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MED8 / Mediator of RNA polymerase II transcription subunit 8 · IHC design guide
Design Immunohistochemistry for MED8
Plan chromogenic MED8 IHC in paraffin sections with the catalog antibody’s documented workflow (datasheet A08094-1). Assess nuclear and cytoplasmic staining, using medium-staining glandular cells as a reference and controls to check the result (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
MED8 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 cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Medium glandular-cell signal; nuclear/cytoplasmic pattern (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A08094-1) | |
| Positive control | Adipose tissue+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 paraffin-section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A08094-1) | |
| Caveat | Staining–RNA agreement has medium consistency; use controls (HPA tissue IHC) | |
| Regulation | No expression regulator annotated (UniProt) | |
| Isoform / epitope | Three isoforms; isoform-specific epitope coverage unknown (UniProt; datasheet A08094-1) |
Section 1
Recommended MED8 IHC & IF Protocols
The catalog antibody protocol uses heat-mediated citrate retrieval at pH 6 (datasheet A08094-1). Two published MED8 IHC protocols provide additional tissue-specific examples (PMC12882893; PMC9086905).
| Sample | Paraffin-embedded mouse testis tissue; fixative not specified (datasheet A08094-1) |
| Fixation | Image fixative and duration unreported (datasheet A08094-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet A08094-1) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A08094-1) |
| Primary antibody | Rabbit anti-MED8, 0.5-1μg/ml (datasheet A08094-1) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A08094-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | MED8-positive staining in adipocytes of adipose tissue (HPA tissue IHC: Medium). HPA tissue profile: Nuclear and cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate retrieval at pH 6 (datasheet A08094-1); the liver study also used heated citrate buffer, without reporting its pH (PMC9086905).
Section 2
What Is the Expected MED8 Staining Pattern?
MED8 is a nuclear component of the Mediator transcription complex and has no transmembrane segment (UniProt Q96G25). In tissue IHC, expect predominantly nuclear staining across many cell types; HPA also reports cytoplasmic staining in most tissues (HPA tissue IHC). Interpret this as a broad pattern with variable intensity: HPA rates the tissue staining “Approved,” with medium consistency against RNA data and external verification pending (HPA tissue IHC).
| Distinct nuclear stain in adrenal glandular cells, bronchial respiratory epithelial cells, or bone marrow hematopoietic cells. | This fits the expected compartment and HPA-reported medium staining in these cells (UniProt Q96G25; HPA tissue IHC). Compare nuclei with the counterstain and include a negative reagent control before scoring a specimen (general IHC practice). |
| Predominantly sharp plasma membrane or extracellular staining, with little nuclear signal. | This conflicts with MED8’s nuclear localization and lack of a transmembrane segment (UniProt Q96G25). HPA does report cytoplasmic tissue staining, so cytoplasmic color alone is not a failure; a dominant membrane or extracellular pattern warrants control review (HPA tissue IHC; general IHC practice). |
| Strong staining in a cell population expected to show only low signal, while nuclei in an HPA-reported medium population are unstained. | Check specificity and detection chemistry before calling this MED8: HPA reports low staining in breast glandular cells, smooth muscle cells, and splenic red pulp cells, but does not designate them negative (HPA tissue IHC). Cross-reactivity or endogenous detection activity is possible (general IHC practice). |
| Even chromogen haze across tissue, stroma, and empty areas obscures nuclear boundaries. | This is difficult to score as compartment-specific MED8 staining (UniProt Q96G25; general IHC practice). Background from detection reagents or incomplete washing is possible; compare with a negative reagent control and inspect whether color follows recognizable cells (general IHC practice). |
| No visible signal in an HPA-reported medium-staining tissue, with readable counterstain and intact morphology. | Treat the run as unresolved, since HPA reports medium signal in several tissues but only Approved IHC reliability and medium consistency with RNA data (HPA tissue IHC). Check antibody and detection controls before interpreting the specimen as MED8 negative (general IHC practice). |
💡Expected MED8 appearanceCall a result consistent with MED8 when nuclear staining is clear in an HPA-reported medium cell population, allowing some cytoplasmic staining; dominant membrane or extracellular color is suspect (UniProt Q96G25; HPA tissue IHC).
| Tissue and cell selection | HPA reports medium staining in adipocytes, adrenal and appendix glandular cells, bronchial epithelium, marrow hematopoietic cells, and selected brain cells (HPA tissue IHC). Its low categories are comparisons, not verified negative controls (HPA tissue IHC). |
| Compartment and topology | UniProt assigns MED8 to the nucleus and lists no transmembrane segment (UniProt Q96G25). HPA tissue IHC describes nuclear and cytoplasmic expression in most tissues, so assess whether the nuclear component remains convincing (HPA tissue IHC). |
| Antibody evidence | Two listed antibodies have Approved IHC status, HPA028377 and HPA028438 (HPA antibodies). The tissue profile has medium consistency with RNA and awaits external verification; do not treat an isolated staining pattern as definitive identification (HPA tissue IHC). |
| Isoforms and processing | UniProt lists three isoforms, one chain spanning residues 1–268, and no signal peptide or propeptide (UniProt Q96G25). No epitope or isoform-specific staining data are supplied, so these entries do not predict a particular IHC intensity. |
| IF/ICC Q: where should signal appear? | A: HPA places MED8 mainly in the nucleoplasm, with additional Golgi localization in ICC-IF (HPA subcellular). This answers the localization question; the tissue IHC profile separately reports nuclear and cytoplasmic staining (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| No nuclear stain in an HPA-reported medium tissue. | The assay may have failed, or the selected section may not show the reported cell population (HPA tissue IHC; general IHC practice). | Identify the relevant cells on the counterstain; check the antibody, retrieval, and detection steps against their validated IHC conditions, then repeat with run controls (general IHC practice). |
| Only diffuse cytoplasmic color is visible. | HPA permits cytoplasmic tissue staining, but loss of a readable nuclear component weakens the match to UniProt localization (HPA tissue IHC; UniProt Q96G25). | Compare matched cells and the negative reagent control; score nuclear and cytoplasmic staining separately, and avoid calling diffuse color alone a convincing positive (general IHC practice). |
| A low-staining HPA population appears strongly positive. | Low does not mean absent; cross-reactivity or endogenous detection activity may also contribute (HPA tissue IHC; general IHC practice). | Compare an HPA-reported medium population in the same run and use a negative reagent control; investigate detection background before assigning the signal to MED8 (HPA tissue IHC; general IHC practice). |
| Chromogen appears without the primary antibody. | A positive negative reagent control points to background from the detection system or tissue activity rather than primary-antibody binding (general IHC practice). | Review the detection reagent and any enzyme-blocking step specified for the detection chemistry; repeat the control before scoring the tissue (general IHC practice). |
| Nuclear stain is visible but tissue-wide haze prevents cell-level scoring. | Insufficient washing or excess detection background can obscure compartment boundaries (general IHC practice). | Review washing and detection conditions, retain the counterstain for nuclear landmarks, and compare the repeat section with its negative reagent control (general IHC practice). |
| Two IHC antibodies give different compartment patterns. | Both HPA028377 and HPA028438 are Approved for IHC, while the tissue profile still has medium RNA consistency and awaits external verification (HPA antibodies; HPA tissue IHC). | Compare the same tissue and cell populations under each antibody’s validated conditions; record the discordance and avoid resolving it by intensity alone (general IHC practice). |
Experiment evidence
IHC & IF images showing MED8 staining
A08094-1 · IHCIHC analysis of MED8 using anti-MED8 antibody (A08094-1). MED8 was detected in paraffin-embedded section of mouse testis tissue. Heat mediat
A08094-1 · IHCIHC analysis of MED8 using anti-MED8 antibody (A08094-1). MED8 was detected in paraffin-embedded section of rat testis tissue. Heat mediated
A08094-1 · IFIF analysis of MED8 and Tubulin beta using anti-MED8 antibody (A08094-1) and anti-Tubulin beta antibody (M03989-3). MED8 and Tubulin beta we
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000159479-MED8/tissue/cerebral+cortex#imid_7459287
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000159479-MED8/tissue/colon#imid_7442334
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000159479-MED8/tissue/liver#imid_7442327
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000159479-MED8/tissue/kidney#imid_7442336
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA028438; green = MED8, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA028438; green = MED8, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for MED8 IHC & IF
🧪Start with bone marrow: hematopoietic cells should show MED8 staining (HPA: Medium in bone marrow hematopoietic cells). HPA detects MED8 in all 45 scored tissues, so there is no supported negative tissue; use no-primary and isotype controls, and treat hematoxylin-only nuclei within the positive section as observational negatives, not as a validated MED8-negative cell type (HPA: no negative rows; UniProt Q96G25: nucleus).
Positive control tissue: Adipose tissue (Adipocytes, HPA Medium)
Negative control tissue: None in HPA: MED8 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 MED8 in A-431, U-251MG, with annotated localisation: Nucleoplasm (approved) (HPA subcellular).
Technical controls: Run a no-primary, secondary-only slide and a concentration-matched, nonimmune rabbit IgG isotype control alongside a MED8 knockout specimen or a validated immunizing-peptide block, if available (standard IHC practice; A08094-1 caption: rabbit primary). Quench endogenous peroxidase and check for chromogen signal in marrow cells on the no-primary slide (standard chromogenic IHC practice; HPA: bone marrow hematopoietic cells).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A08094-1 paraffin-section caption does not state a fixative (A08094-1 caption). Its heat retrieval in citrate buffer at pH 6 for 20 minutes is a practical IHC starting condition, but MED8-specific retrieval dependence is unreported (A08094-1 caption). HPA has ICC-IF images in A-431 and U-251MG cells, supporting an IF localisation check, while the relative ease of frozen-section IHC versus IF is unreported; marrow-cell endogenous peroxidase can complicate chromogenic interpretation (HPA: ICC-IF cell lines; standard IHC practice).
HPA tissue IHC evidence for MED8
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium 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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: MED8 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced MED8 IHC Tips
For MED8 chromogenic IHC, assess nuclear staining alongside controls for retrieval, background, and compartment specificity (UniProt Q96G25; HPA tissue IHC).
How should I adjust retrieval when MED8 staining is weak in paraffin sections?
Start with heat-mediated citrate retrieval at pH 6 for 20 minutes (datasheet A08094-1; A08094-1 IHC caption). The selected paraffin-section example used that treatment before 1 µg/mL primary antibody overnight, so keep detection conditions comparable while assessing retrieval (A08094-1 IHC caption). If nuclear signal remains weak, compare modest changes in heating time on adjacent sections, with matched positive tissue and no-primary controls (standard IHC practice; UniProt Q96G25 localisation). Inspect tissue morphology and background after each change, because stronger staining alone does not establish specificity (standard IHC practice). Record the heating method and cooling conditions so runs remain comparable (standard IHC practice).
What should I check if MED8 staining varies between similarly processed specimens?
MED8-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (A08094-1 IHC caption). Record fixative, time before fixation, specimen thickness, and fixation duration for every block, then compare sections processed together (standard IHC practice). Where formalin fixation is used, keep the concentration and duration consistent across specimens; 10% neutral buffered formalin is a common routine choice, but it has not been established as optimal for MED8 here (standard IHC practice). Recheck citrate pH 6 retrieval and include a shared control section before attributing staining differences to biology (datasheet A08094-1; standard IHC practice).
How should I assess MED8 staining outside the nucleus?
Treat nuclear staining as the principal expected pattern because MED8 is annotated in the nucleus and functions in the Mediator transcription complex (UniProt Q96G25). HPA reports nuclear and cytoplasmic staining across most tissues, while its cell imaging places MED8 mainly in the nucleoplasm with an additional Golgi location (HPA tissue IHC; HPA subcellular). Score nuclear and extranuclear staining separately rather than combining them into one positive category (standard IHC practice). Check whether extranuclear signal follows cell boundaries or diffuse background, and compare it with a no-primary control (standard IHC practice). Do not assign a Golgi location from chromogenic staining alone without an appropriate compartment marker (HPA subcellular; standard IHC practice).
Could MED8 isoforms or epitope accessibility explain inconsistent staining?
MED8 has 3 annotated isoforms, but the supplied product caption does not identify the antibody epitope or establish which isoforms it detects (UniProt Q96G25; A08094-1 IHC caption). A phosphoserine is annotated at residue 82; its effect on this antibody's staining is unknown (UniProt Q96G25). Compare sections using the same retrieval, primary concentration, and detection run before proposing an isoform explanation (standard IHC practice). If epitope information becomes available, map it against the isoform sequences and any relevant modification sites (standard IHC practice; UniProt Q96G25). Treat a changed staining pattern as an observation requiring independent specificity evidence, rather than proof of an isoform switch (standard IHC practice).
How can I investigate MED8 localisation by IF alongside chromogenic IHC?
Use IF as a separate localisation experiment; the selected antibody evidence here describes chromogenic staining of paraffin sections, not IF validation (A08094-1 IHC caption). Multiplex MED8 with a marker for the cell population under study and a nuclear counterstain, then inspect whether MED8 lies in nucleoplasm or an additional compartment (HPA subcellular; standard IF practice). MED8 has no annotated transmembrane segment, so permeabilise cells sufficiently to access its nuclear epitope while preserving morphology (UniProt Q96G25 topology and localisation; standard IF practice). Select fluorophores after checking tissue autofluorescence in unstained controls, and include single-stain controls for channel bleed-through (standard IF practice). Keep IF scoring separate from the IHC example's 1 µg/mL condition (A08094-1 IHC caption; standard IF practice).
What causes diffuse brown staining in a MED8 IHC section?
First compare the affected section with no-primary and detection-only controls to identify background from the detection system (standard IHC practice). The selected example used a biotinylated secondary, streptavidin–biotin complex, and DAB, so examine endogenous biotin and peroxidase activity when that workflow is used (A08094-1 IHC caption; standard IHC practice). Apply a peroxidase block before DAB development and assess whether blocking reduces signal outside cells (standard IHC practice). The example used 10% goat serum and 1 µg/mL primary antibody; use those as documented starting conditions, then titrate if background persists (A08094-1 IHC caption; standard IHC practice). Judge specificity against the expected nuclear pattern (UniProt Q96G25 localisation).
How should I quantify MED8 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue compartment and count only evaluable, intact cells before scoring (standard IHC practice). For nuclear MED8, report the percentage of positive nuclei and an H-score calculated from staining intensity 0–3 times the percentage at each intensity, yielding 0–300 (UniProt Q96G25 localisation; standard IHC practice). If cell density matters, also report positive nuclei per mm² of viable tissue, with the counted area documented (standard IHC practice). Normalise comparisons to the same cell population, tissue area, staining run, and exposure or scan settings (standard IHC practice). Keep cytoplasmic signal in a separate field because HPA reports both nuclear and cytoplasmic tissue staining (HPA tissue IHC).
When is apparent MED8 positivity likely to be artefactual?
Give greatest weight to reproducible staining in intact nuclei, consistent with MED8's nuclear annotation and Mediator role (UniProt Q96G25). Do not automatically dismiss cytoplasmic signal: HPA reports it in tissue, and cell imaging also identifies an additional Golgi location (HPA tissue IHC; HPA subcellular). Scrutinise staining confined to section edges, damaged or necrotic regions, or areas with strong no-primary signal, since these patterns can reflect preparation or detection artefacts (standard IHC practice). With the example's biotin-based DAB workflow, check endogenous enzyme and biotin background before assigning brown deposits to MED8 (A08094-1 IHC caption; standard IHC practice). HPA tissue staining is Approved with medium RNA concordance and pending external verification, so corroborate unusual patterns independently (HPA tissue IHC).
Boster reagents
Best MED8 / Mediator of RNA polymerase II transcription subunit 8 IHC Antibodies
A08094-1 has paraffin-section IHC data in mouse and rat testis (IHC captions) and IF data in U87 cells (IF caption); listed reactivity covers human, mouse and rat (catalog).
Real IHC data
Anti-MED8 Antibody ®
Cat # A08094-1
A08094-1 has IHC images from paraffin-embedded mouse and rat testis (IHC captions). A08094-1 also has an IF image from U87 cells (IF caption), and the catalog lists IF and ICC applications (catalog).
Which to pick: For tissue IHC, choose A08094-1 for paraffin sections based on its mouse and rat testis images (IHC captions); the fixative is unreported (IHC captions). For IF/ICC, A08094-1 is the listed option, with an IF image from U87 cells (IF caption; catalog: IF, ICC). For work across species, A08094-1 lists human, mouse and rat reactivity (catalog), while its tissue IHC images document mouse and rat samples (IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q96G25 (MED8_HUMAN, Mediator of RNA polymerase II transcription subunit 8).
- Human Protein Atlas. MED8 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. MED8 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the Golgi apparatus..
- Human Protein Atlas. MED8 antibody validation summary (2 antibodies).
- Disrupting MED8-dependent epigenetic reprogramming augments avapritinib sensitivity in PDGFRA-driven glioma. Journal of experimental & clinical cancer research : CR 2026 — PMC13360484.
- Mediator complex subunit 8 promotes bladder cancer progression via Stearoyl-CoA desaturase. Cellular and molecular life sciences : CMLS 2026 — PMC12882893.
- Targeting MED8 enhances sorafenib sensitivity in hepatocellular carcinoma by disrupting epithelial-mesenchymal transition mechanisms. Journal of enzyme inhibition and medicinal chemistry 2025 — PMC12541920.
- A Predictive Model for Prognosis and Therapeutic Response in Hepatocellular Carcinoma Based on a Panel of Three MED8-Related Immunomodulators. Frontiers in oncology 2022 — PMC9086905.
- PubMed PMID:12149480 — UniProt-cited evidence.
- PubMed PMID:16710414 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for MED8 (Mediator of RNA polymerase II transcription subunit 8). Expected localisation Nuclear and cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A08094-1). Validated IHC antibody: A08094-1 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); 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 testis tissue; fixative not specified (datasheet A08094-1); 2. Fixation: Image fixative and duration unreported (datasheet A08094-1); 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 retrieval: Citrate pH 6 , 20 min (datasheet A08094-1); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A08094-1); 8. Primary antibody: Rabbit anti-MED8, 0.5-1μg/ml (datasheet A08094-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep paraffin-section fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A08094-1); Caveat: Staining–RNA agreement has medium consistency; use controls (HPA tissue IHC); Isoform / epitope: Three isoforms; isoform-specific epitope coverage unknown (UniProt; datasheet A08094-1); Regulation: No expression regulator annotated (UniProt). Evidence: UniProt Q96G25, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A08094-1, PMC12882893, PMC9086905. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/MED8/immunohistochemistry-ihc-guide.