: High in testis Leydig cells; UniProt Q8TF76 localisation). Treat strong staining restricted to section edges, folds, or necrotic areas as suspect until it persists in well-preserved tissue (standard IHC interpretation practice). Check no-primary and peroxidase-block controls when color appears broadly outside cells or follows tissue structures without matching cell morphology (standard chromogenic IHC practice). Interpret isolated cytoplasmic staining cautiously because HPA reports cytoplasmic signal alongside low consistency between staining and RNA data (HPA tissue IHC: profile and reliability description).
Boster reagents
Best HASPIN / Serine/threonine-protein kinase haspin IHC Antibodies
Anti-HASPIN antibodies have IHC data from formalin-fixed, paraffin-embedded human cancer tissue and IF data from MG63 cells (catalog images). Listed reactivity spans human, mouse, and rat (catalog reactivity).
Real IHC data
Anti-GSG2 Antibody (Center)
Cat # A31862-1
Real IF data
Anti-GSG2/HASPIN Antibody ®
Cat # A31862-2
A31862-1 is listed for human IHC-P and has an image of stained breast carcinoma and hepatocarcinoma sections (catalog applications; catalog IHC image). A31862-2 is listed for IF/ICC and has an image of stained MG63 cells (catalog applications; catalog IF image).
Which to pick: Choose A31862-1 for tissue IHC: it is a rabbit polyclonal antibody listed for IHC-P at 1:50–1:100, with an image from formalin-fixed, paraffin-embedded human cancer tissue (catalog host; catalog dilution; catalog IHC image). Choose A31862-2 for IF/ICC: its MG63 cell image documents 5 μg/mL, while its clonality is unreported (catalog IF image; catalog clone). For mouse or rat work, A31862-2 is the only listed reactive option; the supplied IF image shows MG63 cells (catalog reactivity; catalog IF image).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8TF76 (HASP_HUMAN, Serine/threonine-protein kinase haspin).
- Human Protein Atlas. HASPIN tissue IHC expression (reliability: Approved).
- Human Protein Atlas. HASPIN subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. HASPIN antibody validation summary (2 antibodies).
- GSG2 (Haspin) promotes development and progression of bladder cancer through targeting KIF15 (Kinase-12). Aging 2020 — PMC7288960.
- GSG2 Promotes Development and Predicts Poor Prognosis of Ovarian Cancer. Cancer management and research 2021 — PMC7826093.
- PubMed PMID:11228240 — UniProt-cited evidence.
- PubMed PMID:11311556 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HASPIN (Serine/threonine-protein kinase haspin). Expected localisation Nuclear and cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A31862-1 at 1:50-1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Leydig cells); negative Duodenum. IHC-P workflow on FFPE sections. Protocol steps: 1. Sample: FFPE human cancer tissue (datasheet A31862-1); 2. Fixation: Image formalin-fixed; duration unreported (datasheet A31862-1); 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-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-HASPIN, 1:50-1:100 (datasheet A31862-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Use formalin-fixed paraffin sections (selected-SKU IHC image A31862-1); keep fixation consistent. (standard IHC practice; not target-specific); Caveat: Staining has low concordance with RNA expression (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unspecified (UniProt); Regulation: Detected in proliferating cell lines (UniProt). Evidence: UniProt Q8TF76, HPA tissue IHC · Approved, HPA ICC-IF · nucleoplasm., Datasheet A31862-1, PMC7826093. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HASPIN/immunohistochemistry-ihc-guide.