This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
MAD1L1 / Mitotic spindle assembly checkpoint protein MAD1 · IHC design guide
Design Immunohistochemistry for MAD1L1
Plan paraffin IHC around the nuclear and nuclear membranous staining seen in most tissues (HPA tissue IHC). Start with the catalog antibody's 0.5–1 μg/mL IHC range (datasheet: PB9262) and account for MAD1L1 redistribution during mitosis (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
MAD1L1 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 nuclear membranous in most tissues (HPA tissue IHC) | |
| Staining pattern | Most tissues show nuclear and nuclear membrane staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet PB9262) | |
| Positive control | Cerebellum+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
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 | Mitotic stage can shift MAD1L1 from its nuclear pattern (UniProt) | |
| Regulation | Intensity varies by tissue and cell type (HPA tissue IHC) | |
| Isoform / epitope | 3 isoforms; epitope coverage is unknown (UniProt) |
Section 1
Recommended MAD1L1 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet: PB9262) with one published paraffin-section protocol for MAD1L1 (PMC13163004).
| Sample | Paraffin-embedded mouse intestine tissues; fixative not specified (datasheet PB9262) |
| Fixation | Image fixative and duration unreported (datasheet PB9262); 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 PB9262) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9262) |
| Primary antibody | Rabbit anti-MAD1L1, 0.5-1μg/ml (datasheet PB9262) |
| Primary incubation | Overnight at 4 °C (datasheet PB9262) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9262) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | MAD1L1-positive staining in cells in molecular layer of cerebellum (HPA tissue IHC: High). HPA tissue profile: Nuclear and nuclear membranous expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 heat retrieval (datasheet: PB9262); try the published EDTA pH 9 method if staining needs optimization (PMC13163004).
Section 2
What Is the Expected MAD1L1 Staining Pattern?
MAD1L1 should appear mainly in nuclei and along nuclear membranes across many tissues (HPA: tissue IHC, Supported; medium consistency with RNA). In paraffin sections, assess the named cell populations: glandular cells in duodenum or endometrium, glial cells in hippocampus, or cells in kidney glomeruli show high staining (HPA: tissue IHC). MAD1L1 has no annotated transmembrane segment (UniProt Q9Y6D9: topology).
| Nuclear and nuclear-rim staining in the expected cells, with other compartments less prominent. | This matches the predominant tissue pattern (HPA: tissue IHC). Judge the signal within the named cell population, since HPA reports low tissue specificity and does not imply equal intensity in every cell. |
| Predominantly extracellular, luminal, or uniform cell-surface staining with little nuclear signal. | Treat this as a questionable pattern: HPA reports nuclear and nuclear-membranous tissue staining (HPA: tissue IHC), and UniProt annotates no transmembrane segment (UniProt Q9Y6D9: topology). Check morphology and controls before assigning positivity. |
| Strong staining in cells outside the expected population while the expected cells remain unstained. | Consider cross-reactivity or endogenous detection activity as general IHC possibilities. Compare cells within the same section: HPA names specific high-staining populations, but its low tissue specificity means other positive cells cannot be dismissed solely by identity (HPA: tissue IHC). |
| Weak, diffuse colour across cells and tissue spaces that obscures nuclear boundaries. | This cannot establish the reported MAD1L1 pattern (HPA: tissue IHC). In general IHC practice, nonspecific binding, inadequate blocking, or detection background can reduce contrast; assess a matched negative detection control. |
| No detectable signal in a section containing an expected high-staining population. | This is inconclusive for absence of MAD1L1: HPA reports high staining in several named populations, including duodenal glandular cells and glomerular cells (HPA: tissue IHC). Check that the population is present, then review the IHC workflow and controls. |
💡Expected MAD1L1 appearanceCall a convincing positive result when identifiable nuclei and nuclear rims stain in an HPA high-staining cell population, such as duodenal glandular cells, while diffuse extracellular colour alone is not counted (HPA: tissue IHC).
| Tissue and cell selection | HPA reports high staining in duodenal, endometrial, epididymal, fallopian-tube, and gallbladder glandular cells; cerebellar molecular-layer cells; hippocampal glia; and kidney glomerular cells (HPA: tissue IHC). Its listed low-staining populations are less useful for judging a weak result. |
| Compartment and cell cycle | The dominant tissue readout is nuclear and nuclear-membranous (HPA: tissue IHC). UniProt also annotates kinetochores in prometaphase, centrosomes, spindle structures, and the midbody during mitosis (UniProt Q9Y6D9: subcellular location). Those transient patterns should be interpreted with cell-cycle morphology, not demanded of every cell. |
| Evidence strength | HPA rates tissue IHC Supported with medium consistency between staining and RNA, and lists two antibodies with Supported IHC status (HPA: tissue IHC; HPA: antibodies). This supports a reference pattern, not a guarantee that every antibody, preparation, or section reproduces it. |
| Molecular form and epitope | UniProt lists three isoforms, one annotated chain spanning residues 1–718, and no signal peptide, propeptide, or transmembrane segment (UniProt Q9Y6D9: isoforms, processing, topology). The payload gives no antibody epitope, so isoform coverage and any epitope-dependent staining difference remain unknown. |
| IF/ICC Q: What localisation should a separate IF assay check? | A: Nucleoplasm and nuclear membrane are supported IF locations, with U-251MG listed among imaged cell lines (HPA: subcellular ICC-IF). This is a localisation comparison, not an IHC-P staining score or an IF protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected high-staining cells are present, but chromogenic signal is absent. | The result conflicts with the HPA reference pattern for those cells (HPA: tissue IHC); the cause cannot be assigned from the supplied evidence. | Verify tissue identity and cell morphology, then check the catalog antibody's IHC-P instructions, detection reagents, and a positive control. Review antigen retrieval as general IHC practice; target-specific fixation sensitivity is unreported. |
| Signal is mainly cytoplasmic or extracellular in interphase-appearing cells. | The dominant tissue compartment is nuclear and nuclear-membranous (HPA: tissue IHC). UniProt's additional spindle and centrosome locations vary with mitosis (UniProt Q9Y6D9: subcellular location). | Recheck nuclear boundaries and mitotic morphology. Compare the same run with an HPA high-staining cell population and a matched negative detection control before calling the atypical signal specific. |
| Unexpected cell types stain strongly while expected cells do not. | Cross-reactivity or endogenous detection activity is possible in general IHC practice; HPA's cell-specific high-staining list does not establish that all other cells are negative (HPA: tissue IHC). | Check whether the expected population is actually present. Run appropriate negative detection controls and compare compartment, intensity, and cell identity across the section. |
| Diffuse colour obscures a possible nuclear-rim pattern. | General IHC causes include nonspecific binding or excessive detection background; this appearance cannot be matched confidently to the reported nuclear-membranous pattern (HPA: tissue IHC). | Review blocking, washes, antibody use, and detection conditions under the validated IHC-P workflow. Examine a matched negative detection control and score only distinct cellular signal. |
| A listed low-staining population is weak or negative. | HPA lists salivary glandular cells, liver cholangiocytes, chondrocytes, adipocytes, and splenic red-pulp cells as low staining (HPA: tissue IHC). Weakness there alone is a poor test of assay failure. | Check a section containing a named high-staining population before changing conditions. Compare corresponding cell types rather than whole-tissue averages (HPA: tissue IHC). |
| An IF image shows puncta or mitotic structures that differ from the IHC section. | HPA supports nucleoplasm and nuclear membrane in ICC-IF (HPA: subcellular ICC-IF); UniProt describes cell-cycle-dependent kinetochore and spindle locations (UniProt Q9Y6D9: subcellular location). | Identify mitotic cells and compare like cell states. Interpret IF localisation separately from the paraffin-section chromogenic result; do not transfer an IF appearance or protocol into the IHC-P decision. |
Experiment evidence
IHC & IF images showing MAD1L1 staining
PB9262 · IHCIHC analysis of MAD1 using anti-MAD1 antibody (PB9262). MAD1 was detected in paraffin-embedded section of mouse intestine tissues. Heat medi
PB9262 · IHCIHC analysis of MAD1 using anti-MAD1 antibody (PB9262). MAD1 was detected in paraffin-embedded section of rat intestine tissues. Heat mediat
PB9262 · IFIF analysis of MAD1 using anti-MAD1 antibody (PB9262). MAD1 was detected in immunocytochemical section of U20S cells. Enzyme antigen retriev
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000002822-MAD1L1/tissue/cerebral+cortex#imid_3876968
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000002822-MAD1L1/tissue/colon#imid_3876894
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000002822-MAD1L1/tissue/liver#imid_3876895
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000002822-MAD1L1/tissue/kidney#imid_3876896
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody CAB015338; green = MAD1L1, blue = nucleus, red = microtubules.
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody CAB015338; green = MAD1L1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for MAD1L1 IHC & IF
🧪Run duodenum first and expect glandular cells to stain (HPA: High in duodenum glandular cells). HPA detects MAD1L1 in all 45 scored tissues, so use no-primary and isotype controls for the negative; no internal cell population is validated as negative, and cells without visible staining should be judged against control-slide background (HPA: no negative tissue rows).
Positive control tissue: Cerebellum (Cells in molecular layer, HPA High)
Negative control tissue: None in HPA: MAD1L1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show MAD1L1 in U-251MG, with annotated localisation: Nucleoplasm (supported), Nuclear membrane (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) slide, a matched rabbit IgG isotype control, and, if available, MAD1L1 knockout material or a validated peptide-block control (selected-SKU caption: rabbit primary antibody). For intestinal sections developed with biotin-based detection and DAB, check endogenous biotin and peroxidase background (selected-SKU caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative unreported). The caption uses heat retrieval in citrate buffer at pH 6 for 20 minutes, but does not establish that retrieval is required (selected-SKU caption: citrate retrieval). There is no supplied basis to rank frozen sections against IF for ease; ICC-IF images exist for U-251MG, while intestinal IHC with biotin-based DAB detection warrants background checks (HPA: U-251MG ICC-IF images; selected-SKU caption: SABC and DAB).
HPA tissue IHC evidence for MAD1L1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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 |
|---|---|---|---|---|
| Cerebellum | Cells in molecular layer | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: MAD1L1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced MAD1L1 IHC Tips
Troubleshoot MAD1L1 staining in paraffin sections by checking retrieval, compartment, cell-cycle context and controls before interpreting chromogenic signal.
How should I retrieve MAD1L1 antigen when staining paraffin sections?
Use heat-mediated retrieval in citrate buffer at pH 6 for MAD1L1 paraffin sections (datasheet PB9262). The selected tissue image used 20 minutes of citrate retrieval before staining a mouse intestine section, so use that documented condition as the starting point (caption PB9262). If signal is weak, compare a small retrieval time series on adjacent sections while keeping antibody concentration and detection constant (standard IHC practice). Excess heating can damage section morphology and make nuclear boundaries difficult to score (standard IHC practice). Include a section processed without primary antibody so retrieval-related background is distinguishable from MAD1L1 staining (standard IHC practice).
Could fixation explain weak or uneven MAD1L1 staining?
The selected paraffin-section caption does not report its fixative, and target-specific MAD1L1 fixation sensitivity is unknown from the supplied evidence (caption PB9262). Record each specimen’s fixative, fixation duration and processing history before comparing staining intensity across blocks (standard IHC practice). For troubleshooting, stain adjacent sections from blocks with documented processing using the same citrate pH 6 retrieval and detection conditions (datasheet PB9262; standard IHC practice). Compare nuclear morphology and section integrity alongside signal, since damaged tissue can undermine compartment scoring (standard IHC practice). Do not attribute a fixation effect to MAD1L1 solely because another block stains differently; specimen composition and processing may also differ (standard IHC practice).
Which staining compartments should I expect across interphase and mitosis?
In most tissues, expect predominantly nuclear and nuclear-membranous staining, with nucleoplasm and nuclear membrane supported by subcellular imaging (HPA tissue IHC; HPA subcellular). MAD1L1 also occupies cell-cycle-dependent structures, including centrosomes, spindle regions and the midbody (UniProt Q9Y6D9). Kinetochore localisation is reported during prometaphase but not metaphase, so identify mitotic stage before calling puncta abnormal (UniProt Q9Y6D9). In chromogenic sections, assess puncta against preserved cell and nuclear morphology rather than assigning every brown dot to a spindle structure (standard IHC practice). Persistent diffuse extracellular or luminal colour lacks support from these reported compartments and warrants a background check (UniProt Q9Y6D9; standard IHC practice).
Could isoforms or epitope masking change what the antibody detects?
MAD1L1 has 3 listed isoforms, while the supplied antibody caption does not identify its epitope or establish isoform selectivity (UniProt Q9Y6D9; caption PB9262). Its annotated protein is intracellular and has no transmembrane segment, but those facts do not locate the antibody epitope (UniProt Q9Y6D9). Check the antibody’s epitope documentation before interpreting staining differences as isoform expression, and compare sections under identical retrieval conditions (standard IHC practice). Annotated phosphorylation and acetylation sites could matter only if an epitope overlaps or depends on them; such dependence is untested here (UniProt Q9Y6D9). Report staining as MAD1L1 immunoreactivity unless isoform specificity has been independently established (standard IHC practice).
How can IF help resolve ambiguous MAD1L1 localisation in sections?
For the separate IF/ICC application, nuclear membrane and nucleoplasm are supported locations, with U-251MG listed among imaged cells (HPA subcellular). Multiplex MAD1L1 with a marker identifying the expected cell population, such as glandular cells when examining duodenum, and include a nuclear counterstain to place signal within cells (HPA tissue IHC; standard IF practice). Choose a fluorophore channel after checking unstained tissue autofluorescence, favouring a spectrally separated channel when background overlaps the initial choice (standard IF practice). MAD1L1 has no transmembrane segment and is intracellular, so permeabilisation must permit access to nuclear or cytoskeletal epitopes (UniProt Q9Y6D9; standard IF practice). Check whether permeabilisation preserves nuclear-envelope morphology before comparing IF puncta with chromogenic IHC (standard IF practice).
What should I check when DAB staining looks widespread or granular?
The documented paraffin-section example used 10% goat-serum blocking, a rabbit primary at 1 μg/mL, a biotinylated secondary and DAB detection (caption PB9262). Run a no-primary control through the same detection steps to reveal secondary, biotin-system or chromogen background (standard IHC practice). Apply an endogenous peroxidase block as a general chromogenic IHC step, then compare its effect with a control processed identically apart from that step (standard IHC practice). If background persists, inspect section edges, folds and damaged areas before changing the antibody concentration (standard IHC practice). Genuine MAD1L1 staining should be judged against its predominantly nuclear and nuclear-membranous tissue profile (HPA tissue IHC).
How should I score MAD1L1 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define eligible cells and compartments before scoring, using nuclear or nuclear-membranous staining as the principal tissue readout (HPA tissue IHC; standard IHC practice). Report the percentage of positive eligible cells and, when intensity categories are reproducible, an H-score calculated as 0–300 from percentages at intensities 0–3 (standard IHC practice). For sparse mitotic patterns, report positive-cell density per mm² of evaluable tissue and record mitotic stage separately where morphology permits (UniProt Q9Y6D9; standard IHC practice). Normalise counts to the relevant cell population or evaluable tissue area, excluding folds, necrosis and section loss (standard IHC practice). Keep retrieval, detection and scoring thresholds consistent across compared sections (standard IHC practice).
When is a positive MAD1L1 result convincing rather than artefactual?
A convincing chromogenic result follows the reported nuclear or nuclear-membranous pattern in intact cells and exceeds the no-primary control (HPA tissue IHC; standard IHC practice). Strong glandular-cell staining in duodenum and low staining in salivary-gland glandular cells offer contextual comparisons, although the HPA tissue assessment has medium RNA-staining consistency (HPA tissue IHC). Mitotic puncta require stage-aware interpretation because kinetochore localisation is reported in prometaphase and absent in metaphase (UniProt Q9Y6D9). Colour concentrated at cut edges, folds or necrotic regions should be checked against adjacent intact tissue (standard IHC practice). Signal reproduced without primary antibody suggests detection background, including endogenous enzyme activity, rather than demonstrated MAD1L1 (standard IHC practice).
Boster reagents
Best MAD1L1 / Mitotic spindle assembly checkpoint protein MAD1 IHC Antibodies
PB9262 has IHC images from paraffin sections of mouse and rat intestine and human intestinal cancer, plus IF data in U20S cells (PB9262 image captions).
Real IHC data
Anti-MAD1/MAD1L1 Antibody ®
Cat # PB9262
PB9262 is listed for IHC and IF in human, mouse, and rat samples (PB9262 catalog applications and reactivity). Its images show tissue IHC in mouse and rat intestine and human intestinal cancer, and IF in U20S cells (PB9262 image captions).
Which to pick: For tissue IHC, choose PB9262: its own images show staining in paraffin sections; the fixative is unreported (PB9262 IHC image captions). For IF/ICC, PB9262 is also listed for both applications and has an IF image in U20S cells (PB9262 catalog applications and IF image caption). PB9262 is the cross-species choice here because human, mouse, and rat reactivity is listed; clonality is unreported (PB9262 catalog reactivity and clone field).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y6D9 (MD1L1_HUMAN, Mitotic spindle assembly checkpoint protein MAD1).
- Human Protein Atlas. MAD1L1 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. MAD1L1 subcellular location (ICC-IF): Localized to the nucleoplasm and nuclear membrane..
- Human Protein Atlas. MAD1L1 antibody validation summary (2 antibodies).
- Exosomal transfer of macrophage-derived NEAT1 enhances DNA damage response and confers cisplatin resistance in lung adenocarcinoma via the MAD1L1/p53 axis. International journal of biological sciences 2026 — PMC13086090.
- NFYA-Mediated TTK Up-Regulation Drives Fast Cell Cycle Progression and Its Inhibition Leads to Mitotic Catastrophe in Triple Negative Breast Cancer. Cancers 2026 — PMC13163004.
- Transcriptomic correlates of cell cycle checkpoints with distinct prognosis, molecular characteristics, immunological regulation, and therapeutic response in colorectal adenocarcinoma. Frontiers in immunology 2023 — PMC10749195.
- PubMed PMID:9546394 — UniProt-cited evidence.
- PubMed PMID:10198256 — UniProt-cited evidence.
- PubMed PMID:10049595 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for MAD1L1 (Mitotic spindle assembly checkpoint protein MAD1). Expected localisation Nuclear and nuclear membranous in most tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet PB9262). Validated IHC antibody: PB9262 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (Cells in molecular layer); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded mouse intestine tissues; fixative not specified (datasheet PB9262); 2. Fixation: Image fixative and duration unreported (datasheet PB9262); 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 PB9262); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9262); 8. Primary antibody: Rabbit anti-MAD1L1, 0.5-1μg/ml (datasheet PB9262); 9. Primary incubation: Overnight at 4 °C (datasheet PB9262). Watch for: Fixation: Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific); Caveat: Mitotic stage can shift MAD1L1 from its nuclear pattern (UniProt); Isoform / epitope: 3 isoforms; epitope coverage is unknown (UniProt); Regulation: Intensity varies by tissue and cell type (HPA tissue IHC). Evidence: UniProt Q9Y6D9, HPA tissue IHC · Supported, HPA ICC-IF · nucleoplasm and nuclear membrane., Datasheet PB9262, PMC13163004. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/MAD1L1/immunohistochemistry-ihc-guide.