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ADM / Pro-adrenomedullin · IHC design guide
Design Immunohistochemistry for ADM
Plan chromogenic IHC on paraffin sections around the extracellular staining reported for ADM (HPA tissue IHC). Adrenal glandular cells and kidney glomerular cells are reported as high staining controls (HPA tissue IHC); the catalog antibody A00594 has a 1:100 IHC starting dilution (datasheet: A00594).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ADM 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 | Extracellular staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Extracellular positivity, including placenta and endothelium (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 | Caudate+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 A00594) | |
| Caveat | Secreted ADM may stain away from its source cells (HPA tissue IHC) | |
| Regulation | Staining regulation is not established (UniProt) | |
| Isoform / epitope | No isoforms annotated; peptide processing may affect epitope recognition (UniProt) |
Section 1
Recommended ADM IHC & IF Protocols
The catalog antibody's IHC-P protocol is followed by published ADM protocols for rat spinal cord (PMC5228177), kidney tissue (PMC5802232), and breast tumour sections (PMC2394432).
| Sample | Paraffin-embedded rat kidney tissue; fixative not specified (datasheet A00594) |
| Fixation | Image fixative and duration unreported (datasheet A00594); 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-ADM, 1:100 (datasheet A00594) |
| 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 | ADM-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Extracellular positivity in most tissues, including placenta and endothelial cells. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page antigen retrieval); assess staining with tissue controls.
Section 2
What Is the Expected ADM Staining Pattern?
ADM is a secreted, non-transmembrane propeptide (UniProt P35318 topology), and HPA reports extracellular positivity in most tissues, including placenta and endothelial cells (HPA tissue IHC). High staining is reported in adrenal glandular cells, bronchial basal cells, and kidney glomerular cells (HPA tissue IHC). HPA rates its tissue IHC evidence Approved, with medium consistency against RNA expression; a secreted protein’s tissue RNA and protein locations may differ (HPA reliability description).
| Extracellular staining with strong signal in adrenal glandular cells, bronchial basal cells, or kidney glomerular cells (HPA tissue IHC). | This fits the reported distribution: HPA describes extracellular positivity in most tissues and rates these cell populations High (HPA tissue IHC). Score the stained compartment and cell population separately; extracellular signal need not identify the cell that produced a secreted protein (UniProt P35318 subcellular location; HPA reliability description). |
| Predominantly nuclear staining, especially without the expected extracellular pattern (HPA tissue IHC; UniProt P35318 subcellular location). | An exclusively nuclear pattern lacks support from the supplied localisation evidence (UniProt P35318; HPA tissue IHC). Treat it as suspect and compare a known-positive section and a no-primary control before interpreting it as ADM (general IHC practice). |
| Strong staining in ovarian stromal cells, caudate glial cells, or smooth muscle cells (HPA tissue IHC). | HPA reports ADM as Not detected in these specific populations (HPA tissue IHC). Check for cross-reactivity or endogenous detection activity with appropriate controls (general IHC practice); the HPA result is cell specific and does not make the entire tissue a universal negative control (HPA tissue IHC). |
| Diffuse colour across tissue spaces and unrelated structures, obscuring cell boundaries (general IHC practice). | Because extracellular ADM staining is reported, extracellular colour alone is insufficient to call background (HPA tissue IHC). Compare its distribution with the expected cells and a no-primary control; widespread colour in that control supports a detection or background problem (general IHC practice). |
| No visible signal in adrenal glandular cells or kidney glomerular cells on an otherwise interpretable section (HPA tissue IHC). | These are High populations in HPA tissue IHC, so an absent result warrants a technical check (HPA tissue IHC; general IHC practice). Confirm that the positive control, antibody application, detection reagents, and counterstain allow a readable result before concluding that ADM is absent from the sample (general IHC practice). |
💡Expected ADM appearanceCall a positive result when extracellular staining is present with High signal in an HPA High population such as adrenal glandular or kidney glomerular cells (HPA tissue IHC); isolated nuclear colour or widespread colour in a no-primary control is suspect (UniProt P35318 subcellular location; general IHC practice).
| Secretion and topology (UniProt P35318). | ADM is secreted and has no transmembrane segment (UniProt P35318 topology). Expect extracellular signal and interpret apparent cellular association in the context of nearby extracellular staining (HPA tissue IHC; UniProt P35318 subcellular location). |
| Precursor processing (UniProt P35318). | The 185-aa precursor has a signal peptide and annotated propeptide regions (UniProt P35318 processing). The supplied evidence does not locate the catalog antibody’s epitope, so staining cannot identify which processed ADM-related product it detects (UniProt P35318 processing; supplied evidence). |
| Tissue and cell context (HPA tissue IHC). | HPA reports High staining in several distinct populations, including appendix and duodenal glandular cells and fallopian-tube ciliated-cell rootlets, but Low staining in colon endothelial cells (HPA tissue IHC). Compare like cells in like tissues when judging intensity (general IHC practice). |
| Evidence strength and RNA comparison (HPA tissue IHC). | The tissue IHC assessment is Approved, with medium consistency against RNA data (HPA reliability description). HPA notes that secretion can separate protein location from tissue RNA location, so an RNA mismatch alone does not establish an IHC artefact (HPA reliability description). |
| Antibody validation scope (HPA antibody records). | HPA068955 and CAB016075 are each IHC Approved; neither has an ICC status in the supplied records (HPA antibody records). Their IHC status supports evaluating tissue staining but does not establish an IF/ICC staining pattern or an IF/ICC protocol (HPA antibody records; HPA subcellular record). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in a known-positive population, such as adrenal glandular cells (HPA tissue IHC). | A failed staining run or inadequate detection can give a false negative (general IHC practice). | Run an HPA High tissue as a positive control and check antibody application, detection reagents, and counterstain in the IHC workflow (HPA tissue IHC; general IHC practice). |
| Nuclear staining dominates while extracellular staining is absent (HPA tissue IHC; UniProt P35318). | The compartment disagrees with the reported secreted and extracellular pattern (UniProt P35318; HPA tissue IHC). | Compare with a known-positive section and no-primary control; withhold an ADM-specific call if the discordant pattern persists (HPA tissue IHC; general IHC practice). |
| A cell population reported Not detected stains strongly, such as ovarian stromal cells (HPA tissue IHC). | Cross-reactivity or endogenous detection activity is possible (general IHC practice). | Check a no-primary control and the expected High population in the same run; interpret the unexpected cells separately from other cells in that tissue (HPA tissue IHC; general IHC practice). |
| Colour is widespread and hard to distinguish from reported extracellular positivity (HPA tissue IHC). | Background from the detection workflow can obscure a genuine extracellular pattern (HPA tissue IHC; general IHC practice). | Inspect the no-primary control, then review blocking, washing, and detection conditions as general IHC troubleshooting steps (general IHC practice). |
| Endothelial staining varies between specimens or sites (HPA tissue IHC). | HPA reports extracellular positivity involving endothelial cells, Low colon endothelial staining, and Not detected cerebral-cortex endothelial staining (HPA tissue IHC). | Record the tissue and endothelial population before comparing intensity; avoid applying one site’s HPA level to all endothelium (HPA tissue IHC). |
| IF/ICC question: should fluorescence mark a defined intracellular ADM compartment (HPA subcellular record)? | HPA lists ADM as Secreted but provides no main intracellular location or ICC-IF image cell lines, and the supplied antibodies have no ICC status (HPA subcellular record; HPA antibody records). | Treat a defined intracellular IF/ICC pattern as unverified by these sources; consult the separate IF/ICC guide for assay design (HPA subcellular record; HPA antibody records). |
Experiment evidence
IHC & IF images showing ADM staining
A00594 · IHCImmunohistochemical analysis of paraffin-embedded rat-kidney, antibody was diluted at 1:100
A00594 · IHCImmunohistochemical analysis of paraffin-embedded human-liver, antibody was diluted at 1:100
A00594-1 · IHCImmunohistochemistry (IHC) analyzes of ADM (N100) pAb in paraffin-embedded human breast carcinoma tissue at 1:100.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000148926-ADM/tissue/cerebral+cortex#imid_20927703
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000148926-ADM/tissue/colon#imid_20927629
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000148926-ADM/tissue/liver#imid_20927638
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000148926-ADM/tissue/kidney#imid_20927639
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ADM IHC & IF
🧪Run adrenal gland first and score its glandular cells for staining (HPA: High in adrenal gland glandular cells); use caudate glial cells as the negative tissue comparison (HPA: Not detected in caudate glial cells). On the adrenal slide, use adjacent cells outside the glandular compartment as an internal background reference: they should show counterstain without specific chromogen, but confirm their identity before treating them as a biological negative (HPA: High in glandular cells).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Caudate (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for ADM; 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 no-primary (secondary-only), a concentration-matched isotype control appropriate to the primary antibody’s host species and clonality, and a knockout specimen or peptide-block control if the immunizing peptide is available. For chromogenic IHC of adrenal gland, quench endogenous peroxidase and check blood-rich areas for residual background; assess autofluorescence if using IF (HPA: adrenal gland positive tissue).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the selected A00594 rat-kidney paraffin-section caption does not report a fixative (caption: 1:100; fixative unreported). Retrieval dependency is also unreported; optimize retrieval against the adrenal positive and caudate negative controls, and there is no supplied evidence that frozen sections or IF are easier for ADM (HPA: High in adrenal gland glandular cells; Not detected in caudate glial cells). Because ADM is secreted, extracellular signal can complicate assignment to individual glandular cells, while blood-associated peroxidase can complicate chromogenic scoring in adrenal sections (UniProt P35318: Secreted; standard IHC practice).
HPA tissue IHC evidence for ADM
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. At least one protein variant secreted, tissue location of RNA and protein might differ and correlation is complex.). 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 | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Basal cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cerebral cortex | Endothelial cells | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ADM IHC Tips
Troubleshoot ADM staining in paraffin sections by checking retrieval, tissue context and extracellular signal before assigning cellular expression (UniProt P35318; HPA tissue IHC).
What retrieval should I try first for ADM in paraffin sections?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval specification). Let sections cool in the buffer, then compare staining with a section processed identically except for retrieval; this checks whether heating exposes signal or increases background (standard IHC practice). Keep the catalog antibody at the captioned 1:100 dilution during that comparison, while recognizing that the caption documents staining in paraffin-embedded rat kidney without reporting its retrieval method (A00594 caption). If signal remains weak, evaluate an alternative retrieval condition on adjacent sections and retain it only if tissue morphology and localization remain credible (standard IHC practice; HPA tissue IHC).
How should I troubleshoot variable ADM staining after tissue fixation?
The supplied ADM evidence does not establish target-specific fixation sensitivity, and the paraffin-embedded rat-kidney caption does not state a fixative (A00594 caption). Record the actual fixative, fixation duration and processing history for each specimen, then compare sections with matched histories under the same retrieval and detection conditions (standard IHC practice). When a batch is weak, include a previously staining section and assess whether other antigens also fail, which helps separate processing problems from ADM staining variation (standard IHC practice). Do not attribute differences to ADM secretion, precursor processing or amidation without direct fixation comparisons for the antibody and specimens being tested (UniProt P35318; standard IHC practice).
Where should convincing ADM signal appear in a paraffin section?
Interpret ADM as a secreted protein without a transmembrane segment; an exclusively crisp plasma-membrane outline is therefore not an expected topology-based pattern (UniProt P35318 subcellular annotation and topology). Extracellular positivity across many tissues is reported, including placenta and endothelial cells, while kidney glomerular cells and adrenal glandular cells show high staining in the supplied tissue profile (HPA tissue IHC). Compare cellular and extracellular staining separately against those reference patterns, because secreted protein can appear away from the cells that produced it (HPA tissue IHC reliability description; standard IHC practice). If staining is confined to a surprising compartment, inspect adjacent sections and detection controls before calling it ADM (standard IHC practice).
Could precursor processing explain different ADM staining patterns?
Yes: the 185-residue ADM precursor has an annotated signal peptide at residues 1–21 and propeptide regions at 45–92 and 148–185 (UniProt P35318 processing). Its record also lists amidated residues at 41 and 146, but no annotated isoforms or glycosylation sites (UniProt P35318 modified residues and isoforms). Determine which sequence the catalog antibody recognizes before interpreting staining as precursor, ADM or another processed peptide; the supplied caption does not identify its epitope (A00594 caption; standard IHC practice). Compare sections stained with antibodies against distinct documented epitopes when available, and describe discordance as epitope-dependent staining until specificity is established (standard IHC practice).
How can I assess ADM by IF alongside a cell-type marker?
For the separate IF/ICC workflow, pair ADM with a marker appropriate to the cell population under study and assess extracellular signal independently of marker overlap (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and place the weaker signal in a channel with low measured tissue autofluorescence; include single-stain and unstained controls for bleed-through and autofluorescence (standard IF practice). ADM is secreted and has no transmembrane segment, so determine the antibody’s epitope before choosing permeabilisation: intracellular precursor epitopes require access across the cell membrane, whereas extracellular antigen should be assessed with minimal disruption (UniProt P35318 topology; standard IF practice). The supplied catalog caption documents paraffin-section IHC at 1:100, not an IF/ICC validation or fixation condition (A00594 caption).
What should I check when ADM staining looks diffuse or widespread?
Some diffuse extracellular staining may be biologically plausible because ADM is secreted and the tissue profile reports extracellular positivity in most tissues (UniProt P35318 subcellular annotation; HPA tissue IHC). Compare no-primary controls, reagent-only areas and intact tissue away from cut edges; broad staining shared by controls or concentrated at edges supports a technical source (standard IHC practice). For peroxidase-based chromogenic detection, check the peroxidase block and examine whether endogenous pigment or enzyme activity could mimic the chosen chromogen (standard IHC practice). Adjust blocking, washing and antibody concentration one variable at a time, using the captioned 1:100 as the documented catalog reference rather than treating it as universally optimal (A00594 caption; standard IHC practice).
How should I score ADM when extracellular and cellular staining coexist? ⚠ ANSWER MARKED FOR VERIFICATION
Define regions and compartments before scoring, and report cellular and extracellular staining separately because ADM is secreted and extracellular positivity is common in the supplied profile (UniProt P35318 subcellular annotation; HPA tissue IHC). For identifiable cells, record the percentage positive and intensity, or calculate an H-score from percentages at each intensity grade; use density per mm² for defined extracellular deposits (standard IHC practice). Normalize cellular counts to eligible cells in the same compartment and area-based measurements to viable tissue area, excluding folds, edges and necrosis by a prespecified rule (standard IHC practice). Apply identical thresholds, counterstain settings and sampling rules across specimens, and document how background-control signal affected the cutoff (standard IHC practice).
How can I distinguish credible ADM staining from artefact?
A credible pattern should be reproducible across intact regions and compatible with secreted ADM, including possible extracellular signal rather than a required membrane rim (UniProt P35318 subcellular annotation and topology; standard IHC practice). High staining in adrenal glandular cells or kidney glomerular cells fits the supplied tissue profile, but neither location alone proves antibody specificity (HPA tissue IHC; standard IHC practice). Flag signal restricted to section edges, necrotic areas or structures positive in a no-primary control, and investigate endogenous peroxidase when using chromogenic detection (standard IHC practice). Interpret RNA–protein disagreement cautiously because the tissue profile reports medium consistency and notes that secretion can separate protein location from the site of RNA expression (HPA tissue IHC reliability description).
Boster reagents
Best ADM / Pro-adrenomedullin IHC Antibodies
Two anti-ADM antibodies have paraffin-section IHC figures from rat kidney, human liver, and human breast carcinoma (IHC image captions); catalog reactivity spans human, mouse, and rat (catalog: reactivity).
Real IHC data
Anti-ADM/Adrenomedullin Antibody
Cat # A00594
Real IHC data
Anti-ADM (N100) Antibody
Cat # A00594-1
A00594 lists IHC and IF for human and rat, with paraffin-section IHC images from rat kidney and human liver (catalog: applications and reactivity; A00594 IHC image captions). A00594-1 lists IHC for human, mouse, and rat, with a paraffin-section IHC image from human breast carcinoma (catalog: applications and reactivity; A00594-1 IHC image caption).
Which to pick: For human breast carcinoma IHC or listed mouse reactivity, choose A00594-1 at an IHC starting dilution within 1:50–1:200 (A00594-1 IHC image caption; catalog: reactivity and IHC dilution). For IF, choose A00594 at the listed 1:50 dilution; ICC validation is unreported (catalog: applications and IF dilution). Both IHC captions show paraffin sections, and neither reports the fixative (A00594 and A00594-1 IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P35318 (ADML_HUMAN, Pro-adrenomedullin).
- Human Protein Atlas. ADM tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ADM subcellular location (ICC-IF): Secreted.
- Human Protein Atlas. ADM antibody validation summary (2 antibodies).
- Expression of adrenomedullin in rats after spinal cord injury and intervention effect of recombinant human erythropoietin. Experimental and therapeutic medicine 2016 — PMC5228177.
- Adrenomedullin serves a role in the humoral pathway of delayed remote ischemic preconditioning via a hypoxia-inducible factor-1α-associated mechanism. Molecular medicine reports 2018 — PMC5802232.
- Sex differences in expression of CGRP family of receptors and ligands in the rat trigeminal system. The journal of headache and pain 2024 — PMC11545840.
- Tissue and plasma expression of the angiogenic peptide adrenomedullin in breast cancer. British journal of cancer 2003 — PMC2394432.
- PubMed PMID:7688224 — UniProt-cited evidence.
- PubMed PMID:8074714 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ADM (Pro-adrenomedullin). Expected localisation Extracellular staining in most tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A00594 at 1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Caudate. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded rat kidney tissue; fixative not specified (datasheet A00594); 2. Fixation: Image fixative and duration unreported (datasheet A00594); 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-ADM, 1:100 (datasheet A00594); 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 A00594); Caveat: Secreted ADM may stain away from its source cells (HPA tissue IHC); Isoform / epitope: No isoforms annotated; peptide processing may affect epitope recognition (UniProt); Regulation: Staining regulation is not established (UniProt). Evidence: UniProt P35318, HPA tissue IHC · Approved, HPA ICC-IF · Secreted, Datasheet A00594, PMC5228177, PMC5802232. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ADM/immunohistochemistry-ihc-guide.