MAD1L1 / Mitotic spindle assembly checkpoint protein MAD1 · IHC design guide

Design Immunohistochemistry for MAD1L1

Plan MAD1L1 chromogenic IHC in paraffin sections around the nuclear and nuclear membranous tissue pattern (HPA tissue IHC). This guide covers fixation consistency, the catalog antibody’s 0.5–1 μg/mL IHC dilution (datasheet PB9262), and interpretation of mitotic redistribution (UniProt).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for MAD1L1 (IHC for MAD1L1): expected localisation Nuclear and nuclear membranous tissue staining (HPA tissue IHC), antibody PB9262, validated IHC image, and IHC protocol steps — Printable **MAD1L1** IHC protocol sheet — expected localisation Nuclear and nuclear membranous tissue staining (HPA tissue IHC), antibody PB9262, controls and protocol steps. Open the full MAD1L1 IHC guide →

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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 tissue staining (HPA tissue IHC)
	Staining pattern	Most tissues show nuclear and nuclear rim 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 consistent across sections. (standard IHC practice; not target-specific)
	Caveat	Mitotic redistribution can change the nuclear pattern (UniProt)
	Regulation	Cell-cycle stage changes signal distribution (UniProt)
	Isoform / epitope	3 isoforms; epitope coverage is undetermined (UniProt)

Section 1

Recommended MAD1L1 IHC & IF Protocols

The catalog antibody uses citrate pH 6 retrieval (datasheet: PB9262). One published MAD1L1 IHC protocol uses EDTA pH 9.0 (PMC13163004).

Recommended immunohistochemistry (IHC-P) protocol parameters
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 for the catalog antibody (datasheet: PB9262); compare EDTA pH 9.0 if optimizing retrieval (PMC13163004).

Section 2

What Is the Expected MAD1L1 Staining Pattern?

MAD1L1 staining is expected mainly in nuclei and along nuclear membranes in most tissues (HPA tissue IHC: Supported; medium consistency with RNA). In paraffin sections, examine the named cell populations rather than treating every cell as equally positive: high staining is reported in duodenal glandular cells and hippocampal glial cells, among others (HPA tissue IHC). MAD1L1 also has cell cycle dependent locations, including kinetochores and the spindle (UniProt Q9Y6D9). It has no transmembrane segment (UniProt Q9Y6D9 topology).

What am I looking at on my slide?
Nuclear and nuclear rim staining in the expected cell population, with discernible cell boundaries and little signal outside cells.	This fits the tissue IHC profile of nuclear and nuclear membranous expression in most tissues (HPA tissue IHC). Compare cells within the section and a matched control before judging intensity; HPA reports a tissue pattern, not an intensity threshold for every paraffin specimen (HPA tissue IHC; general IHC practice).
A small number of dividing cells show staining at kinetochores or spindle structures rather than the usual interphase pattern.	Such a pattern can fit MAD1L1 trafficking during mitosis (UniProt Q9Y6D9 subcellular location). Kinetochore localisation is reported in prometaphase but not metaphase, so a mitotic figure needs cell cycle context (UniProt Q9Y6D9). A broad membrane or extracellular deposit does not follow the reported locations (UniProt Q9Y6D9).
Strong, widespread surface, extracellular, or diffuse cytoplasmic colour dominates while nuclei remain indistinct.	Treat this as a possible artefact and inspect detection controls and slide morphology (general IHC practice). MAD1L1 has no transmembrane segment, and the HPA tissue IHC profile is nuclear and nuclear membranous (UniProt Q9Y6D9 topology; HPA tissue IHC). A cytoplasmic spot alone is insufficient to identify a mitotic structure (UniProt Q9Y6D9 subcellular location).
Signal is concentrated in an unexpected cell population while the documented high staining population is weak.	Review cell identification and consider antibody cross-reactivity or endogenous chromogen activity (general IHC practice). For example, HPA reports high staining in kidney glomerular cells and low staining in adipocytes; low is not an absence claim (HPA tissue IHC). One unexpected cell population cannot establish a new tissue distribution by itself (general IHC practice).
No clear staining appears in a section selected for an HPA high staining cell population.	First confirm that the relevant cells are present, then check the matched positive control and staining run (general IHC practice). HPA reports high staining in duodenal glandular cells, but its overall tissue IHC reliability is Supported with medium consistency between staining and RNA data; an HPA observation does not guarantee every specimen will stain (HPA tissue IHC).

💡Expected MAD1L1 appearanceCall the IHC result positive when the appropriate cells show discernible nuclear or nuclear rim chromogen, potentially high in documented populations such as duodenal glandular cells; dominant extracellular or broad surface staining is suspect (HPA tissue IHC; UniProt Q9Y6D9 topology).

How each factor affects the staining
Tissue and cell choice	HPA reports high staining in cerebellar molecular layer cells, glandular cells of several named tissues, hippocampal glial cells, and kidney glomerular cells (HPA tissue IHC). Its low entries, including liver cholangiocytes, are low observations rather than validated negative controls (HPA tissue IHC).
Cell cycle stage	MAD1L1 is reported at the nuclear envelope during interphase and at kinetochores in prometaphase; its location changes through mitosis (UniProt Q9Y6D9 subcellular location). Interpret isolated mitotic patterns against nearby cell morphology before scoring the entire section (general IHC practice).
Antibody evidence	HPA lists IHC as Supported for HPA003635 and CAB015338; that status does not specify an intensity cutoff for a new specimen (HPA antibodies; HPA tissue IHC). Record the antibody and controls used when comparing patterns (general IHC practice).
Protein forms and target access	UniProt lists three MAD1L1 isoforms and a chain spanning residues 1–718, with no signal peptide or propeptide annotated (UniProt Q9Y6D9). The supplied record gives no antibody epitope, so it cannot predict which isoforms an individual antibody detects or a target-specific retrieval response (UniProt Q9Y6D9; HPA antibodies).
What should IF/ICC show?	The separate IF/ICC interpretation is nucleoplasm and nuclear membrane localisation; HPA lists U-251MG among cell lines with images (HPA subcellular ICC-IF). This IHC section supplies no IF/ICC protocol or dilution; the ICC evidence does not set a paraffin-section chromogen threshold (HPA subcellular ICC-IF; HPA tissue IHC).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Known high staining cells are present, but the whole IHC run is blank.	A failed staining or detection step is possible (general IHC practice); HPA high staining is an observation, not a guaranteed result for each run (HPA tissue IHC).	Check the run control, antibody identity, reagent sequence, and detection readout; repeat with documented IHC conditions if the control failed (general IHC practice).
The run control stains, but the expected nuclear or nuclear rim pattern is faint.	Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation.	Check tissue preservation and perform a controlled retrieval and antibody dilution comparison using the antibody's IHC instructions (general IHC practice).
Chromogen covers much of the section, including spaces between cells.	Excess background, nonspecific antibody binding, or endogenous detection activity may obscure compartments (general IHC practice).	Review the no-primary and detection controls; adjust blocking, washing, or development time according to the detection system (general IHC practice).
Surface or cytoplasmic staining is stronger than nuclear staining.	The dominant compartment conflicts with the HPA tissue IHC profile; a few mitotic MAD1L1 structures can have other locations (HPA tissue IHC; UniProt Q9Y6D9 subcellular location).	Inspect nuclear morphology and mitotic figures, then compare with an IHC-supported antibody and controls before assigning the signal to MAD1L1 (HPA antibodies; general IHC practice).
Only an unexpected cell population stains strongly.	Cell identification, cross-reactivity, or endogenous activity may explain the mismatch (general IHC practice); HPA reports distinct levels by cell population (HPA tissue IHC).	Verify the cell type on the counterstain and compare a documented high staining population in the same run; use detection controls to investigate the unexpected signal (HPA tissue IHC; general IHC practice).
Two antibodies give different tissue patterns.	HPA marks both listed antibodies IHC Supported, while the tissue profile has medium consistency with RNA; this does not resolve a specimen-level disagreement (HPA antibodies; HPA tissue IHC).	Compare the same cell populations and controls under documented conditions, and report the discordance rather than assigning an unsupported positive or negative call (general IHC practice).

Source: UniProt Q9Y6D9 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing MAD1L1 staining

IHC analysis of MAD1 using anti-MAD1 antibody (PB9262). MAD1 was detected in paraffin-embedded section of mouse intestine tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-MAD1 Antibody (PB9262) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

PB9262 · IHCIHC analysis of MAD1 using anti-MAD1 antibody (PB9262). MAD1 was detected in paraffin-embedded section of mouse intestine tissues. Heat medi

IHC analysis of MAD1 using anti-MAD1 antibody (PB9262). MAD1 was detected in paraffin-embedded section of rat intestine tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-MAD1 Antibody (PB9262) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

PB9262 · IHCIHC analysis of MAD1 using anti-MAD1 antibody (PB9262). MAD1 was detected in paraffin-embedded section of rat intestine tissues. Heat mediat

IF analysis of MAD1 using anti-MAD1 antibody (PB9262). MAD1 was detected in immunocytochemical section of U20S cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL rabbit anti-MAD1 Antibody (PB9262) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

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.

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Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for MAD1L1 IHC & IF

🧪Run duodenum first and expect staining in its glandular cells (HPA: High in duodenal glandular cells). HPA detects MAD1L1 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and treat any unstained neighboring cells as a within-slide background comparison rather than a validated negative (HPA: no negative tissue rows; standard IHC practice).

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, an isotype control matched to the primary antibody’s host and clonality, and MAD1L1-knockout material as a biological specificity control if available (standard IHC practice). Block endogenous peroxidase and check for endogenous biotin background in duodenum because the selected IHC caption uses biotin-based detection with DAB (caption: PB9262; standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state a fixative (caption: PB9262). The caption uses citrate retrieval at pH 6 for 20 minutes, but does not establish that retrieval is required under other conditions (caption: PB9262). Frozen sections are not established as easier by the supplied evidence; IF/ICC images in U-251MG support assessing nucleoplasmic and nuclear-membrane localization in its separate guide, while duodenal biotin background may complicate the caption’s IHC detection method (HPA: subcellular localization and U-251MG ICC-IF images; caption: PB9262; standard IHC practice).

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, controls and scoring before interpreting biological differences.

How should I retrieve MAD1L1 when nuclear staining is weak?

Start with heat-mediated citrate buffer at pH 6 for 20 minutes on paraffin sections (datasheet PB9262). The selected tissue image used this retrieval before overnight primary-antibody incubation, so it is the starting condition for this catalog antibody (datasheet PB9262). If staining remains weak, compare a longer heating interval or an alternative retrieval buffer on adjacent sections while holding detection conditions constant (standard IHC practice). Inspect tissue morphology and background alongside nuclear signal, because excessive retrieval can damage sections or increase nonspecific staining (standard IHC practice). Include a known positive section in each comparison to distinguish retrieval failure from low expression in the sample (HPA tissue IHC; standard IHC practice).

Could fixation explain inconsistent MAD1L1 staining across paraffin blocks?

Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative, and the supplied evidence does not compare fixation conditions (datasheet PB9262). Record the fixative, fixation duration and processing history for each block before attributing staining differences to MAD1L1 biology (standard IHC practice). Compare sections processed together, using the same pH 6 citrate retrieval and detection run to limit technical variation (datasheet PB9262; standard IHC practice). If blocks differ, assess nuclear morphology and staining in a reference tissue from each processing batch (standard IHC practice). Do not infer a target-specific fixation effect from its nuclear localisation or modified residues (UniProt Q9Y6D9).

Where should convincing MAD1L1 staining appear in tissue sections?

Expect predominantly nuclear and nuclear-membranous staining across many tissues, with the nucleoplasm and nuclear membrane supported by subcellular imaging (HPA tissue IHC; HPA subcellular). Interpret focal kinetochore, centrosome or spindle-associated signal in the context of mitotic stage, since MAD1L1 changes location through mitosis (UniProt Q9Y6D9). In particular, kinetochore localisation is reported during prometaphase but absent during metaphase (UniProt Q9Y6D9). Use a nuclear counterstain and inspect cell boundaries before assigning faint chromogen to a compartment (standard IHC practice). Predominantly diffuse extracellular staining does not match the supplied localisation evidence and warrants a background-control comparison (UniProt Q9Y6D9; standard IHC practice).

Could isoforms or epitope masking change the MAD1L1 IHC pattern?

MAD1L1 has three annotated isoforms, so establish which sequence region the antibody recognises before treating every staining difference as altered protein abundance (UniProt Q9Y6D9; standard IHC practice). The supplied material does not map the catalog antibody's epitope or establish which isoforms it detects (datasheet PB9262). MAD1L1 also has annotated phosphorylation and acetylation sites, but no supplied evidence shows that these modifications alter this antibody's staining (UniProt Q9Y6D9; datasheet PB9262). Compare matched sections under identical retrieval and detection conditions, then confirm a disputed compartment or cell population with an independently validated epitope when available (standard IHC practice). Record any epitope uncertainty alongside the interpretation (standard IHC practice).

How can IF help check a puzzling chromogenic MAD1L1 pattern?

Use IF as a separate corroborating readout: subcellular images support nucleoplasmic and nuclear-membrane MAD1L1, including images from U-251MG cells (HPA subcellular). Multiplex with a validated marker for the cell population being assessed and a nuclear stain, then compare colocalisation with the chromogenic assignment (standard IF practice). Choose fluorophores away from the tissue's strongest autofluorescence and include unstained and single-label controls before interpreting weak puncta (standard IF practice). MAD1L1 has no annotated transmembrane segment; for a nuclear epitope, optimise permeabilisation to permit nuclear access while preserving morphology, since this antibody's epitope is unspecified (UniProt Q9Y6D9; standard IF practice). IF conditions require their own validation (standard IF practice).

What should I check when MAD1L1 chromogen appears throughout the section?

First compare a no-primary control with the test section to identify signal from detection reagents or endogenous activity (standard IHC practice). The selected workflow used 10% goat serum, a biotinylated secondary, streptavidin-biotin detection and DAB, so check blocking and detection background in that configuration (datasheet PB9262; standard IHC practice). Include an endogenous-peroxidase block before DAB and assess whether biotin-based detection contributes staining in the tissue under study (standard IHC practice). Titrate the primary from the reported 1 μg/ml condition if diffuse signal persists, keeping retrieval and development time fixed (datasheet PB9262; standard IHC practice). Compare background against the expected nuclear pattern (HPA tissue IHC).

How should I score MAD1L1 across sections with different cell mixtures? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and score nuclear staining separately from nuclear-membranous staining before reviewing outcomes (HPA tissue IHC; standard IHC practice). For each compartment, record the percentage of positive eligible cells and staining intensity; an H-score can combine those two measures (standard IHC practice). Normalise positive counts to the number of evaluable cells in the same population, or report positive-cell density per mm² of evaluable tissue when cell counts are impractical (standard IHC practice). Exclude folds, damaged edges and necrotic regions using a prespecified rule, and keep chromogen exposure and scoring thresholds consistent across batches (standard IHC practice). Report mitotic figures separately if assessing spindle-associated localisation (UniProt Q9Y6D9; standard IHC practice).

How do I distinguish genuine MAD1L1 signal from staining artefact?

Treat nuclear or nuclear-membranous staining in intact cells as more consistent with the tissue profile than diffuse extracellular deposit (HPA tissue IHC; standard IHC practice). Confirm the stained cell type against tissue morphology: HPA reports high staining in duodenal glandular cells and low staining in salivary-gland glandular cells, without implying every cell must follow that pattern (HPA tissue IHC). Recheck isolated mitotic puncta against mitotic stage because kinetochore localisation is reported in prometaphase, but not metaphase (UniProt Q9Y6D9). Discount signal confined to section edges or necrosis after comparison with adjacent intact tissue (standard IHC practice). Use a no-primary control and an endogenous-peroxidase block to investigate DAB signal unrelated to the primary antibody (standard IHC practice).

Boster reagents

Best MAD1L1 / Mitotic spindle assembly checkpoint protein MAD1 IHC Antibodies

The IHC-validated antibody PB9262 has paraffin-section images from mouse and rat intestine and human intestinal cancer, plus an IF image from U20S cells (PB9262 image captions).

Real IHC data

Anti-MAD1/MAD1L1 Antibody ®

Cat # PB9262

View product →

PB9262 is listed for IHC in human, mouse and rat; its paraffin-section images show mouse and rat intestine and human intestinal cancer (PB9262 catalog applications, reactivity and IHC captions). PB9262 is also listed for IF/ICC, with an IF image from U20S cells (PB9262 catalog applications and IF caption).

Which to pick: Choose PB9262 for paraffin-section IHC: its tissue captions report citrate retrieval at pH 6 for 20 minutes and primary antibody at 1 μg/ml (PB9262 IHC captions); the fixative is unreported (PB9262 IHC captions). For IF/ICC, PB9262 has an IF image from U20S cells (PB9262 IF caption); for cross-species IHC, it is listed as reactive with human, mouse and rat (PB9262 catalog reactivity), with tissue images for all three (PB9262 IHC captions). Its host is rabbit, and clonality is unreported (PB9262 catalog host and clone fields).

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.