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MC4R / Melanocortin receptor 4 · IHC design guide
Design Immunohistochemistry for MC4R
Plan MC4R paraffin IHC with the catalog antibody and score cytoplasmic and membranous staining (HPA tissue IHC; datasheet A00619-2). Interpret positive staining cautiously: HPA rates its tissue IHC evidence uncertain because staining and RNA expression show very low consistency (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
MC4R 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 membranous tissue staining (HPA tissue IHC) | |
| Staining pattern | Most tissues show cytoplasmic and membranous staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A00619-2) | |
| Positive control | Bone marrow+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Poor staining–RNA concordance; verify specificity (HPA tissue IHC) | |
| Regulation | Expression regulation is not specified (UniProt) | |
| Isoform / epitope | No annotated isoforms; epitope side matters across 7 transmembrane segments (UniProt) |
Section 1
Recommended MC4R IHC & IF Protocols
The catalog antibody has an IHC-P protocol; the published protocol below uses fluorescent staining of MC4R-GFP mouse brain sections (PMC3652326).
| Sample | Paraffin-embedded human rectal cancer tissue; fixative not specified (datasheet A00619-2) |
| Fixation | Image fixative and duration unreported (datasheet A00619-2); 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 A00619-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A00619-2) |
| Primary antibody | Rabbit anti-MC4R, 0.5-1μg/ml (datasheet A00619-2) |
| Primary incubation | Overnight at 4 °C (datasheet A00619-2) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A00619-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | MC4R-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and membranous expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet A00619-2). The published cryosection protocol does not establish retrieval conditions for paraffin sections (PMC3652326).
Section 2
What Is the Expected MC4R Staining Pattern?
MC4R is a 7-transmembrane receptor assigned to the cell membrane and cilium membrane (UniProt P32245 topology and subcellular location). For paraffin-section IHC, assess membranous staining in HPA-reported cells such as placental trophoblasts and kidney tubular cells; HPA also describes cytoplasmic staining (HPA tissue IHC: High in both cell types; cytoplasmic and membranous profile). Treat the pattern cautiously: tissue IHC reliability is Uncertain, with very low agreement between antibody staining and RNA expression (HPA tissue IHC).
| Cell outlines stain in placental trophoblasts or kidney tubular cells, with some cytoplasmic signal. | Membrane localisation fits receptor topology (UniProt P32245 topology). These cells have High HPA staining, and HPA describes both membranous and cytoplasmic signal (HPA tissue IHC). Reproducible cell-associated staining is encouraging, but the HPA reliability rating is Uncertain (HPA tissue IHC). |
| Strong nuclear staining dominates while cell outlines are indistinct. | Nuclear dominance conflicts with the annotated cell and cilium membrane locations (UniProt P32245 subcellular location). Review the negative control and detection background before calling it MC4R; the available HPA tissue pattern has Uncertain reliability (HPA tissue IHC; general IHC practice). |
| Adipocytes or prostate glandular cells stain as strongly as the selected positive cells. | HPA lists those cell types as Not detected, while its tissue staining overall is Uncertain (HPA tissue IHC). Compare staining with a negative control and assess cross-reactivity or endogenous detection activity; staining alone cannot identify which cause applies (general IHC practice). |
| Color spreads across stroma, lumens, or the whole section without clear cell boundaries. | A diffuse field is difficult to assign to the reported membranous and cytoplasmic cell-associated profile (HPA tissue IHC). Check control slides for background from blocking, secondary detection, or chromogen development before scoring cells (general IHC practice). |
| No signal appears in a placental trophoblast or kidney tubular cell section. | Both are HPA High examples, so absence warrants a run-level check, although they are not independently verified positive controls here (HPA tissue IHC: High; reliability Uncertain). Check section quality, primary incubation, detection reagents, and counterstain, then compare a control processed in the same run (general IHC practice). |
💡Expected MC4R appearanceCall a result provisionally positive when cell-associated membrane staining, potentially accompanied by cytoplasmic staining, appears in HPA High trophoblasts or kidney tubular cells (UniProt P32245 subcellular location; HPA tissue IHC: profile and High); dominant nuclear or diffuse acellular color is suspect (general IHC practice), and HPA rates the tissue pattern Uncertain (HPA tissue IHC).
| Compartment and epitope orientation | MC4R has 7 membrane spans and annotated extracellular and cytoplasmic regions (UniProt P32245 topology). Interpret a membrane-associated pattern in that context. The supplied sources do not locate this antibody's epitope, so they do not establish which side of the receptor its stain should favor. |
| Tissue selection | HPA reports High staining in placental trophoblasts, kidney tubular cells, small-intestinal glandular cells, and several other cell groups (HPA tissue IHC). Use a named cell population when judging a section; the High label describes reported staining, while the overall tissue profile remains Uncertain (HPA tissue IHC). |
| Negative comparison | Adipocytes, prostate glandular cells, and liver cholangiocytes are listed as Not detected (HPA tissue IHC). They can help flag broadly distributed signal, but an unstained cell is not proof of antibody specificity, particularly given the HPA Uncertain tissue reliability (HPA tissue IHC; general IHC practice). |
| Protein processing and glycosylation | UniProt lists one 332-aa chain, no signal peptide or propeptide, and three glycosylation sites at residues 3, 17, and 26 (UniProt P32245 processing and glycosylation). These annotations do not establish antibody epitope recognition or a target-specific antigen-retrieval effect. |
| Antigen retrieval in paraffin sections | Retrieval is a general IHC workflow variable; judge any change against a matched control and tissue morphology (general IHC practice). Neither source supplied here reports MC4R-specific fixation sensitivity or a retrieval condition, so a stronger signal after retrieval alone does not verify identity. |
| IF/ICC Q&A: should the same appearance be expected? | Membrane localisation remains the biological expectation (UniProt P32245 subcellular location). HPA's ICC-IF summary says Membrane, but gives no main-location assignment or cell-line images; its listed antibody has no ICC status (HPA subcellular; HPA antibodies). Assess IF/ICC on its own guide page. |
| Situation | Likely cause | Next action |
|---|---|---|
| A suspected positive section is blank. | The HPA High designation is an uncertain staining observation, or the IHC run may have failed (HPA tissue IHC: High; reliability Uncertain; general IHC practice). | Verify the named cell population, section integrity, primary incubation, and detection with a same-run control; review retrieval as a general workflow variable, without assuming MC4R-specific fixation sensitivity (general IHC practice). |
| Nuclei stain more strongly than cell edges. | That distribution disagrees with the annotated membrane locations (UniProt P32245 subcellular location); nonspecific nuclear color is a possible technical cause (general IHC practice). | Compare a negative control and inspect chromogen development and counterstain; score membrane and cell-associated signal separately (general IHC practice). |
| Adipocytes or other HPA Not detected cells stain. | Broad staining may reflect cross-reactivity or endogenous detection activity; HPA itself rates its tissue profile Uncertain (HPA tissue IHC; general IHC practice). | Check a negative control, examine where color sits within each cell, and compare the suspect population with HPA High cells on the same run (HPA tissue IHC; general IHC practice). |
| The entire section looks hazy. | Diffuse color can arise from insufficient blocking, secondary detection background, or extended chromogen development (general IHC practice). | Inspect negative controls and reduce the background-producing step identified there; interpret only resolvable cell-associated staining (general IHC practice). |
| Only cytoplasmic staining is visible. | HPA does report cytoplasmic as well as membranous staining, but membrane localisation is annotated by UniProt and the HPA profile is Uncertain (HPA tissue IHC; UniProt P32245 subcellular location). | Record the compartment explicitly, compare cell outlines and controls, and avoid calling cytoplasmic-only signal confirmed MC4R from these sources alone (general IHC practice; HPA tissue IHC: reliability Uncertain). |
| Staining differs between sections or runs. | Technical variation is possible (general IHC practice), and the HPA tissue result has very low antibody-staining versus RNA-expression consistency (HPA tissue IHC). | Compare the same named cell type using matched controls and consistent scoring; report the observed compartment and the HPA Uncertain reliability alongside any result (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing MC4R staining
A00619-2 · IHCIHC analysis of MC4R using anti-MC4R antibody (A00619-2). MC4R was detected in paraffin-embedded section of human rectal cancer tissue. Heat
A00619-2 · IFIF analysis of MC4R using anti-MC4R antibody (A00619-2). MC4R was detected in immunocytochemical section of A431 cells. Enzyme antigen retri
A00619 · IHCImmunohistochemistry of MC4R in human brain tissue with MC4R antibody at 2.5 μg/mL.
A00619 · IFImmunofluorescence of MC4R in Human Brain cells with MC4R antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000166603-MC4R/tissue/cerebral+cortex#imid_4213988
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000166603-MC4R/tissue/colon#imid_4213922
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000166603-MC4R/tissue/liver#imid_4213923
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000166603-MC4R/tissue/kidney#imid_4213916
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for MC4R IHC & IF
🧪Run placenta first: trophoblastic cells should stain (HPA: High in trophoblastic cells). Use adipose tissue adipocytes as the biological tissue negative (HPA: Not detected in adipocytes); on the placenta slide, assess morphologically distinct nontrophoblastic cells for background staining, without assuming their MC4R status (HPA: trophoblastic cells are High).
Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for MC4R; derive a cell-line control from the positive tissue's cell type (Hematopoietic cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the rabbit primary’s clonality when known (caption: rabbit anti-MC4R antibody; standard IHC practice). A verified MC4R knockout sample is the stronger biological negative; peptide competition can supplement it if the immunizing peptide is available (standard IHC practice). For placenta, quench endogenous peroxidase and check for endogenous biotin before using the caption’s biotin-based DAB detection (caption: biotinylated secondary, SABC and DAB; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected paraffin-section caption does not state a fixative (caption: fixative unreported). The documented chromogenic IHC example uses heat retrieval in EDTA at pH 8.0 and a 1 μg/ml primary incubation overnight at 4°C; it does not establish whether retrieval is required for other preparations (caption: retrieval and incubation conditions). Whether frozen sections or IF would be easier is unreported in the supplied evidence; placental endogenous biotin and peroxidase warrant control checks for the documented biotin-based DAB method (caption: SABC and DAB; standard IHC practice).
HPA tissue IHC evidence for MC4R
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Very low consistency between antibody staining and RNA expression data. Pending external verification.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Kidney | Cells in tubules | High | Protein (IHC) | HPA → |
| Nasopharynx | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Pancreas | Exocrine glandular cells | High | Protein (IHC) | HPA → |
| Placenta | Trophoblastic cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MC4R IHC Tips
Troubleshoot MC4R chromogenic IHC in paraffin sections by checking retrieval, cellular localisation, controls and scoring before interpreting staining.
What retrieval should I use when MC4R staining is weak in paraffin sections?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A00619-2). The selected tissue image used that retrieval before incubation with 1 μg/ml antibody overnight at 4°C, so it provides a defined starting condition for troubleshooting (caption A00619-2). If staining remains weak, compare controlled changes in heating duration while keeping section thickness, antibody concentration and detection conditions matched (standard IHC practice). Include a known staining control and a no-primary control in each comparison; stronger colour alone cannot establish MC4R specificity, particularly given the uncertain tissue-IHC reliability (HPA: Uncertain).
Could fixation explain inconsistent MC4R staining across paraffin blocks?
The selected MC4R image identifies a paraffin section but does not report its fixative (caption A00619-2). Target-specific sensitivity to fixation is therefore unknown from the supplied evidence; record the fixative and processing history for each block before comparing staining (caption A00619-2; standard IHC practice). Keep retrieval in EDTA at pH 8.0 and the antibody incubation at 1 μg/ml overnight at 4°C constant while assessing blocks with different processing histories (caption A00619-2). Compare matched tissue regions and controls, because differences between blocks cannot by themselves identify fixation as the cause of a signal change (standard IHC practice).
How should I judge membranous versus cytoplasmic MC4R staining?
Prioritise staining that outlines the cell membrane, while recognising that MC4R is also annotated at the cilium membrane (UniProt P32245: subcellular location). The HPA describes cytoplasmic and membranous staining in most tissues, but rates its tissue-IHC reliability as uncertain because staining and RNA expression show very low consistency (HPA: tissue IHC). Score membrane and cytoplasm separately rather than treating diffuse cytoplasmic colour as equivalent to a membrane pattern (UniProt P32245: subcellular location; standard IHC practice). Check adjacent sections, cell morphology and no-primary controls before interpreting a punctate or apical signal as ciliary MC4R; chromogenic staining alone does not establish that localisation (standard IHC practice).
Does the antibody epitope change how I interpret MC4R staining?
Confirm the antibody's immunogen or mapped epitope before assigning an absent or altered staining pattern to MC4R biology (standard IHC practice). MC4R has a 332-residue annotated chain and no listed isoforms, so the supplied record does not support an isoform-specific explanation for staining differences (UniProt P32245: processing and isoforms). Its residues 1–47 are extracellular, residues 303–332 are cytoplasmic, and glycosylation sites occur at 3, 17 and 26 (UniProt P32245: topology and glycosylation). Without a mapped antibody epitope, neither the accessible side of the membrane nor an effect of those glycosylation sites on staining can be assigned (UniProt P32245: topology; standard IHC practice).
How can IF help assess an ambiguous MC4R IHC pattern?
Use IF as a separate localisation check, alongside the paraffin-section chromogenic IHC result, with controls appropriate to each detection method (standard IHC/IF practice). Multiplex MC4R with a marker for the expected cell type; trophoblastic cells in placenta are one reported IHC-positive population, although that tissue-IHC evidence is rated uncertain (HPA: placenta High; HPA: Uncertain). Choose a fluorophore channel after checking tissue autofluorescence, and include single-colour and no-primary controls when evaluating apparent overlap (standard IF practice). If the antibody epitope is cytoplasmic, assess permeabilisation; if it is extracellular, assess staining without permeabilisation first, because the mapped epitope is not supplied here (UniProt P32245: topology; standard IF practice).
What causes widespread brown signal in MC4R chromogenic IHC?
Compare a no-primary section with the stained section to identify colour produced independently of the MC4R primary antibody (standard IHC practice). The selected workflow used 10% goat serum, a biotinylated secondary antibody, a streptavidin–biotin complex and DAB, so assess nonspecific binding and biotin-dependent background under those same conditions (caption A00619-2; standard IHC practice). Include an appropriate endogenous peroxidase block and inspect pigmented, damaged and section-edge regions before calling brown deposits positive (standard IHC practice). If background persists, titrate the primary around the documented 1 μg/ml condition while retaining matched controls and the same EDTA pH 8.0 retrieval (caption A00619-2; standard IHC practice).
How should I quantify MC4R staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, and report membranous and cytoplasmic staining separately because their interpretation differs for MC4R (UniProt P32245: subcellular location; standard IHC practice). For each compartment, record the percentage of positive cells and intensity, or calculate an H-score from 0–300 using fixed thresholds (standard IHC practice). For sparse positive cells, report positive-cell density per mm² of evaluable tissue rather than per whole image (standard IHC practice). Normalise comparisons to the same eligible cell population or tissue area, exclude damaged regions by a preset rule, and retain matched controls because HPA tissue-IHC reliability is uncertain (HPA: Uncertain; standard IHC practice).
When is apparent MC4R positivity more likely to be artefact?
Treat isolated nuclear colour, strong section-edge staining and signal concentrated in necrotic areas as suspect until morphology and controls support the result (UniProt P32245: membrane localisation; standard IHC practice). MC4R is annotated at the cell and cilium membranes, while HPA reports both membranous and cytoplasmic tissue staining with uncertain reliability (UniProt P32245: subcellular location; HPA: tissue IHC). Verify that positive cells belong to the population being scored; HPA reports high staining in placental trophoblastic cells but no detected staining in adipocytes (HPA: placenta High; HPA: adipose tissue Not detected). A matching no-primary signal implicates endogenous enzyme or detection-system background rather than specific antibody binding (standard IHC practice).
Boster reagents
Best MC4R / Melanocortin receptor 4 IHC Antibodies
Both anti-MC4R antibodies have human tissue IHC and human cell IF images (catalog image captions); both list human, mouse and rat reactivity (catalog reactivity).
Real IHC data
Anti-MC4-R/MC4R Antibody ®
Cat # A00619-2
Real IHC data
Anti-Melanocortin receptor 4 MC4R Antibody
Cat # A00619
A00619-2 shows IHC in a human rectal cancer paraffin section and IF/ICC in A431 cells (A00619-2 image captions; catalog applications: IF, ICC). A00619 shows IHC in human brain tissue and IF in human brain cells (A00619 image captions; catalog applications: IHC-P, IF).
Which to pick: Choose A00619-2 for paraffin-section IHC or A431 cell IF/ICC, matching its images (A00619-2 image captions); its IHC caption does not report the fixative (A00619-2 IHC caption). Choose A00619 for human brain tissue IHC-P or human brain cell IF, matching its images (A00619 image captions); its IHC caption also does not report the fixative (A00619 IHC caption). For cross-species work, both list human, mouse and rat reactivity, but their IHC and IF images show human samples only (catalog reactivity; image captions); clonality is unreported for both (catalog clone fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P32245 (MC4R_HUMAN, Melanocortin receptor 4).
- Human Protein Atlas. MC4R tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. MC4R subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. MC4R antibody validation summary (1 antibodies).
- Neuroanatomy of melanocortin-4 receptor pathway in the lateral hypothalamic area. The Journal of comparative neurology 2012 — PMC3652326.
- PubMed PMID:8392067 — UniProt-cited evidence.
- PubMed PMID:7854347 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for MC4R (Melanocortin receptor 4). Expected localisation Cytoplasmic and membranous tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A00619-2). Validated IHC antibody: A00619-2 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human rectal cancer tissue; fixative not specified (datasheet A00619-2); 2. Fixation: Image fixative and duration unreported (datasheet A00619-2); 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 A00619-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A00619-2); 8. Primary antibody: Rabbit anti-MC4R, 0.5-1μg/ml (datasheet A00619-2); 9. Primary incubation: Overnight at 4 °C (datasheet A00619-2). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Poor staining–RNA concordance; verify specificity (HPA tissue IHC); Isoform / epitope: No annotated isoforms; epitope side matters across 7 transmembrane segments (UniProt); Regulation: Expression regulation is not specified (UniProt). Evidence: UniProt P32245, HPA tissue IHC · Uncertain, HPA ICC-IF · Membrane, Datasheet A00619-2, PMC3652326. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/MC4R/immunohistochemistry-ihc-guide.