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MELK / Maternal embryonic leucine zipper kinase · IHC design guide
Design Immunohistochemistry for MELK
Plan paraffin-section MELK IHC using the catalog antibody (datasheet A04038-2). Assess cytoplasmic and nuclear staining with appropriate controls, and interpret tissue patterns cautiously because staining has low consistency with RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
MELK Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic and nuclear staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic and nuclear signal across diverse tissue cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A04038-2) | |
| Positive control | Adrenal gland+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 | Tissue staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Cell-cycle role; effect on staining intensity unknown (UniProt) | |
| Isoform / epitope | 8 isoforms; epitope coverage unknown; no TM segment (UniProt) |
Section 1
Recommended MELK IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by 4 published MELK IHC protocols from glioma, cervical tissue, and lung adenocarcinoma studies (PMC9637824; PMC6246930; PMC8495384; PMC7471708).
| Sample | Paraffin-embedded mouse ovary tissue; fixative not specified (datasheet A04038-2) |
| Fixation | Image fixative and duration unreported (datasheet A04038-2); 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 A04038-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A04038-2) |
| Primary antibody | Rabbit anti-MELK, 2-5 μg/ml (datasheet A04038-2) |
| Primary incubation | Overnight at 4 °C (datasheet A04038-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A04038-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | MELK-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nuclear expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat retrieval in EDTA pH 8.0 (datasheet: A04038-2); the cited studies also report citrate pH 6.0 and Tris/EDTA pH 9.0 (PMC6246930; PMC8495384; PMC7471708).
Section 2
What Is the Expected MELK Staining Pattern?
MELK IHC is expected to show cytoplasmic and nuclear staining in cells across many tissues, including glandular and hematopoietic cells (HPA: tissue IHC profile; High in glandular and hematopoietic cells). UniProt also lists MELK at the cell membrane, although it has no transmembrane segment (UniProt Q14680: subcellular location and topology). Interpret a positive slide cautiously: the HPA tissue IHC assessment is Uncertain because antibody staining and RNA expression have low consistency (HPA: reliability).
| Cytoplasmic and nuclear chromogen appears in glandular cells or bone marrow hematopoietic cells. | This matches the reported compartments and cell types (HPA: tissue IHC profile; High in glandular and hematopoietic cells). It is a plausible positive pattern, not proof of specificity, because the tissue IHC assessment is Uncertain (HPA: reliability). |
| Signal is confined to luminal material or tissue edges, with no convincing cellular staining. | This does not match HPA's reported cytoplasmic and nuclear pattern (HPA: tissue IHC profile). Treat it as possible artefact and check slide morphology and controls (general IHC practice). Membrane association alone does not establish luminal staining (UniProt Q14680: location and topology). |
| Strong staining dominates adipocytes or smooth muscle cells while expected cellular staining is absent. | HPA reports Low staining in those cells (HPA: Low in adipocytes and smooth muscle cells). Consider cross-reactivity or endogenous detection activity; compare no-primary and detection controls before assigning the signal to MELK (general IHC practice). |
| Brown haze covers nuclei, cytoplasm and empty regions with little cell-to-cell distinction. | A noncellular haze cannot be scored against HPA's cellular pattern (HPA: tissue IHC profile). In chromogenic IHC, diffuse background can arise from nonspecific binding, detection reagents or incomplete washing; assess a no-primary control (general IHC practice). |
| No cellular signal appears in a bone marrow section expected to serve as a positive reference. | HPA reports High staining in bone marrow hematopoietic cells, but its tissue IHC reliability is Uncertain (HPA: Bone marrow; reliability). Check section quality, retrieval, antibody incubation and detection with controls before concluding MELK is absent (general IHC practice). |
💡Expected MELK appearanceCall a result consistent with MELK when distinguishable cytoplasmic and nuclear staining appears in HPA-reported cell types, potentially at High levels in glandular or bone marrow hematopoietic cells; isolated edge, luminal or diffuse staining is suspect (HPA: tissue IHC profile; High cell entries; general IHC practice).
| Reported localization and topology | HPA describes cytoplasmic and nuclear tissue staining; UniProt lists cell-membrane localization without a transmembrane segment (HPA: tissue IHC profile; UniProt Q14680: location and topology). Assess the cellular pattern as a whole; membrane labeling alone is not established as the diagnostic IHC pattern. |
| Strength of IHC evidence | HPA assigns tissue IHC an Uncertain rating because staining and RNA expression have low consistency; its listed antibody HPA017214 is also IHC Uncertain (HPA: reliability; antibody validation). A High tissue staining entry is an observation, not independent confirmation of specificity. |
| Variation across sampled cells | HPA reports that transcript expression varies with the cell cycle (HPA: subcellular summary). This supports caution when comparing cells, but does not establish a particular phase-specific IHC pattern or explain a specific stained cell on a slide. |
| Isoforms and epitope coverage | UniProt lists 8 MELK isoforms (UniProt Q14680: isoforms). Whether the catalog antibody detects each isoform depends on its epitope, which is not supplied; do not interpret a negative section as evidence that all MELK isoforms are absent. |
| IF/ICC: can it confirm the IHC compartment? | HPA supplies no main ICC-IF location or cell-line images for MELK (HPA: subcellular record). Its tissue IHC pattern therefore cannot be treated as an independently confirmed IF/ICC pattern; evaluate IF/ICC with its own controls and guide (general IF practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Cytoplasmic and nuclear staining is present, but its intensity differs between cells. | HPA reports staining across most tissues and notes cell-cycle-correlated transcript variation; neither establishes the reason for intensity differences on this slide (HPA: tissue IHC profile; subcellular summary). | Score the observed compartment and cell type separately from intensity. Compare cells within the same section and retain the HPA Uncertain qualification when reporting the pattern (HPA: reliability; general IHC practice). |
| Only a thin membrane-like outline stains strongly. | UniProt lists a cell-membrane location without a transmembrane segment, while HPA describes mainly cytoplasmic and nuclear tissue staining (UniProt Q14680: location and topology; HPA: tissue IHC profile). | Review whether cellular cytoplasm or nuclei also stain. Use morphology and a no-primary control before interpreting an isolated outline as MELK (general IHC practice). |
| Smooth muscle or adipocytes appear stronger than neighboring expected cells. | Those cell types are listed as Low by HPA; unexpected chromogen could reflect nonspecific binding or endogenous detection activity (HPA: Low cell entries; general IHC practice). | Inspect a no-primary control and the detection system, then compare staining with the section's cell morphology. Report the discordance instead of scoring those cells as a validated MELK-positive population (general IHC practice). |
| The section has widespread brown background that obscures cell boundaries. | Diffuse deposition can result from nonspecific reagent binding, inadequate washing or endogenous enzyme activity in chromogenic IHC (general IHC practice). | Check a no-primary control, blocking and wash steps, and the detection controls appropriate to the chromogen. Reassess only interpretable cellular staining against the HPA pattern (general IHC practice; HPA: tissue IHC profile). |
| A bone marrow reference section shows no detectable hematopoietic-cell staining. | HPA reports High staining in these cells, yet rates the tissue IHC evidence Uncertain; a negative run could also reflect a workflow failure (HPA: Bone marrow; reliability; general IHC practice). | Check tissue preservation and section morphology, then verify the established antigen-retrieval, primary-antibody and detection steps with appropriate controls. Do not infer a MELK-specific fixation sensitivity from these data (general IHC practice). |
| The slide is positive, but the result is being used to claim confirmed MELK specificity. | HPA reports Low consistency between antibody staining and RNA expression and assigns Uncertain reliability; HPA017214 is IHC Uncertain (HPA: reliability; antibody validation). | Describe the observed staining and its match to HPA's reported cells and compartments. Qualify the interpretation as provisional unless the experiment supplies independent specificity evidence (HPA: tissue IHC profile and reliability; general IHC practice). |
Experiment evidence
IHC & IF images showing MELK staining
A04038-2 · IHCIHC analysis of MELK using anti-MELK antibody (A04038-2). MELK was detected in a paraffin-embedded section of mouse ovary tissue. Heat media
A04038-2 · IHCIHC analysis of MELK using anti-MELK antibody (A04038-2). MELK was detected in a paraffin-embedded section of rat ovary tissue. Heat mediate
A04038-2 · IFIF analysis of MELK using anti-MELK antibody (A04038-2). MELK was detected in an immunocytochemical section of A549 cells. Enzyme antigen re
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000165304-MELK/tissue/cerebral+cortex#imid_4737108
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000165304-MELK/tissue/colon#imid_4737050
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000165304-MELK/tissue/liver#imid_4737051
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000165304-MELK/tissue/kidney#imid_4737044
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for MELK IHC & IF
🧪Run bone marrow first and assess staining in hematopoietic cells (HPA: High in bone marrow hematopoietic cells). HPA lists no negative tissue and detects MELK in all 45 scored tissues, so no-primary and isotype controls carry the negative comparison; use cells within the positive section as internal background comparators only if their MELK-negative status is independently verified (HPA: no negative tissue rows).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: None in HPA: MELK 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 carries no ICC-IF cell line for MELK; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control, a rabbit IgG isotype control matched to the primary antibody’s clonality where known, and a biological specificity control such as MELK knockout material or peptide blocking when the immunizing peptide is available (selected-SKU caption: rabbit anti-MELK antibody; standard IHC practice). Block endogenous peroxidase in bone marrow before HRP/DAB detection because hematopoietic cells can produce peroxidase background (HPA: High in bone marrow hematopoietic cells; selected-SKU caption: HRP/DAB detection).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A04038-2 paraffin-section caption does not state the fixative (selected-SKU caption: fixative unreported). That caption uses heat retrieval in EDTA at pH 8.0, but it does not establish that MELK detection depends on that retrieval condition (selected-SKU caption: EDTA retrieval at pH 8.0). Neither frozen sections nor IF can be judged easier from the supplied evidence; HPA lists no ICC-IF cell-line images, while bone marrow IHC requires attention to endogenous peroxidase background (HPA subcellular: no ICC-IF cell-line images; HPA: High in bone marrow hematopoietic cells; standard IHC practice).
HPA tissue IHC evidence for MELK
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: MELK is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced MELK IHC Tips
Troubleshoot MELK staining in paraffin sections using the selected antibody’s IHC conditions, matched controls, and compartment-aware interpretation.
What retrieval should I try first when MELK staining is weak in paraffin sections?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 (datasheet A04038-2). The selected mouse ovary image used this retrieval, followed by 10% goat-serum block and 2 μg/ml primary antibody overnight at 4°C (caption A04038-2). Keep section thickness, heating, and cooling consistent between test and control sections so retrieval variation does not distort comparisons (standard IHC practice). If staining remains weak, compare an alternative retrieval condition on matched sections while retaining EDTA pH 8.0 as the reference (standard IHC practice). Select the condition that preserves morphology and improves cellular signal without increasing diffuse DAB staining (standard IHC practice).
Could fixation explain variable MELK staining across my paraffin sections?
MELK-specific sensitivity to fixation is unknown from the supplied evidence; the selected paraffin-section caption does not state a fixative (caption A04038-2). Record fixative, time before fixation, fixation duration, and processing conditions for each specimen before comparing staining intensity (standard IHC practice). Compare similarly processed sections using the same EDTA pH 8.0 retrieval and antibody incubation conditions (datasheet A04038-2; standard IHC practice). If a processing difference tracks weak signal, test matched sections across controlled processing conditions and assess both staining and tissue morphology (standard IHC practice). Do not attribute that result to a MELK-specific fixation effect without direct validation (standard IHC practice).
How should I judge cytoplasmic, nuclear, or membrane-associated MELK staining?
Assess cytoplasmic and nuclear staining separately, because both appear in the tissue staining profile (HPA: cytoplasmic and nuclear expression in most tissues). MELK also has a cell-membrane annotation, but no transmembrane segment is listed; membrane-edge signal alone therefore needs corroboration (UniProt Q14680: subcellular location and topology). Compare staining within intact cells against a no-primary control and the same tissue’s background before assigning a compartment (standard IHC practice). Record compartment, cell type, and staining intensity instead of pooling every brown pixel into one score (standard IHC practice). Treat unexpected, uniformly sharp tissue edges as possible processing artefact until matched sections reproduce the pattern (standard IHC practice).
Could MELK isoforms or epitope changes explain discordant staining?
MELK has 8 listed isoforms and a protein kinase domain at residues 11–263 plus a KA1 domain at 602–651 (UniProt Q14680: isoforms and domains). The supplied caption gives no epitope map for A04038-2, so its coverage of individual isoforms cannot be established here (caption A04038-2). MELK has reported autophosphorylation sites, including residues 56, 163, and 167, but their effect on this antibody’s staining is unknown (UniProt Q14680: modified residues; caption A04038-2). If results disagree, compare antibody epitope documentation with isoform sequences and test an independent epitope on matched sections (standard IHC practice). Keep retrieval and detection conditions aligned during that comparison (standard IHC practice).
How can I check a MELK IHC pattern using multiplex immunofluorescence?
Pair MELK with a validated marker for the cell population being assessed, then examine whether signals occupy the same cells (standard IF practice). Choose fluorophores after inspecting each tissue’s autofluorescence and include single-label controls to check spectral bleed-through (standard IF practice). For an intracellular epitope, titrate permeabilisation after fixation; for an accessible membrane-facing epitope, assess whether permeabilisation is necessary (standard IF practice). The supplied evidence does not map the A04038-2 epitope, while MELK has a membrane annotation without a transmembrane segment (caption A04038-2; UniProt Q14680: topology). Interpret any IF pattern as a separate assay result and compare its cellular distribution with the paraffin-section IHC pattern (standard IF/IHC practice).
What should I change when MELK DAB staining is widespread or granular?
Inspect a no-primary section first to separate secondary-reagent or detection background from primary-antibody staining (standard IHC practice). In the selected image, sections received 10% goat-serum block, 2 μg/ml primary overnight at 4°C, and peroxidase-based DAB detection (caption A04038-2). Check peroxidase blocking, wash steps, primary concentration, and DAB development time one variable at a time on matched sections (standard IHC practice). Also inspect unstained pigment, folds, edges, and damaged areas before calling granular deposits cellular MELK (standard IHC practice). Because the tissue profile includes broad cytoplasmic and nuclear staining, diffuse signal requires cellular and control-based review rather than a brightness threshold alone (HPA: tissue profile; standard IHC practice).
How should I quantify MELK staining when cell types and compartments vary? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, then report the percentage of positive cells and an intensity-weighted H-score where appropriate (standard IHC practice). For scattered populations, positive cells per mm² can supplement an H-score if the analysed tissue area is recorded (standard IHC practice). Normalise counts to evaluable cells or area within the same annotated compartment, and exclude folds, necrosis, and poorly preserved regions (standard IHC practice). Keep retrieval, DAB development, imaging, and scoring thresholds consistent across compared sections (standard IHC practice). Report cytoplasmic and nuclear scores separately because both occur in the tissue profile, and note its uncertain staining-to-RNA consistency (HPA: profile and reliability).
How can I distinguish convincing MELK staining from a tissue artefact?
Look for reproducible cellular staining in intact tissue and compare it with a no-primary control and a similarly processed reference section (standard IHC practice). Cytoplasmic and nuclear patterns are reported, while the tissue staining assessment has uncertain consistency with RNA expression (HPA: profile and reliability). Check whether staining follows identifiable cells rather than section edges, folds, necrotic material, or areas with endogenous peroxidase signal (standard IHC practice). A membrane-only rim needs independent support because MELK has a cell-membrane annotation but no listed transmembrane segment (UniProt Q14680: location and topology). Interpret differences between cell populations alongside morphology and controls, without treating DAB intensity alone as proof of MELK abundance (standard IHC practice).
Boster reagents
Best MELK / Maternal embryonic leucine zipper kinase IHC Antibodies
A04038-2 has paraffin-section IHC images from mouse and rat ovary and an IF/ICC image from A549 cells; listed reactivity includes human, mouse, and rat (catalog image captions; catalog reactivity).
Real IHC data
Anti-MELK Antibody ®
Cat # A04038-2
A04038-2 has documented IHC staining in paraffin sections of mouse and rat ovary (A04038-2 IHC captions). The same SKU has documented IF staining in A549 cells (A04038-2 IF caption).
Which to pick: Choose A04038-2 for tissue IHC in paraffin sections: its own ovary images used EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody (A04038-2 IHC captions). For IF/ICC, A04038-2 has an A549-cell IF image using 5 μg/ml primary antibody (A04038-2 IF caption). For cross-species planning, A04038-2 lists human, mouse, and rat reactivity and IHC, IF, and ICC applications; clonality is unspecified, and the IHC captions do not report the tissue fixative (catalog reactivity and applications; catalog clone field; A04038-2 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q14680 (MELK_HUMAN, Maternal embryonic leucine zipper kinase).
- Human Protein Atlas. MELK tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. MELK subcellular location (ICC-IF): Variation in transcript expression is correlated to the cell cycle..
- Human Protein Atlas. MELK antibody validation summary (1 antibodies).
- MELK is a prognostic biomarker and correlated with immune infiltration in glioma. Frontiers in neurology 2022 — PMC9637824.
- Maternal embryonic leucine zipper kinase: A novel biomarker and a potential therapeutic target of cervical cancer. Cancer medicine 2018 — PMC6246930.
- Up-regulation of MELK by E2F1 promotes the proliferation in cervical cancer cells. International journal of biological sciences 2021 — PMC8495384.
- Maternal embryonic leucine zipper kinase: A novel biomarker and a potential therapeutic target in lung adenocarcinoma. Oncology letters 2020 — PMC7471708.
- PubMed PMID:8724849 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15164053 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for MELK (Maternal embryonic leucine zipper kinase). Expected localisation Cytoplasmic and nuclear staining in most tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A04038-2). Validated IHC antibody: A04038-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); 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 ovary tissue; fixative not specified (datasheet A04038-2); 2. Fixation: Image fixative and duration unreported (datasheet A04038-2); 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 A04038-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A04038-2); 8. Primary antibody: Rabbit anti-MELK, 2-5 μg/ml (datasheet A04038-2); 9. Primary incubation: Overnight at 4 °C (datasheet A04038-2). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Tissue staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 8 isoforms; epitope coverage unknown; no TM segment (UniProt); Regulation: Cell-cycle role; effect on staining intensity unknown (UniProt). Evidence: UniProt Q14680, HPA tissue IHC · Uncertain, HPA cell-line RNA · Variation in transcript expression is correlated to the cell cycle., Datasheet A04038-2, PMC9637824, PMC6246930. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/MELK/immunohistochemistry-ihc-guide.