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MEGF8 / Multiple epidermal growth factor-like domains protein 8 · IHC design guide
Design Immunohistochemistry for MEGF8
Plan MEGF8 paraffin IHC around the reported cytoplasmic tissue pattern (HPA tissue IHC). Compare staining with membrane topology (UniProt), and check the antibody’s epitope and documented IHC dilution before scoring (UniProt; datasheet).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
MEGF8 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 tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in several tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Adrenal gland+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 paraffin sections (standard IHC practice; not target-specific) | |
| Caveat | Splice/transcript discrepancy may complicate interpretation (HPA tissue IHC) | |
| Regulation | No specific expression regulator annotated (UniProt) | |
| Isoform / epitope | 2 isoforms; check whether the epitope is extracellular or cytoplasmic (UniProt) |
Section 1
Recommended MEGF8 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with one published mouse brain cryosection IHC method (PMC10778434).
| Sample | Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A12840-1) |
| Fixation | Image fixative and duration unreported (datasheet A12840-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane 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-MEGF8, 1:50-1:200 (datasheet A12840-1) |
| 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 | MEGF8-positive staining in glandular cells of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in several different tissue types. No signal in the no-primary control. |
💡Decision noteFor paraffin sections, start with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval rule). The published cryosection method does not report retrieval (PMC10778434).
Section 2
What Is the Expected MEGF8 Staining Pattern?
MEGF8 is a membrane protein with a large extracellular region and a cytoplasmic tail (UniProt Q7Z7M0 topology). In paraffin section IHC, expect cytoplasmic staining across several tissue types, including glandular cells, kidney tubule cells and cardiomyocytes (HPA tissue IHC: Approved; Medium). HPA flags a splice or transcript discrepancy, so assess the observed tissue pattern alongside the antibody’s validation status (HPA tissue IHC: reliability description).
| Moderate staining in colon or duodenal glandular cells, kidney tubule cells or cardiomyocytes. | These match HPA’s Medium IHC observations in the named cells (HPA tissue IHC). Compare staining within the relevant cell population; a positive result need not appear in every cell of a section (general IHC practice). |
| Predominantly crisp nuclear staining, with little staining in the expected cell population. | A nuclear-only pattern conflicts with HPA’s reported cytoplasmic tissue profile and UniProt’s membrane assignment (HPA tissue IHC; UniProt Q7Z7M0). Check counterstain, detection and primary-antibody controls before assigning it to MEGF8 (general IHC practice). |
| Strong staining in adipocytes or bone-marrow hematopoietic cells, while the expected positive cells are unstained. | HPA reports MEGF8 as Not detected in those cell populations (HPA tissue IHC). The discordance raises concern for cross-reactivity or endogenous detection activity; it does not prove either cause (general IHC practice). |
| Broad, even color over cells and surrounding section structures, with no cell-specific pattern. | This is difficult to score as MEGF8 because HPA describes a cellular staining pattern (HPA tissue IHC). Diffuse signal can reflect background from blocking, washes or detection chemistry (general IHC practice). |
| No staining in colon glandular cells or kidney tubule cells that are suitable for assessment. | Both are Medium HPA IHC examples, so an absent signal warrants a run check (HPA tissue IHC). Confirm section integrity, primary-antibody application, retrieval and detection with appropriate controls; HPA provides no MEGF8-specific retrieval condition (general IHC practice). |
💡Expected MEGF8 appearanceCall a section positive when the expected cells show discernible cytoplasmic staining at approximately HPA’s Medium level; staining in HPA Not detected cells, isolated nuclear color or uniform background needs control review before interpretation (HPA tissue IHC; general IHC practice).
| Compartment and topology | HPA describes cytoplasmic tissue staining, whereas UniProt assigns MEGF8 to the membrane, with residues 28–2647 extracellular and 2669–2845 cytoplasmic (HPA tissue IHC; UniProt Q7Z7M0 topology). These descriptions need not yield a sharply outlined membrane in chromogenic sections; do not infer the antibody’s epitope from them. |
| Antibody validation and transcript caveat | HPA labels tissue IHC Approved for HPA049248 and flags a splice or transcript discrepancy (HPA tissue IHC; HPA antibodies). Approved is the supplied status; independent-antibody reproduction or an Enhanced IHC designation is not established by this payload. |
| Isoforms and epitope knowledge | UniProt lists two MEGF8 isoforms, while the supplied antibody record gives no epitope position (UniProt Q7Z7M0; HPA antibodies). An isoform-specific staining prediction therefore cannot be made from this evidence. |
| Processing and glycosylation | UniProt lists a signal peptide at residues 1–27, a mature chain beginning at residue 28 and six glycosylation sites (UniProt Q7Z7M0). These annotations describe the protein; they do not establish shedding, an IHC staining difference or an antigen-retrieval requirement. |
| IF/ICC: what localisation is supported? | HPA summarizes MEGF8 as membrane-localized for ICC-IF but supplies no main-location assignment or cell-line images here (HPA subcellular). Treat that summary as context for IF/ICC interpretation; this IHC section supplies no IF/ICC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells show no chromogenic signal. | An IHC run or reagent problem is possible; HPA reports Medium staining in colon glands and kidney tubules (HPA tissue IHC). | Use a suitable HPA-positive tissue as a run control and verify primary-antibody application, retrieval and detection steps (HPA tissue IHC; general IHC practice). |
| The section is uniformly dark. | Background can arise from insufficient blocking or washing, or from endogenous detection activity (general IHC practice). | Review a no-primary control, blocking and wash steps; assess endogenous activity relevant to the detection chemistry (general IHC practice). |
| Only nuclei appear positive. | A nuclear-only result conflicts with the supplied cytoplasmic tissue profile and membrane annotation (HPA tissue IHC; UniProt Q7Z7M0). | Inspect counterstain and no-primary controls, then compare the expected cell populations before calling MEGF8 positive (general IHC practice; HPA tissue IHC). |
| Adipocytes or bone-marrow hematopoietic cells appear strongly positive. | HPA lists those cells as Not detected; cross-reactivity or endogenous activity is possible, but neither is proven (HPA tissue IHC; general IHC practice). | Compare an HPA Medium-positive cell population and detection controls on the same run; avoid treating the discordant color alone as MEGF8 (HPA tissue IHC; general IHC practice). |
| Staining varies between glandular samples. | HPA reports Medium staining in several glandular sites but Not detected staining in cervix, epididymis and fallopian-tube glands (HPA tissue IHC). | Identify the specific tissue and cell population before judging consistency; compare each result with its matching HPA entry (HPA tissue IHC). |
| The signal looks cytoplasmic rather than like a thin membrane rim. | That appearance can agree with HPA’s tissue IHC profile despite UniProt’s membrane topology (HPA tissue IHC; UniProt Q7Z7M0). | Score the expected cells and controls together; reserve a membrane-rim requirement until the antibody and assay establish one (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing MEGF8 staining
A12840-1 · IHCImmunohistochemistry (IHC) analyzes of EGFL4 (R1274) pAb in paraffin-embedded human breast carcinoma tissue at 1:50.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000105429-MEGF8/tissue/cerebral+cortex#imid_13327819
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000105429-MEGF8/tissue/colon#imid_13316897
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000105429-MEGF8/tissue/liver#imid_13316898
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000105429-MEGF8/tissue/kidney#imid_13316899
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for MEGF8 IHC & IF
🧪Run colon first: its glandular cells should stain at the HPA Medium level (HPA: Colon, glandular cells, Medium). Use cervix glandular cells as the negative tissue (HPA: Cervix, glandular cells, Not detected); on the colon slide, stromal cells can serve as candidate internal negatives if they show counterstain without specific chromogen, but their negativity needs confirmation because HPA scores only the glandular cells in the supplied row (HPA: Colon, glandular cells, Medium).
Positive control tissue: Adrenal gland (Glandular cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for MEGF8; 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 concentration-matched isotype control appropriate to the primary antibody’s host species and clonality; and, where available, a MEGF8 knockout specimen or validated immunizing-peptide block (standard IHC practice). Quench endogenous peroxidase and inspect colon gland lumens for nonspecific chromogen before scoring glandular staining (standard chromogenic IHC practice).
⚠️Feasibility: A MEGF8-specific fixation window or fixation effect is unreported, and the selected A12840-1 paraffin-section caption does not state its fixative (caption: fixative not stated). Antigen retrieval dependence is also unreported, so compare retrieval conditions on paired paraffin sections; the supplied evidence does not establish that frozen sections or IF are easier (caption: paraffin-embedded tissue; HPA: no ICC-IF images). Colon gland lumens can complicate chromogen interpretation, so score staining against cell morphology and the control slides (standard IHC practice).
HPA tissue IHC evidence for MEGF8
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Caution, Splice and/or transcript discrepancy exists.). 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 | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Neuropil | Medium | Protein (IHC) | HPA → |
| Colon | Glandular cells | Medium | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Medium | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MEGF8 IHC Tips
Troubleshoot MEGF8 chromogenic IHC in paraffin sections using the page retrieval setting, the selected antibody image, and tissue and topology evidence.
How should I adjust retrieval when MEGF8 staining is weak or uneven?
Start MEGF8 paraffin-section IHC with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval rule). If staining is weak, first check section adhesion, complete dewaxing, temperature, and the actual time at temperature before changing retrieval conditions (standard IHC practice). If those checks pass, compare a shorter and a longer heating interval on adjacent sections while keeping the antibody and detection steps fixed; excessive heating can damage morphology (standard IHC practice). Record whether signal changes in glandular cells or kidney tubules, which have medium HPA staining, alongside any change in background (HPA: tissue IHC; standard IHC practice).
Could fixation explain weak or patchy MEGF8 staining?
MEGF8-specific sensitivity to fixation is unknown from the supplied evidence; the selected image describes paraffin-embedded breast carcinoma but does not state a fixative (A12840-1 caption). Record the fixative, fixation duration, processing history, and section age for each specimen so a weak result can be compared with a similarly handled section (standard IHC practice). Compare specimens processed together before attributing a staining difference to biology, and inspect morphology for poorly preserved or unevenly processed regions (standard IHC practice). If fixation history varies, repeat the same citrate pH 6.0, 20 min retrieval and detection workflow across sections; do not infer a MEGF8-specific fixation effect from tissue patterns (page retrieval rule; standard IHC practice).
Should MEGF8 appear at the membrane or in the cytoplasm?
MEGF8 is annotated as a membrane protein with an extracellular region at residues 28–2647, a transmembrane segment at 2648–2668, and a cytoplasmic region at 2669–2845 (UniProt Q7Z7M0 topology). HPA tissue IHC reports cytoplasmic expression across several tissue types, so score a reproducible cytoplasmic pattern while noting any membrane accentuation separately (HPA: tissue IHC). Check whether the signal follows intact cell outlines or instead fills damaged tissue, folds, and section edges (standard IHC practice). An intracellular signal cannot by itself identify which side of MEGF8 the antibody recognizes; epitope information and an appropriate specificity control are needed (UniProt Q7Z7M0 topology; standard IHC practice).
Could isoforms or epitope placement change the MEGF8 IHC pattern?
MEGF8 has 2 annotated isoforms, and HPA marks its tissue IHC as Approved with a splice or transcript discrepancy caution (UniProt Q7Z7M0 isoforms; HPA: reliability). Confirm the antibody's immunogen or mapped epitope and determine whether it is shared by both isoforms before comparing specimens (standard IHC practice; UniProt Q7Z7M0 isoforms). The large extracellular region contains EGF-like and other domains and has 6 annotated glycosylation sites, so document epitope location when interpreting retrieval-dependent staining (UniProt Q7Z7M0 domains, topology, glycosylation). A different pattern with another antibody may reflect epitope or isoform coverage; assess each reagent with its own controls rather than assigning a cellular mechanism from staining alone (standard IHC practice).
How can I check a MEGF8 pattern by multiplex IF?
Treat IF as a separate validation experiment because the supplied selected-antibody image is paraffin-section chromogenic IHC and HPA lists no cell lines with MEGF8 ICC/IF images (A12840-1 caption; HPA: subcellular). Multiplex MEGF8 with an independently validated marker for the cell population being scored, and include single-stain controls to assess channel bleed-through (standard IF practice). Choose a fluorophore and imaging channel after measuring tissue autofluorescence, then hold exposure settings constant across comparison samples (standard IF practice). Select permeabilisation based on the mapped epitope: intracellular access may require it, whereas an extracellular epitope should be assessed without unnecessary membrane disruption (UniProt Q7Z7M0 topology; standard IF practice).
What should I check when MEGF8 DAB staining is diffuse?
Run a no-primary control through the same secondary detection and DAB steps to identify signal from the detection system, and inspect the peroxidase-block step (standard chromogenic IHC practice). Compare stained regions with tissue folds, damaged edges, pigment, and necrotic areas before calling diffuse brown signal MEGF8-positive (standard IHC practice). The selected A12840-1 image used 1:50 in paraffin-embedded breast carcinoma; treat that concentration as a tested image condition, not a universal dilution (A12840-1 caption). Titrate antibody concentration against a consistently stained reference compartment, such as kidney tubules, while checking background in HPA-undetected cell populations on suitable sections (HPA: tissue IHC; standard IHC practice).
How should I score MEGF8 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and scoring compartment before analysis, then use an H-score based on staining intensity and the percentage of positive cells, or report percentage positive alone (standard IHC practice). For a spatial question, count positive cells per mm² of viable tissue and report the analyzed area (standard IHC practice). Normalize comparisons to the same cell type and viable area, with matched staining and image settings across specimens; exclude folds, edges, and necrosis consistently (standard IHC practice). Keep glandular-cell, tubular-cell, and neuropil measurements separate because HPA reports these as distinct MEGF8-positive compartments (HPA: tissue IHC).
How do I distinguish convincing MEGF8 staining from artefact?
A convincing result is reproducible in intact cells, follows a defined compartment, and differs from the no-primary control under the same detection conditions (standard IHC practice). HPA reports cytoplasmic expression and medium staining in several compartments, including kidney tubules and adrenal glandular cells; use those observations as context rather than proof for an individual section (HPA: tissue IHC). Reconsider staining concentrated at cut edges, folds, necrosis, or areas with residual endogenous peroxidase activity, since these can produce misleading chromogenic signal (standard IHC practice). The selected breast carcinoma image establishes staining at 1:50 in paraffin sections but does not establish fixative, epitope, or clinical specificity (A12840-1 caption).
Boster reagents
Best MEGF8 / Multiple epidermal growth factor-like domains protein 8 IHC Antibodies
For MEGF8, A12840-1 has an IHC image from paraffin-embedded human breast carcinoma (IHC image caption); A12840 lists IHC and IF for human, mouse and rat (catalog: A12840).
Real IHC data
Anti-EGFL4 (R1274) MEGF8 Antibody
Cat # A12840-1
A12840-1 will render with its own IHC image of paraffin-embedded human breast carcinoma stained at 1:50 (A12840-1 IHC image caption). A12840 lists IHC and IF applications and human, mouse and rat reactivity, but has no IHC or IF image in the supplied catalog (catalog: A12840).
Which to pick: For tissue IHC, choose A12840-1 when its pictured paraffin-section example is relevant; the caption does not report the fixative (A12840-1 IHC image caption). For IF/ICC planning, choose A12840 because IF is listed at 1:50, though ICC-specific validation is unreported; A12840 is polyclonal, and A12840-1 is described as a pAb (catalog: A12840 applications and dilution; A12840-1 IHC image caption). For cross-species work, both list human, mouse and rat reactivity, while the supplied A12840-1 IHC image documents human tissue only (catalog: A12840 and A12840-1 reactivity; A12840-1 IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q7Z7M0 (MEGF8_HUMAN, Multiple epidermal growth factor-like domains protein 8).
- Human Protein Atlas. MEGF8 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. MEGF8 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. MEGF8 antibody validation summary (1 antibodies).
- Immunohistochemical and Immunoelectron Microscopical Distribution of MEGF8 in the Mouse Central Nervous System. Cells 2023 — PMC10778434.
- MEGF8 is a modifier of BMP signaling in trigeminal sensory neurons. eLife 2013 — PMC3776557.
- PubMed PMID:9693030 — UniProt-cited evidence.
- PubMed PMID:15057824 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for MEGF8 (Multiple epidermal growth factor-like domains protein 8). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A12840-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A12840-1); 2. Fixation: Image fixative and duration unreported (datasheet A12840-1); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 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-MEGF8, 1:50-1:200 (datasheet A12840-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections (standard IHC practice; not target-specific); Caveat: Splice/transcript discrepancy may complicate interpretation (HPA tissue IHC); Isoform / epitope: 2 isoforms; check whether the epitope is extracellular or cytoplasmic (UniProt); Regulation: No specific expression regulator annotated (UniProt). Evidence: UniProt Q7Z7M0, HPA tissue IHC · Approved, HPA ICC-IF · Membrane, Datasheet A12840-1, PMC10778434. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/MEGF8/immunohistochemistry-ihc-guide.