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MYC / Myc proto-oncogene protein · IHC design guide
Design Immunohistochemistry for MYC
Plan chromogenic MYC IHC on paraffin sections using nuclear and nucleolar tissue staining as the benchmark (HPA tissue IHC). Start with the IHC-validated total-MYC antibody M00026-1 at 1:50 (datasheet M00026-1), and assess glial and stromal cells separately (HPA tissue IHC).
Evidence assembled Sep 2026 · For research use; verify linked source records and product datasheet before use
MYC 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 nucleolar staining in tissue (HPA tissue IHC) | |
| Staining pattern | Nuclear and nucleolar staining in most cells; glia and stroma spared (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Caudate+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; overfixation may mask epitopes (standard IHC practice) | |
| Caveat | Staining and RNA expression have medium consistency (HPA tissue IHC) | |
| Regulation | No expression inducer is annotated (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope differences are unspecified (UniProt) |
Section 1
Recommended MYC IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by published MYC IHC methods for mantle cell lymphoma, breast cancer specimens, and large B-cell lymphoma (PMC3871984; PMC3276715; PMC6365198).
| Sample | FFPE tissue sections (standard) |
| Fixation | 10% neutral-buffered formalin, 24 h, standard processing (standard) |
| 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-MYC, 1:200-1:1000 (datasheet M00026-6) |
| 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 | MYC-positive staining in neuronal cells of caudate (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear and nucleolar expression in essentially all cells except in glial- and stroma cells. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 min (page antigen retrieval); adjust using the published retrieval conditions if staining needs optimization (PMC3871984; PMC3276715).
Section 2
What Is the Expected MYC Staining Pattern?
MYC is mainly nuclear, including the nucleoplasm and nucleolus (UniProt P01106). HPA reports nuclear and nucleolar staining in essentially all cells except glial and stromal cells; its tissue IHC rating is Approved, with medium consistency against RNA data (HPA tissue IHC). MYC has no transmembrane segment (UniProt P01106 topology).
| Nuclear/nucleolar stain in cervix squamous cells (HPA: High). | Fits the expected MYC pattern (HPA tissue IHC; UniProt P01106 localization). |
| Predominantly membranous staining. | Investigate artefact: MYC has no transmembrane segment (UniProt P01106 topology). |
| Strong staining confined to glial or stromal cells. | Check cross-reactivity or endogenous activity (HPA tissue IHC profile; IHC practice). |
| Diffuse colour across cells and tissue background. | Check blocking, washing and chromogen background (IHC practice). |
| No nuclear stain in cervix squamous cells (HPA: High). | Check retrieval, antibody dilution and detection controls (IHC practice). |
💡Expected MYC appearanceNuclear/nucleolar staining in cervix squamous cells should be High (HPA); broad membranous colour flags artefact (UniProt P01106 topology; IHC practice).
| Fixation and cross-linking (IHC practice) | Overfixation may mask nuclear epitopes; compare fixation conditions (IHC practice). |
| Antigen retrieval (IHC practice) | Insufficient retrieval can weaken nuclear staining in paraffin sections (IHC practice). |
| 3 isoforms (UniProt P01106) | Confirm which isoforms the antibody recognizes; epitope coverage is unspecified (UniProt P01106; IHC practice). |
| Endogenous peroxidase (IHC practice) | Unblocked activity can produce misleading chromogenic colour (IHC practice). |
| Species reactivity (IHC practice) | Confirm antibody reactivity for the specimen species (IHC practice). |
| IF/ICC: where should signal appear? | Primarily in the nucleoplasm (HPA ICC-IF: enhanced nucleoplasmic localization). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in cervix squamous cells (HPA: High). | Masked epitope or weak detection (IHC practice). | Optimize retrieval and check antibody and detection controls (IHC practice). |
| Weak nuclear signal (HPA tissue IHC: nuclear pattern). | Fixation or retrieval may reduce epitope access (IHC practice). | Review fixation and optimize retrieval (IHC practice). |
| Membranous colour dominates. | Possible nonspecific signal; MYC lacks a membrane span (UniProt P01106 topology). | Check antibody specificity and detection controls (IHC practice). |
| Diffuse tissue background. | Incomplete blocking or excess antibody (IHC practice). | Improve blocking and washing; titrate antibody (IHC practice). |
| Colour appears without primary antibody. | Endogenous enzyme activity or detection background (IHC practice). | Block peroxidase and inspect the no-primary control (IHC practice). |
| Strong glial or stromal staining (HPA tissue IHC profile). | Possible cross-reactivity or endogenous activity (IHC practice). | Compare no-primary control and a known-positive tissue (IHC practice). |
Experiment evidence
IHC & IF images showing MYC staining
M00026-6 · IHCHuman tonsil was stained with Anti-c-Myc rabbit antibody
M00026-6 · IHCHuman lung was stained with Anti-c-Myc rabbit antibody
M00026-1 · IHCImmunohistochemical analysis of paraffin-embedded human lung cancer, using c-Myc Antibody.
M00026-1 · IFImmunofluorescent analysis using the Antibody at 1:50 dilution.
P00026-2 · IHCImmunohistochemical analysis of paraffin-embedded human lung carcinoma, using Phospho-c-Myc (S62) Antibody .
A00026S62-1 · IFICC staining Phospho-c-Myc(S62) in Hela cells (green). The nuclear counter stain is DAPI (blue). Cells were fixed in paraformaldehyde, perme
PB9092 · IFIF analysis of c-Myc using anti-c-Myc antibody (PB9092) and anti-Beta Tubulin antibody (M01857-3). c-Myc was detected in immunocytochemical
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000136997-MYC/tissue/cerebral+cortex#imid_3467304
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000136997-MYC/tissue/colon#imid_3467230
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000136997-MYC/tissue/liver#imid_3467247
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000136997-MYC/tissue/kidney#imid_3467232
HPA · ICC-IF PC-3Human Protein Atlas immunofluorescence, PC-3, antibody HPA055893; green = MYC, blue = nucleus, red = microtubules.
HPA · ICC-IF PC-3Human Protein Atlas immunofluorescence, PC-3, antibody HPA055893; green = MYC, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for MYC IHC & IF
🧪Run cervix first: its squamous epithelial cells should show MYC staining (HPA: High in cervical squamous epithelial cells). HPA detects MYC in all 45 scored tissues, so use no-primary and isotype controls for the negative comparison; other cells on the positive slide may stain, while empty tissue spaces should remain clear (HPA: no negative tissue; standard IHC practice).
Positive control tissue: Caudate (Neuronal cells, HPA High)
Negative control tissue: None in HPA: MYC 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 MYC in PC-3, U-251MG, U2OS, HEK293, SK-MEL-30, HeLa BAC 5379, HeLa , with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Run a no-primary, secondary-only slide and a matched-host, matched-clonality isotype control; a matched MYC-knockout specimen provides a biological specificity control (standard IHC practice). For chromogenic IHC, quench endogenous peroxidase and check for background at the cervical epithelial surface (standard IHC practice).
⚠️Feasibility: Fix cervical sections promptly in buffered formalin and keep fixation times consistent across controls; optimize heat-induced antigen retrieval because fixation can mask epitopes in paraffin sections (standard IHC practice). Score predominantly nuclear staining, using the nucleoplasm as the main localization reference (HPA: nucleoplasm enhanced; UniProt P01106: nuclear localization). Frozen-section IF may reduce retrieval needs but can sacrifice tissue morphology; check cervical surface staining for nonspecific signal before scoring (standard IHC/IF practice).
HPA tissue IHC evidence for MYC
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 |
|---|---|---|---|---|
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | High | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | High | Protein (IHC) | HPA → |
| Lung | Alveolar cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: MYC is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced MYC IHC Tips
Troubleshoot MYC staining by checking nuclear localisation, specimen handling and controls before comparing chromogenic IHC scores (UniProt P01106; HPA tissue IHC).
How should I optimise retrieval when MYC nuclear staining is weak in paraffin sections?
Use Tris-EDTA, pH 9.0, heat-induced retrieval at 95–98 °C for 20 min as the starting condition (page retrieval specification). Allow sections to cool in retrieval buffer, then compare a known positive control with the test section under the same detection conditions (standard IHC practice). MYC is chiefly nuclear and nucleoplasmic, so judge retrieval by restored nuclear signal rather than stronger diffuse cytoplasmic colour (UniProt P01106; HPA subcellular). If staining remains weak, test a milder citrate buffer at pH 6.0 on adjacent sections and assess tissue preservation alongside signal (standard IHC practice). Keep antibody concentration and chromogen development constant during that comparison (standard IHC practice).
Could fixation explain weak or uneven MYC staining across my paraffin sections?
Record fixative, time before fixation and fixation duration for each specimen before comparing MYC intensity (standard IHC practice). For newly prepared material, a consistent 10% neutral buffered formalin workflow with a documented 6–24 h fixation window provides a practical starting point, adjusted to specimen size (standard histology practice). Underfixation can impair morphology, while prolonged fixation can reduce epitope access; compare affected sections with a similarly processed positive control (standard IHC practice). Because MYC is detected mainly in nuclei and nucleoplasm, examine nuclear detail before calling pale staining biological absence (UniProt P01106; HPA subcellular). Change retrieval only after confirming that the fixation history and control staining are credible (standard IHC practice).
What MYC staining pattern should I expect, and when is cytoplasmic signal concerning?
Prioritise nuclear staining: MYC is annotated in the nucleus and nucleoplasm, with nucleolar, chromosomal and cytoplasmic locations also recorded (UniProt P01106). Tissue IHC describes widespread nuclear and nucleolar expression, with glial and stromal cells noted as exceptions in its overall profile (HPA tissue IHC). Score convincing cytoplasmic staining separately from nuclear staining, particularly when nuclei are faint or the negative control also develops colour (standard IHC practice). Chromatin association can fall with hyperphosphorylation, and nucleolar localisation depends on HEATR1, so compartment differences need biological and technical review (UniProt P01106). Compare adjacent sections using the same 20 min retrieval and chromogen development before interpreting a shift (page retrieval specification; standard IHC practice).
How can epitope selection affect MYC detection after paraffin processing?
Check the antibody's stated immunogen or epitope against the MYC sequence before comparing staining across experiments (standard IHC practice). UniProt lists 3 MYC isoforms, a bHLH domain at residues 369–421, and multiple modified residues, so epitope location may matter for recognition (UniProt P01106). The record alone does not establish which isoforms a particular antibody detects or whether a modification blocks binding (UniProt P01106). If an epitope is disclosed, compare its location with the listed isoforms and modifications, then use matched positive and negative controls in paraffin sections (UniProt P01106; standard IHC practice). Treat a change after retrieval or fixation as an accessibility finding until independent evidence supports an isoform-specific interpretation (standard IHC practice).
How can I use IF to check an ambiguous MYC IHC localisation pattern?
Use IF as a complementary localisation check while keeping chromogenic paraffin IHC as the scoring assay (standard IHC practice). MYC should be assessed against a nuclear counterstain, and multiplexing with a marker for the expected cell population can test whether the signal belongs to the cells being scored (UniProt P01106; standard IF practice). Choose a fluorophore channel with low tissue autofluorescence and include single-colour and no-primary controls to separate bleed-through and background from MYC signal (standard IF practice). MYC has no transmembrane segment and is primarily nuclear, so permeabilisation must admit antibody to intracellular nuclear epitopes; titrate detergent exposure to preserve morphology (UniProt P01106; standard IF practice). Compare compartment patterns with IHC rather than equating fluorescence intensity with DAB intensity (standard IHC/IF practice).
How do I reduce diffuse or misleading brown background in MYC IHC?
Inspect a no-primary control and a positive control processed beside the test section before changing the MYC antibody concentration (standard IHC practice). Block endogenous peroxidase before HRP-based chromogenic detection, and use a protein block and thorough washes to limit nonspecific signal (standard IHC practice). Shorten chromogen development if brown colour appears equally in negative controls, extracellular spaces or damaged tissue (standard IHC practice). Because MYC is mainly nucleoplasmic, diffuse brown colour without corresponding nuclear detail is insufficient evidence of specific staining (HPA subcellular; standard IHC practice). Titrate the catalog antibody in adjacent sections while holding the 20 min retrieval and detection steps constant (page retrieval specification; standard IHC practice).
What is a defensible way to score MYC in chromogenic paraffin IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and count evaluable nuclei before assigning a MYC score (standard IHC practice). Report the percentage of positive nuclei and, when intensity matters, an H-score from 0–300 using percentages at 0, 1+, 2+ and 3+ intensity (standard IHC scoring practice). Normalise positive counts to all evaluable nuclei in the same annotated compartment; for spatial comparisons, report positive nuclei per mm² of viable tissue (standard image analysis practice). Exclude folds, necrosis and edge artefacts using rules fixed before scoring (standard IHC practice). Keep staining and analysis thresholds consistent because MYC tissue staining is broadly nuclear, while some stromal and glial populations are exceptions in the HPA profile (HPA tissue IHC; standard IHC practice).
How can I distinguish a true MYC-positive cell from staining artefact?
Call a cell positive when brown signal resolves to an intact nucleus within the expected cell population and exceeds the matched negative-control background (UniProt P01106; standard IHC practice). HPA reports high staining in caudate neuronal cells and cerebellar Purkinje cells, while its overall profile notes limited staining in glial and stromal cells; use that contrast cautiously because HPA rates staining consistency as medium (HPA tissue IHC). Review cytoplasmic-only colour, section edges, folds and necrotic areas for artefact before scoring (UniProt P01106; standard IHC practice). A no-primary control helps reveal endogenous enzyme activity or detection-system background in chromogenic IHC (standard IHC practice). Confirm disputed patterns on an adjacent section with preserved morphology and the same 20 min retrieval (page retrieval specification; standard IHC practice).
Boster reagents
Best MYC / Myc proto-oncogene protein IHC Antibodies
IHC figures show MYC staining in human tonsil and lung tumors; IF/ICC figures show HeLa cells (figure captions). Catalog-listed reactivity includes human, mouse, and rat (catalog: reactivity).
Real IHC data
Anti-c-Myc Rabbit Monoclonal Antibody
Cat # M00026-6
Real IHC data
Anti-c-Myc Monoclonal Antibody
Cat # M00026-1
Real IHC data
Anti-Phospho-c-Myc (S62) Rabbit Monoclonal Antibody
Cat # P00026-2
Real IF data
Anti-c-Myc (Phospho-S62) Antibody
Cat # A00026S62-1
Real IF data
Anti-c-Myc Antibody ®
Cat # PB9092
M00026-6 has an IHC figure from human tonsil, M00026-1 from paraffin-embedded human lung cancer, and P00026-2 from paraffin-embedded human lung carcinoma stained for phospho-S62 MYC (figure captions). A00026S62-1 has a phospho-S62 IF/ICC figure in HeLa cells, and PB9092 has a MYC IF/ICC figure in HeLa cells (figure captions).
Which to pick: For FFPE IHC, choose M00026-1 for total MYC or P00026-2 for phospho-S62 MYC; both are rabbit monoclonals with paraffin-embedded human lung tumor figures (catalog: titles; figure captions). For IF/ICC, choose A00026S62-1 for phospho-S62 or PB9092 for total MYC; both have HeLa cell figures and list IF/ICC applications (catalog: titles/applications; figure captions). For work across human, mouse, and rat, M00026-6 is a recombinant rabbit monoclonal listed for IHC and IF in all three species, with an IHC figure from human tonsil (catalog: description/applications/reactivity; figure caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P01106 (MYC_HUMAN, Myc proto-oncogene protein).
- Human Protein Atlas. MYC tissue IHC expression (reliability: Approved).
- Human Protein Atlas. MYC subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. MYC antibody validation summary (4 antibodies).
- Immunohistochemical evaluation of MYC expression in mantle cell lymphoma. Histopathology 2013 — PMC3871984.
- MYC gene amplification is often acquired in lethal distant breast cancer metastases of unamplified primary tumors. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 2012 — PMC3276715.
- Comparison of evaluation techniques, including digital image analysis, for MYC protein expression by immunohistochemical stain in aggressive B-cell lymphomas. Human pathology 2019 — PMC6365198.
- Double-hit lymphomas: clinical, morphological, immunohistochemical and cytogenetic study in a series of Brazilian patients with high-grade non-Hodgkin lymphoma. Diagnostic pathology 2017 — PMC5219810.
- PubMed PMID:6414718 — UniProt-cited evidence.
- PubMed PMID:6321164 — UniProt-cited evidence.
- PubMed PMID:6298632 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for MYC (Myc proto-oncogene protein). Expected localisation Nuclear and nucleolar staining in tissue (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: M00026-6 at 1:200-1:1000 (the product's own IHC validation image is shown). Controls (HPA): positive Caudate (Neuronal cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. Protocol steps: 1. Sample: FFPE tissue sections (standard); 2. Fixation: 10% neutral-buffered formalin, 24 h , standard processing (standard); 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-MYC, 1:200-1:1000 (datasheet M00026-6); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent; overfixation may mask epitopes (standard IHC practice); Caveat: Staining and RNA expression have medium consistency (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope differences are unspecified (UniProt); Regulation: No expression inducer is annotated (UniProt). Evidence: UniProt P01106, HPA tissue IHC · Approved, HPA ICC-IF · nucleoplasm., Datasheet M00026-6, PMC3871984, PMC3276715. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/MYC/immunohistochemistry-ihc-guide.