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MED28 / Mediator of RNA polymerase II transcription subunit 28 · IHC design guide
Design Immunohistochemistry for MED28
Plan MED28 chromogenic IHC around the low nuclear and cytoplasmic staining reported across several tissues (HPA tissue IHC). The IHC-validated antibody A08611 has a starting concentration of 2.5 μg/mL for human tissue (datasheet).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
MED28 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 | Low nuclear and cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Low nuclear/cytoplasmic staining; medium in neuronal cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Breast+4 more · see all | |
| Negative control | Adrenal gland+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A08611) | |
| Caveat | Staining and RNA data show low consistency (HPA tissue IHC) | |
| Regulation | Higher expression in vascular tissues (UniProt) | |
| Isoform / epitope | One 1–178 chain; no isoforms annotated (UniProt) |
Section 1
Recommended MED28 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with two published MED28 IHC workflows (PMC2907343; PMC9444419).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A08611); 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 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-MED28, 2.5 μg/mL (datasheet A08611) |
| 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 | MED28-positive staining in myoepithelial cells of breast (HPA tissue IHC: Medium). HPA tissue profile: Low nuclear and cytoplasmic expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 at 95–98 °C for 20 min (page antigen retrieval); citrate pH 6 is a published alternative (PMC2907343).
Section 2
What Is the Expected MED28 Staining Pattern?
MED28 is reported in the nucleus, cytoplasm and at the membrane, despite having no transmembrane segment (UniProt Q9H204 localization and topology). In paraffin-section IHC, expect generally low nuclear and cytoplasmic staining, with medium staining reported in selected epithelial and neuronal cell populations (HPA tissue IHC). Treat the pattern as provisional: HPA rates its tissue IHC evidence Uncertain because antibody staining and RNA expression show low consistency (HPA tissue IHC reliability).
| Low nuclear and cytoplasmic staining across several tissues; medium staining in breast myoepithelial cells or bronchial respiratory epithelial cells. | This fits the reported tissue pattern (HPA tissue IHC: low nuclear and cytoplasmic expression; Medium in those cell populations). Score the named cells and compartments separately. A medium signal is an observed IHC result, not a definitive specificity control, because HPA rates the tissue IHC evidence Uncertain (HPA tissue IHC reliability). |
| Staining is confined to extracellular deposits or another compartment without an expected cellular pattern. | Investigate artefact before assigning that signal to MED28. Nuclear and cytoplasmic staining is reported in tissue IHC (HPA tissue IHC); UniProt also lists membrane localization and describes mainly membrane-associated cytoplasmic protein in one cited study (UniProt Q9H204 localization). Membrane-adjacent signal alone therefore does not establish an incorrect compartment. |
| Strong staining appears in cells listed as Not detected, such as adrenal glandular cells or bone-marrow hematopoietic cells. | Check cell identity, cross-reactivity and endogenous detection activity using appropriate controls (general IHC practice). HPA reports those specific cell populations as Not detected (HPA tissue IHC). That label is cell-specific and comes from IHC evidence rated Uncertain; it does not establish that every cell in either tissue must be negative (HPA tissue IHC reliability). |
| Diffuse chromogen covers tissue structures or obscures the nuclear and cytoplasmic pattern. | Treat the result as uninterpretable until background is resolved (general IHC practice). Review the no-primary control, blocking, detection chemistry, antibody concentration and wash conditions (general IHC practice). Diffuse background cannot be used to infer MED28 localization or tissue abundance from HPA's cell-specific observations (HPA tissue IHC). |
| No signal appears in a breast myoepithelial or cerebellar Purkinje cell population selected as a positive comparator. | Both populations have reported Medium staining (HPA tissue IHC). Confirm that the relevant cells are present and assess the staining run and detection controls (general IHC practice). A blank comparator warrants investigation, but one negative run does not overturn a target pattern whose tissue IHC reliability is Uncertain (HPA tissue IHC reliability). |
💡Expected MED28 appearanceA plausible positive result is low nuclear and cytoplasmic staining, reaching medium intensity in reported populations such as breast myoepithelial cells; diffuse deposits or strong staining in an HPA-listed Not detected cell population require control checks (HPA tissue IHC; general IHC practice).
| Tissue IHC confidence | HPA describes low nuclear and cytoplasmic expression in several tissues, yet rates its tissue IHC evidence Uncertain because staining and RNA expression have low consistency (HPA tissue IHC). Use reported patterns as comparators, not absolute pass/fail thresholds. Both listed antibodies have Uncertain IHC status (HPA antibodies: HPA035900, HPA035901). |
| Cell population | A tissue name alone can conceal different cell results. HPA reports Medium staining in breast myoepithelial cells and bronchial respiratory epithelial cells, Low staining in lung alveolar cells, and Not detected in liver cholangiocytes (HPA tissue IHC). Compare like cells when judging intensity; do not transfer a cell-specific result to its whole tissue. |
| Localization and topology | Nuclear, cytoplasmic and membrane localizations are listed for MED28, while no transmembrane segment is annotated (UniProt Q9H204 localization and topology). HPA tissue IHC emphasizes low nuclear and cytoplasmic staining (HPA tissue IHC). Interpret a membrane-associated appearance cautiously against morphology and controls; topology alone does not predict an IHC compartment or retrieval condition. |
| Protein forms | The supplied record annotates one chain spanning residues 1–178, no isoforms, no signal peptide, no propeptide and no glycosylation sites (UniProt Q9H204 processing and isoforms). These annotations offer no basis to expect a distinct processed or shed staining pattern. They also do not identify the catalog antibody's epitope or establish fixation sensitivity. |
| IF/ICC question: should fluorescence match tissue IHC? | HPA reports supported nucleoplasmic localization in ICC-IF images from A-431, U-251MG and U2OS, while its tissue IHC profile is low nuclear and cytoplasmic with Uncertain reliability (HPA subcellular; HPA tissue IHC). Use the ICC-IF result as compartment context; assess IF/ICC workflow on its separate guide page. |
| Situation | Likely cause | Next action |
|---|---|---|
| The positive comparator is blank. | The selected cell population may be absent from the section, or the IHC run may have failed (general IHC practice). | Confirm the target cells on the counterstained section, then inspect run controls and detection steps (general IHC practice). Choose a population with reported Medium staining, such as breast myoepithelial cells, while retaining HPA's Uncertain reliability caveat (HPA tissue IHC). |
| A Not detected comparator stains strongly. | Possible cross-reactivity, endogenous detection activity or misidentified cells (general IHC practice); HPA lists adrenal glandular cells as Not detected (HPA tissue IHC). | Compare the no-primary and detection controls and verify the stained cell type (general IHC practice). Report the discrepancy by cell population rather than declaring the whole tissue positive; HPA's IHC assessment remains Uncertain (HPA tissue IHC reliability). |
| The whole section has diffuse background. | Excess antibody, insufficient blocking or wash carryover are possible general IHC causes (general IHC practice). | Review the no-primary control, blocking, wash steps and antibody titration (general IHC practice). Re-score MED28 only after cell boundaries and nuclear versus cytoplasmic staining can be resolved against the reported pattern (HPA tissue IHC). |
| Signal appears exclusively at cell edges. | MED28 has a reported membrane-associated cytoplasmic localization, but HPA tissue IHC describes low nuclear and cytoplasmic staining (UniProt Q9H204 localization; HPA tissue IHC). | Check morphology and controls before calling the edge signal specific (general IHC practice). Record its compartment separately and avoid assuming a transmembrane pattern: no transmembrane segment is annotated (UniProt Q9H204 topology). |
| Reported Medium populations stain only weakly. | The observed run may differ from HPA's examples, whose tissue IHC reliability is Uncertain (HPA tissue IHC reliability). | Compare equivalent cells and review run controls, antibody titration and detection conditions (general IHC practice). Document the observed intensity; do not force a Medium score solely because HPA reports Medium staining in that population (HPA tissue IHC). |
| A researcher expects the ICC-IF nucleoplasmic result to appear identically in paraffin IHC. | HPA supports nucleoplasmic localization in ICC-IF, while tissue IHC reports low nuclear and cytoplasmic staining and carries Uncertain reliability (HPA subcellular; HPA tissue IHC). | Interpret each assay against its own reported pattern and controls (general IHC practice). Use the separate IF/ICC guide for fluorescence workflow; do not treat ICC-IF support for HPA035900 as IHC validation (HPA antibodies: HPA035900 ICC Supported, IHC Uncertain). |
Experiment evidence
IHC & IF images showing MED28 staining
A08611 · IHCImmunohistochemistry of MED28 in human brain tissue with MED28 antibody at 2.5 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000118579-MED28/tissue/cerebral+cortex#imid_9358640
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000118579-MED28/tissue/colon#imid_9358574
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000118579-MED28/tissue/liver#imid_9358559
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000118579-MED28/tissue/kidney#imid_9358560
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA035900; green = MED28, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA035900; green = MED28, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for MED28 IHC & IF
🧪Run cerebral cortex first; neuronal cells should stain at a medium level (HPA: Medium in cerebral cortex neuronal cells). Use heart muscle as the negative tissue, with cardiomyocytes at background (HPA: Not detected in heart muscle cardiomyocytes); within the cortex slide, assess cells without the expected neuronal signal against the no-primary control rather than assigning an unlisted cell type as negative.
Positive control tissue: Breast (Myoepithelial cells, HPA Medium)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show MED28 in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) control, a host-species-matched isotype control appropriate to the primary antibody’s clonality, and MED28-knockout material as a biological specificity control (standard IHC practice). Quench endogenous peroxidase for chromogenic IHC and distinguish brain pigment from chromogen; for IF/ICC, check autofluorescence with an unstained control (standard IHC/IF practice).
⚠️Feasibility: A MED28-specific fixation window and retrieval dependency are unreported in the supplied evidence; the selected A08611 brain IHC caption also leaves the fixative unreported (selected A08611 tissue-IHC caption). There is no supplied comparison showing that frozen sections or IF are easier than paraffin IHC; IF/ICC images support nucleoplasmic localization in A-431, U-251MG and U2OS cells (HPA: Nucleoplasm, supported). In brain sections, pigment may complicate chromogenic interpretation, so compare the positive slide with its no-primary control (standard IHC practice).
HPA tissue IHC evidence for MED28
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — 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 |
|---|---|---|---|---|
| Breast | Myoepithelial cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | Purkinje cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced MED28 IHC Tips
Troubleshoot MED28 staining in paraffin sections by checking retrieval, compartment-specific signal and cell-level controls before interpreting chromogenic intensity.
How should I retrieve MED28 when staining is weak in paraffin sections?
Start with Tris-EDTA at pH 9.0, heated to 95–98 °C for 20 minutes (page retrieval setting). Let sections cool in the retrieval buffer, then compare a retrieval-treated section with an otherwise matched control; this helps separate insufficient unmasking from later staining problems (standard IHC practice). The selected A08611 human brain image reports antibody at 2.5 µg/mL but does not report its retrieval conditions, so that image cannot establish an optimal retrieval method (A08611 caption). If signal remains weak, compare a milder or alternative buffer condition on serial sections while holding antibody concentration, detection and imaging constant, and reject conditions that increase diffuse background or damage morphology (standard IHC practice).
Could fixation explain inconsistent MED28 staining across paraffin blocks?
MED28-specific sensitivity to fixation is unknown from the supplied evidence; the selected human brain caption gives no fixative, and tissue staining patterns do not establish a fixation effect (A08611 caption; HPA tissue IHC). Record each block’s fixative, fixation duration and processing history, then compare serial sections stained in the same run at the same antibody concentration (standard IHC practice). Start with the page’s Tris-EDTA pH 9.0 retrieval at 95–98 °C for 20 minutes, and change one condition at a time if morphology or signal differs (page retrieval setting; standard IHC practice). Use hematoxylin morphology and matched control tissue to judge whether a changed signal reflects tissue preservation or staining performance (standard IHC practice).
Should MED28 staining appear nuclear, cytoplasmic or membrane associated?
Assess nuclear and cytoplasmic signal separately: MED28 is annotated in the nucleus, cytoplasm and membrane, while the HPA tissue profile describes low nuclear and cytoplasmic expression (UniProt Q9H204 subcellular; HPA tissue IHC). Supported nucleoplasmic localisation in ICC/IF makes a well-defined nuclear pattern plausible, but it does not prove every chromogenic tissue signal is nuclear (HPA subcellular). A reported cytoplasmic, mainly membrane-associated pool also makes a restricted peripheral pattern biologically plausible (UniProt Q9H204 subcellular). Because MED28 has no annotated transmembrane segment, treat a continuous membrane outline as a finding to verify with controls and cell morphology, rather than assuming it marks a membrane-spanning protein (UniProt Q9H204 topology; standard IHC practice).
How can I evaluate an unexpected MED28 staining pattern without an epitope map?
The supplied record lists a single 1–178 chain, no annotated isoforms, and no annotated glycosylation or modified residues (UniProt Q9H204 processing; UniProt Q9H204 isoforms; UniProt Q9H204 PTMs). Those annotations do not identify the catalog antibody’s binding site or show how paraffin processing affects it, so check the antibody’s epitope information before assigning an apparent compartment-specific loss to epitope masking (standard IHC practice). Compare adjacent sections across retrieval conditions while keeping antibody concentration and chromogenic detection constant (standard IHC practice). If an independent MED28 antibody with a documented distinct epitope is available, concordant cell-level localisation strengthens interpretation; discordance calls for specificity controls and review of staining artefacts (standard IHC practice).
How should I investigate MED28 localisation by multiplex immunofluorescence?
Use IF as a separate localisation check: supported nucleoplasmic staining has been reported in A-431, U-251MG and U2OS images (HPA subcellular). Pair MED28 with a marker identifying the expected cell population, such as a neuronal marker when examining a neuronal region, and keep a nuclear counterstain in a separate channel (HPA tissue IHC; standard IF practice). Choose a fluorophore channel with low tissue autofluorescence and include unstained and single-stain controls before interpreting overlap (standard IF practice). For intracellular MED28, test mild permeabilisation after fixation; its annotated cytoplasmic and membrane-associated pools do not establish which side of a membrane the antibody epitope faces, so optimise permeabilisation empirically (UniProt Q9H204 subcellular; UniProt Q9H204 topology; standard IF practice).
What should I change when MED28 chromogenic staining is diffuse or widespread?
First compare the stained section with a no-primary control and inspect whether the diffuse colour follows tissue edges, damaged areas or endogenous pigment (standard IHC practice). Quench endogenous peroxidase before HRP/DAB detection, block nonspecific binding, and titrate the antibody downward if the no-primary control is clean but the test section remains broadly stained (standard IHC practice). The selected A08611 image used 2.5 µg/mL in human brain; that is a reported image condition, not a validated concentration for every paraffin specimen (A08611 caption). Interpret weak widespread signal cautiously because HPA reports low expression across several tissues and rates its tissue IHC reliability as uncertain (HPA tissue IHC).
How should I score MED28 staining across samples with different cell composition? ⚠ ANSWER MARKED FOR VERIFICATION
Score MED28 within a prespecified cell population and compartment, separating nuclear from cytoplasmic staining rather than combining them into one intensity value (UniProt Q9H204 subcellular; standard IHC practice). For each region, report the percentage of positive target cells and an intensity-based H-score from 0–300, using the same threshold and imaging settings across samples (standard IHC practice). When cell abundance varies, normalise positive-cell counts to the number of eligible cells; for spatial comparisons, also report positive-cell density per mm² of viable tissue (standard IHC practice). Exclude folds, edges and necrotic areas, and document the compartment and cell-type rules before scoring because HPA tissue IHC reliability is uncertain (HPA tissue IHC; standard IHC practice).
How do I distinguish convincing MED28 staining from a chromogenic artefact?
Give greatest weight to reproducible staining in morphologically intact cells with a defined nuclear or cytoplasmic pattern, consistent with MED28 localisation annotations and the low nuclear/cytoplasmic HPA tissue profile (UniProt Q9H204 subcellular; HPA tissue IHC). Compare cell identity: HPA reports medium staining in cerebral cortex neuronal cells and cerebellar Purkinje cells, while several other listed populations are not detected (HPA tissue IHC). Discount edge-only staining, necrotic regions and colour reproduced in a no-primary or peroxidase-control section, which suggest processing or endogenous-enzyme artefact (standard IHC practice). Treat agreement with a positive tissue pattern as supporting evidence rather than proof, because HPA labels its MED28 tissue IHC reliability uncertain owing to low consistency with RNA expression (HPA tissue IHC).
Boster reagents
Best MED28 / Mediator of RNA polymerase II transcription subunit 28 IHC Antibodies
A08611 has a human brain tissue IHC image (A08611 image alt). A08611-2 lists human IF/ICC applications, but no IF image is supplied (catalog applications; image alts).
Real IHC data
Anti-MED28 Antibody
Cat # A08611
The only rendered card is A08611, which lists IHC-P and shows human brain tissue IHC at 2.5 μg/mL (catalog applications; A08611 image alt). A08611-2 lists human IF/ICC applications but has no IHC or IF figure in the payload, so it has no rendered card (catalog applications; image alts).
Which to pick: Choose A08611 for paraffin-section tissue IHC: IHC-P is listed, and its own image shows human brain tissue; the caption does not report the fixative (catalog applications; A08611 image alt). For human IF/ICC, A08611-2 lists both applications at 5 μg/mL, although no IF image is supplied (catalog applications; datasheet: 5 μg/mL; image alts). For mouse samples, A08611 lists Mouse reactivity, while A08611-2 lists Human only; neither antibody has a clone listed, and mouse IHC validation is unreported (catalog reactivity; catalog clone fields; A08611 image alt).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9H204 (MED28_HUMAN, Mediator of RNA polymerase II transcription subunit 28).
- Human Protein Atlas. MED28 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. MED28 subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. MED28 antibody validation summary (2 antibodies).
- Elevated MED28 expression predicts poor outcome in women with breast cancer. BMC cancer 2010 — PMC2907343.
- Bioactive Vitamin D Attenuates MED28-Mediated Cell Growth and Epithelial-Mesenchymal Transition in Human Colorectal Cancer Cells. BioMed research international 2022 — PMC9444419.
- PubMed PMID:11779215 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:15175163 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for MED28 (Mediator of RNA polymerase II transcription subunit 28). Expected localisation Low nuclear and cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A08611 at 2.5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Breast (Myoepithelial cells); negative Adrenal gland. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A08611); 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 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-MED28, 2.5 μg/mL (datasheet A08611); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A08611); Caveat: Staining and RNA data show low consistency (HPA tissue IHC); Isoform / epitope: One 1–178 chain; no isoforms annotated (UniProt); Regulation: Higher expression in vascular tissues (UniProt). Evidence: UniProt Q9H204, HPA tissue IHC · Uncertain, HPA ICC-IF · nucleoplasm., Datasheet A08611, PMC2907343, PMC9444419. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/MED28/immunohistochemistry-ihc-guide.