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ADM / Pro-adrenomedullin · IHC design guide
Design Immunohistochemistry for ADM
Plan chromogenic IHC for ADM in paraffin sections using its extracellular tissue staining profile (HPA tissue IHC). Compare positive and negative tissue controls while accounting for secretion and peptide processing (HPA tissue IHC; UniProt).
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 many tissues (HPA tissue IHC) | |
| Staining pattern | Extracellular signal, including placenta and endothelial cells (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 | Keep fixation conditions consistent across samples. (standard IHC practice; not target-specific) | |
| Caveat | Secreted protein may stain away from its RNA source (HPA tissue IHC) | |
| Regulation | Expression regulators not specified (UniProt) | |
| Isoform / epitope | No isoforms annotated; processing yields distinct peptides (UniProt) |
Section 1
Recommended ADM IHC & IF Protocols
The catalog antibody's IHC-P protocol (datasheet) is accompanied by a published breast tumour IHC method (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 retrieval at 95–98 °C for 20 min (page retrieval rule); the published method does not specify retrieval (PMC2394432).
Section 2
What Is the Expected ADM Staining Pattern?
ADM is secreted and has no transmembrane segment (UniProt P35318: subcellular location and topology). In paraffin sections, expect extracellular positivity across many tissues, with high staining reported in specified glandular, basal, ciliated and glomerular cell populations (HPA: tissue IHC). HPA rates the tissue pattern Approved, with medium consistency against RNA data; secretion can separate the site of protein staining from the site of expression (HPA: reliability description).
| Extracellular staining accompanies adrenal glandular cells or kidney glomerular cells. | This fits a secreted target (UniProt P35318: secreted) and named high staining populations (HPA: adrenal gland and kidney, High). Score the compartment and cell population separately: nearby extracellular signal need not mark the cell that made ADM (HPA: reliability description). |
| Strong, uniform nuclear staining dominates the section. | That compartment conflicts with the secreted annotation and extracellular tissue profile (UniProt P35318: secreted; HPA: tissue profile). Treat it as a suspect pattern, then inspect the negative control and repeat with detection background controlled (general IHC practice). |
| An unexpected cell population stains strongly, including smooth muscle cells. | Smooth muscle cells are reported as not detected (HPA: smooth muscle), so test for cross-reactivity or endogenous detection activity (general IHC practice). Interpret local extracellular deposits cautiously: secreted ADM can appear away from its expressing cell (HPA: reliability description). |
| Brown signal spreads across stroma and tissue edges without a coherent cellular pattern. | Some extracellular signal is plausible (HPA: tissue profile), but a uniform haze obscuring anatomy suggests assay background (general IHC practice). Compare a negative reagent control and assess whether deposits follow recognizable extracellular structures before scoring ADM. |
| No signal appears in a section with a named high staining population. | An adrenal glandular or kidney glomerular section provides a reference pattern (HPA: High in those populations). Check tissue identity and assay controls, then review retrieval, primary antibody dilution and detection (general IHC practice); a blank reference section cannot establish true ADM absence. |
💡Expected ADM appearanceCall positive when staining is mainly extracellular and associated with a reported high staining population, such as adrenal glandular or kidney glomerular cells (HPA: tissue IHC); dominant uniform nuclear colour is suspect (UniProt P35318: secreted; general IHC practice).
| Secretion and topology (UniProt P35318). | ADM is secreted and lacks a transmembrane segment (UniProt P35318). Interpret extracellular deposits alongside cell-associated staining; do not require a crisp membrane rim (HPA: extracellular tissue profile). |
| Precursor processing (UniProt P35318). | The precursor has a signal peptide and annotated propeptide regions (UniProt P35318: processing). Antibody epitope location is unspecified here, so the record cannot establish which processed ADM species its stain detects. |
| Tissue and cell context (HPA: tissue IHC). | High staining is reported in bronchial basal cells, fallopian tube ciliary rootlets and kidney glomerular cells (HPA: High). Endothelial staining varies: colon is Low, while cerebral cortex is Not detected (HPA: tissue IHC). |
| Reliability and RNA comparison (HPA: Approved). | HPA reports medium agreement between antibody staining and RNA expression (HPA: reliability description). Because ADM is secreted, a protein deposit and its producing cell may occupy different locations; RNA alone cannot adjudicate the slide. |
| Antibody evidence (HPA: HPA068955 and CAB016075). | Both listed antibodies have Approved IHC status (HPA: antibody validation). Neither is marked Enhanced in this payload, so do not describe this pattern as reproduced by independent antibodies or orthogonal data. |
| IF/ICC expectation? (HPA: subcellular and antibody records). | The subcellular summary says Secreted, but gives no main location or cell-line images; neither listed antibody has an ICC status (HPA: subcellular and antibody records). The separate IF/ICC guide must establish its own visual pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| Adrenal glandular cells and kidney glomerular cells are both blank. | A failed assay is possible because both populations are reported High (HPA: tissue IHC); absence alone does not identify the failed step. | Confirm the expected cells are present, inspect the positive control, and review retrieval, primary dilution and chromogenic detection in sequence (general IHC practice). |
| Nearly every structure is brown, including tissue outside the expected cell populations. | Nonspecific antibody or detection background can obscure the extracellular pattern (general IHC practice; HPA: tissue profile). | Compare a primary-omission control, review blocking and washes, and adjust primary antibody concentration according to its documented IHC guidance (general IHC practice). |
| Nuclei stain more strongly than extracellular spaces or reported positive cells. | A dominant nuclear pattern conflicts with secreted ADM and HPA extracellular positivity (UniProt P35318: location; HPA: tissue profile). | Check the reagent control and examine a named high staining population before assigning ADM positivity; review detection background if nuclear colour persists (general IHC practice). |
| Smooth muscle cells appear strongly positive. | This conflicts with the Not detected entry for smooth muscle cells (HPA: smooth muscle); cross-reactivity or endogenous activity is possible (general IHC practice). | Verify cell identity, compare controls, and distinguish cellular colour from adjacent extracellular deposits before scoring (general IHC practice; HPA: secreted-protein reliability caveat). |
| An endothelial result differs between colon and cerebral cortex. | HPA reports Low staining in colon endothelial cells and Not detected in cerebral cortex endothelial cells (HPA: tissue IHC). | Use the tissue-specific reference when scoring. Check controls if a strong cerebral cortex endothelial result persists (general IHC practice; HPA: cerebral cortex, Not detected). |
| A positive IHC result is expected to reproduce as a defined IF/ICC cell pattern. | HPA supplies a Secreted summary but no main IF location, cell-line images or ICC status for the listed antibodies (HPA: subcellular and antibody records). | Treat IF/ICC localisation as unestablished by these records; assess it using the separate IF/ICC guide and appropriate imaging controls (general IF practice). |
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: glandular cells should stain for ADM (HPA: High in adrenal gland glandular cells). Use caudate glial cells as the negative tissue (HPA: Not detected in caudate glial cells); on the adrenal slide, cells outside the glandular population should lack convincing cell-associated staining, while staining beyond cell boundaries needs cautious interpretation because ADM is secreted (UniProt P35318: Secreted).
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 a no-primary, secondary-only control and a host-matched nonimmune IgG control for a polyclonal antibody or subclass-matched isotype control for a monoclonal antibody; confirm specificity with knockout tissue or a validated peptide-block control (standard IHC practice). For chromogenic adrenal IHC, block endogenous peroxidase and check for residual signal in the no-primary control (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported; the selected A00594 rat-kidney paraffin IHC caption gives a 1:100 dilution but does not report the fixative (A00594 tissue-IHC caption). Antigen-retrieval dependence is unreported, and the supplied evidence does not establish whether frozen sections or IF are easier (A00594 tissue-IHC caption; HPA subcellular: no ICC-IF image cell lines listed). Diffuse or extracellular signal can complicate assignment of staining to adrenal glandular cells because ADM is secreted (UniProt P35318: Secreted).
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 by tracking retrieval, tissue handling, compartment, and controls before interpreting chromogenic signal (UniProt P35318; HPA tissue IHC).
How should I adjust retrieval when ADM staining is weak or diffuse?
Start with citrate pH 6.0 heat-induced retrieval at 95–98 °C for 20 min (page retrieval rule). If staining is weak, compare a modestly longer exposure or an alternative buffer on adjacent sections, keeping antibody dilution and detection constant (standard IHC practice). Score glandular or glomerular signal alongside extracellular staining, because ADM is secreted and HPA reports both cellular positivity and extracellular positivity (UniProt P35318; HPA tissue IHC). Excessive retrieval can damage section morphology, so reject conditions that improve color at the expense of tissue integrity (standard IHC practice). Record the selected condition with each staining run so differences in retrieval are not mistaken for biological differences (standard IHC practice).
Could fixation explain inconsistent ADM staining between tissue blocks?
ADM-specific fixation sensitivity is unknown from the supplied evidence; the selected antibody image identifies paraffin-embedded rat kidney and 1:100 dilution but does not state a fixative (caption A00594). Record fixative, time to fixation, fixation duration, and processing history for each block before comparing staining intensity (standard IHC practice). Test differently handled blocks in the same staining run with identical retrieval and detection, using preserved morphology to judge section quality (standard IHC practice). A difference between blocks can reflect tissue handling or biology, so do not assign it to ADM epitope damage without a controlled comparison (standard IHC practice). HPA staining patterns provide tissue context, not fixation sensitivity measurements (HPA tissue IHC).
Should ADM stain producer cells, extracellular space, or both?
Expect localisation to require compartment-specific interpretation: ADM is a secreted precursor with no annotated transmembrane segment (UniProt P35318). HPA describes extracellular positivity in most tissues, including placenta and endothelial regions, and reports high staining in adrenal glandular cells and kidney glomerular cells (HPA tissue IHC). Score intracellular and extracellular chromogenic deposits separately, with the cell boundary and surrounding tissue visible on the counterstained section (standard IHC practice). Do not treat extracellular staining beside a cell as proof that the adjacent cell produced ADM, because secreted protein and its source can occupy different locations (UniProt P35318; HPA tissue IHC). Compare the pattern with a matched negative control before assigning faint deposits to either compartment (standard IHC practice).
Can this stain distinguish mature ADM from its precursor or PAMP?
Do not identify the detected molecular species from chromogenic staining alone: the record describes a 185-amino-acid precursor, processing regions, and amidated residues (UniProt P35318). ADM and the proadrenomedullin N-terminal peptide PAMP arise from this precursor, while no isoforms are annotated in the supplied record (UniProt P35318). Check the catalog antibody's stated immunogen or epitope before making a mature-ADM or PAMP-specific claim; that epitope is not supplied here (catalog antibody evidence supplied). If the epitope is undocumented, report staining as ADM-related immunoreactivity and retain the observed cellular or extracellular compartment in the result (standard IHC reporting practice). An appropriate peptide-competition or independent-epitope comparison can test specificity, subject to reagent availability (standard IHC practice).
How should I check ADM localisation in a companion IF experiment?
For companion IF/ICC, pair ADM with a marker of the expected cell population, such as an endothelial marker where that population is being examined (HPA tissue IHC; standard IF practice). Choose fluorophores after checking tissue autofluorescence in unstained controls, and place a weak ADM signal in a channel with low background (standard IF practice). Set permeabilisation according to the antibody epitope's accessibility: intracellular epitopes may require it, whereas extracellular epitopes can be assessed without it (standard IF practice). The supplied record does not identify this antibody's epitope or provide IF/ICC fixation evidence, so establish those choices with matched controls (catalog antibody evidence supplied). Keep channel exposures fixed when comparing samples and inspect single-color controls for bleed-through (standard IF practice).
How can I reduce brown background without losing extracellular ADM signal?
First inspect a no-primary control and the tissue edge to separate detection background from the extracellular positivity reported for ADM (HPA tissue IHC; standard IHC practice). Use an appropriate protein block, wash consistently, and titrate primary antibody against a matched positive section rather than relying on stronger counterstaining (standard IHC practice). The selected paraffin rat-kidney image used 1:100, which is a documented example for SKU A00594, not a validated dilution for every specimen (caption A00594). With peroxidase-based chromogenic detection, include a peroxidase block and check whether signal persists without primary antibody (standard IHC practice). Preserve comparable development times across sections so changes in diffuse brown deposit remain interpretable (standard IHC practice).
What should I measure when ADM staining spans cells and extracellular space? ⚠ ANSWER MARKED FOR VERIFICATION
Define the compartment before scoring, because HPA reports extracellular ADM positivity as well as high staining in selected cell populations (HPA tissue IHC). For cell-associated signal, record the percentage of positive cells and an H-score using prespecified intensity categories; normalise to the eligible cells counted in each region (standard IHC scoring practice). For extracellular signal, measure positive area or optical density per mm² of analysable tissue, excluding folds and necrosis by a fixed rule (standard image-analysis practice). Keep retrieval, detection development, counterstain, illumination, and threshold settings consistent across the comparison (standard IHC practice). Report cellular and extracellular values separately rather than combining them into one ADM score (standard IHC scoring practice).
What makes an apparent ADM-positive IHC result convincing?
A convincing result has a reproducible anatomical pattern with interpretable cellular or extracellular staining and a clean matched negative control (standard IHC practice). HPA reports high staining in adrenal glandular cells and kidney glomerular cells, while ADM is secreted and extracellular positivity is common (HPA tissue IHC; UniProt P35318). Treat isolated nuclear staining as a compartment mismatch, and scrutinise edge staining, necrotic areas, and endogenous peroxidase signal before calling a section positive (UniProt P35318; standard IHC practice). A cell type reported as undetected by HPA requires additional validation if strongly stained, because HPA rates tissue staining and RNA agreement as medium consistency (HPA tissue IHC). Confirm disputed patterns with an independent specificity control and document the compartment scored (standard IHC practice).
Boster reagents
Best ADM / Pro-adrenomedullin IHC Antibodies
Anti-ADM IHC images show paraffin-embedded rat kidney, human liver, and human breast carcinoma (catalog IHC captions); IF is listed for A00594 in human and rat (catalog applications and reactivity).
Real IHC data
Anti-ADM/Adrenomedullin Antibody
Cat # A00594
Real IHC data
Anti-ADM (N100) Antibody
Cat # A00594-1
A00594 will render with rat-kidney IHC at 1:100; its catalog also lists human-liver IHC and IF for human and rat (catalog IHC captions; catalog applications and reactivity). A00594-1 will render with human breast carcinoma IHC at 1:100 and lists IHC reactivity in human, mouse, and rat (catalog IHC caption; catalog applications and reactivity).
Which to pick: For paraffin-section IHC, choose A00594 for rat kidney or human liver (A00594 IHC captions), or A00594-1 for human breast carcinoma (A00594-1 IHC caption); the fixative is unreported in both captions. For IF, choose A00594 at the listed 1:50 dilution (A00594 applications and catalog dilution); ICC validation is unreported (catalog applications). For mouse tissue IHC, choose A00594-1 because it lists mouse reactivity and IHC, while A00594 lists human and rat reactivity (catalog applications and reactivity).
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 many 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: Keep fixation conditions consistent across samples. (standard IHC practice; not target-specific); Caveat: Secreted protein may stain away from its RNA source (HPA tissue IHC); Isoform / epitope: No isoforms annotated; processing yields distinct peptides (UniProt); Regulation: Expression regulators not specified (UniProt). Evidence: UniProt P35318, HPA tissue IHC · Approved, HPA ICC-IF · Secreted, Datasheet A00594, PMC2394432. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ADM/immunohistochemistry-ihc-guide.