This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
PAM / Peptidyl-glycine alpha-amidating monooxygenase · IHC design guide
Design Immunohistochemistry for PAM
Plan PAM IHC on paraffin sections using the catalog antibody’s validated conditions (datasheet A00332-2). Compare staining with the cytoplasmic pattern reported in neuroendocrine cells, islets and heart muscle (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
PAM 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 | Observed cytoplasmic (HPA tissue IHC); secretory granule membrane expected (UniProt) | |
| Staining pattern | Cytoplasmic in neuroendocrine cells, islets and heart muscle (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A00332-2) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | A secreted variant may weaken RNA–protein concordance (HPA tissue IHC) | |
| Regulation | No expression regulator specified in the record (UniProt) | |
| Isoform / epitope | 6 isoforms; map the epitope against processing and TM sites (UniProt) |
Section 1
Recommended PAM IHC & IF Protocols
The catalog antibody protocol uses EDTA retrieval (datasheet: A00332-2). One published chromogenic PAM IHC protocol provides a comparison (PMC7331689).
| Sample | Paraffin-embedded human prostate cancer tissue; fixative not specified (datasheet A00332-2) |
| Fixation | Image fixative and duration unreported (datasheet A00332-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A00332-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A00332-2) |
| Primary antibody | Rabbit anti-PAM, 2-5 μg/ml (datasheet A00332-2) |
| Primary incubation | Overnight at 4 °C (datasheet A00332-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A00332-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | PAM-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in neuroendocrine cells, islets of Langerhans and heart muscle. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA at pH 8.0 for the catalog antibody (datasheet: A00332-2); the published protocol used sodium citrate at pH 6.0 (PMC7331689).
Section 2
What Is the Expected PAM Staining Pattern?
PAM should show predominantly cytoplasmic staining in neuroendocrine cells, islets of Langerhans and heart muscle (HPA tissue IHC). Secretory granule and membrane localization is consistent with an intragranular region spanning residues 31–863 and a transmembrane segment at 864–887 (UniProt P19021 topology). HPA rates tissue IHC reliability Enhanced, with medium consistency between staining and RNA data (HPA tissue IHC).
| Cytoplasmic signal in endocrine cells or cardiomyocytes, with clearer staining than surrounding cells. | This fits the reported cellular distribution (HPA tissue IHC: high in appendix, colon and duodenal endocrine cells; high in cardiomyocytes). Score the named cell populations rather than treating every cell in a positive tissue as positive. |
| Predominantly nuclear staining, without the expected cytoplasmic signal. | Treat this as a localization mismatch requiring investigation: the reported tissue pattern is cytoplasmic (HPA tissue IHC), and UniProt places PAM in secretory vesicles, membranes and secreted compartments (UniProt P19021 subcellular location). Review controls and staining conditions before assigning it to PAM. |
| Strong staining in adipocytes or esophageal squamous epithelial cells. | These cell populations are reported as not detected (HPA tissue IHC: adipose adipocytes; esophageal squamous epithelial cells). Consider nonspecific antibody binding or endogenous chromogen activity, and check a detection-only control. A negative call applies to the listed cells, not necessarily every cell in either tissue. |
| Uniform haze across cells and surrounding tissue, with little cellular distinction. | Diffuse background does not reproduce the cell-selective tissue profile (HPA tissue IHC). In chromogenic IHC, incomplete blocking, excess detection reagent or residual endogenous enzyme activity can contribute (general IHC practice); assess controls before interpreting weak staining. |
| No signal in an expected positive cell population. | Check that the section contains the relevant cells: endocrine cells and cardiomyocytes are reported high (HPA tissue IHC). Then review antibody application, detection controls and retrieval conditions (general IHC practice). Absence in one section alone cannot establish absence of PAM. |
💡Expected PAM appearanceCall a result positive when cytoplasmic staining is clear in the relevant endocrine cells or cardiomyocytes (HPA tissue IHC: high); diffuse haze, isolated nuclear signal or staining confined to reported negative cell populations warrants control review (HPA tissue IHC; UniProt P19021 subcellular location).
| Cell identity and tissue choice | Use a positive population such as heart cardiomyocytes or intestinal endocrine cells alongside a reported negative population such as adipose adipocytes (HPA tissue IHC). Interpret each at the cell level: HPA's high and not-detected calls name specific cells. |
| Epitope location and processing | PAM has a signal peptide at residues 1–20, an intragranular region at 31–863 and a cytoplasmic tail at 888–973 (UniProt P19021 processing and topology). Antibody epitope location is not supplied, so these features cannot predict which processed form the assay detects. |
| Secreted forms and RNA comparison | At least one protein variant is secreted, so protein location may differ from RNA location and their correlation is complex (HPA tissue IHC reliability note). Secreted PAM is also annotated by UniProt (UniProt P19021 subcellular location); do not require a strict RNA-to-staining match. |
| Isoforms | Six isoforms are listed (UniProt P19021 isoforms). Without an antibody epitope or isoform-reactivity record, the staining cannot be assigned to a particular isoform. Avoid explaining a negative or unexpected pattern through isoform selectivity alone. |
| Antigen retrieval | Retrieval conditions can affect IHC staining in general (general IHC practice). Neither supplied source reports a PAM-specific fixation or retrieval effect; optimize against positive and negative cell populations without predicting which retrieval condition will increase PAM signal. |
| IF/ICC question: should the same appearance be expected? | IF/ICC shows approved Golgi apparatus and cytosol localization (HPA subcellular ICC-IF). That supports intracellular staining but is a separate assay from paraffin IHC; use the tissue IHC profile to judge chromogenic sections (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells show no chromogen. | The section may lack the relevant cells, or staining or detection may have failed (HPA tissue IHC: cell-specific high calls; general IHC practice). | Confirm cell identity and section quality; check the positive control, detection reagents and retrieval conditions (general IHC practice). |
| Only nuclei stain strongly. | This conflicts with the cytoplasmic tissue profile and secretory-vesicle annotation (HPA tissue IHC; UniProt P19021 subcellular location). | Review detection-only controls, antibody concentration and morphology; withhold a PAM-positive call until cytoplasmic staining is supported (general IHC practice). |
| Adipocytes or esophageal squamous cells stain. | These named cell populations are reported not detected (HPA tissue IHC); nonspecific binding or endogenous detection activity is possible (general IHC practice). | Check a detection-only control and blocking, then compare with a reported positive cell population on a suitable section (general IHC practice; HPA tissue IHC). |
| The entire section has diffuse brown background. | Poor cell-level contrast can arise from incomplete blocking, excess reagent or residual endogenous enzyme activity (general IHC practice). | Inspect a detection-only control, review blocking and reagent conditions, and score only distinguishable cellular staining (general IHC practice). |
| Signal differs from tissue RNA abundance. | HPA reports medium staining-to-RNA consistency and notes that a secreted variant can make tissue locations differ (HPA tissue IHC reliability note). | Check the named cell population and IHC controls; interpret the protein stain on its own terms rather than forcing an RNA-level match (HPA tissue IHC). |
| Golgi-like IF staining seems different from paraffin IHC. | Approved Golgi and cytosol localization comes from ICC-IF, while the tissue IHC profile describes cytoplasmic cell populations (HPA subcellular ICC-IF; HPA tissue IHC). | Judge the chromogenic section against the tissue IHC profile and its controls; consult the separate IF/ICC guide for that assay. |
Experiment evidence
IHC & IF images showing PAM staining
A00332-2 · IHCIHC analysis of PAM using anti-PAM antibody (A00332-2). PAM was detected in a paraffin-embedded section of human prostate cancer tissue. Hea
A00332-2 · IHCIHC analysis of PAM using anti-PAM antibody (A00332-2). PAM was detected in a paraffin-embedded section of mouse heart tissue. Heat mediated
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000145730-PAM/tissue/cerebral+cortex#imid_15276074
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000145730-PAM/tissue/colon#imid_15276016
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000145730-PAM/tissue/liver#imid_15276009
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000145730-PAM/tissue/kidney#imid_15276010
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB026119; green = PAM, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB026119; green = PAM, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for PAM IHC & IF
🧪Run adrenal gland first: its glandular cells must stain for PAM (HPA: High in adrenal gland glandular cells). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); on the adrenal slide, assess cells outside the glandular population for background without assuming they are PAM-negative (HPA: glandular-cell staining only).
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show PAM in A-431, U-251MG, U2OS, with annotated localisation: Golgi apparatus (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide, a rabbit IgG isotype control matched to the primary antibody’s clonality, and PAM-knockout tissue as a biological negative where available (caption: rabbit anti-PAM primary; standard IHC practice). For adrenal DAB sections, quench endogenous peroxidase and check blood-cell-associated background before scoring (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A00332-2 paraffin-section caption does not state a fixative (selected-SKU caption: fixative unreported). The caption reports heat-mediated retrieval in EDTA at pH 8.0, but does not establish whether retrieval is required under other conditions (selected-SKU caption). Frozen sections and IF cannot be ranked as easier from the supplied evidence; for adrenal DAB sections, endogenous peroxidase can complicate interpretation (HPA: ICC-IF images available; standard IHC practice).
HPA tissue IHC evidence for PAM
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 | Endocrine cells | High | Protein (IHC) | HPA → |
| Bronchus | Ciliated cells (cilia axoneme) | High | Protein (IHC) | HPA → |
| Colon | Endocrine cells | High | Protein (IHC) | HPA → |
| Duodenum | Endocrine cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PAM IHC Tips
Troubleshoot PAM staining in paraffin sections by checking retrieval, compartment, cell type and controls before interpreting chromogenic signal.
What retrieval should I start with if PAM staining is weak?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A00332-2). This is the retrieval used for the catalog antibody’s human prostate cancer tissue image; the caption does not report a heating time (datasheet A00332-2). If staining is weak, compare heating durations on matched sections while keeping antibody concentration, incubation and detection constant (standard IHC practice). The reported primary incubation was 2 μg/ml overnight at 4°C, so check those conditions before changing retrieval chemistry (datasheet A00332-2). Include a positive control section in each comparison to distinguish poor retrieval from a low-expressing specimen (standard IHC practice).
Could fixation explain weak or uneven PAM staining?
The selected paraffin-section caption does not state a fixative, so PAM-specific fixation sensitivity is unknown for this antibody (datasheet A00332-2). Record the fixative, fixation interval and processing history for every section before comparing staining across specimens (standard IHC practice). If a section stains weakly, compare it with a positive control processed under the same conditions, using the reported EDTA retrieval at pH 8.0 (datasheet A00332-2; standard IHC practice). Examine tissue preservation and edge-to-center variation before increasing antibody concentration, because processing differences can affect chromogenic IHC signal (standard IHC practice). Do not assign a PAM-specific fixation effect without a controlled comparison (datasheet A00332-2).
Where should convincing PAM staining appear in a tissue section?
Expect a predominantly intracellular pattern: PAM is associated with secretory vesicle membranes and granules, while HPA reports cytoplasmic tissue staining (UniProt P19021 localisation; HPA tissue IHC). HPA also assigns Golgi apparatus and cytosol localisation in its subcellular dataset (HPA subcellular). Its annotated transmembrane segment spans residues 864–887, with residues 31–863 intragranular and 888–973 cytoplasmic (UniProt P19021 topology). Compare the stained compartment with nearby unstained cells and a matched positive control; diffuse nuclear signal alone would not fit these annotations (UniProt P19021 localisation; standard IHC practice). Extracellular signal needs careful interpretation because at least one variant is secreted (HPA tissue IHC).
How should I interpret staining when the antibody epitope is unspecified?
Do not assume the catalog antibody detects every PAM product: the record lists 6 isoforms, while the supplied caption does not map its epitope (UniProt P19021 isoforms; datasheet A00332-2). PAM has a signal peptide at residues 1–20 and a propeptide at 21–30, so epitope position matters when considering processed protein (UniProt P19021 processing). The transmembrane segment at 864–887 separates an intragranular region from a cytoplasmic tail that includes annotated phosphorylation sites (UniProt P19021 topology and modified residues). A glycosylation site is annotated at residue 762, but the supplied evidence does not show whether it affects this antibody (UniProt P19021 glycosylation; datasheet A00332-2). Compare independent epitopes or orthogonal evidence before assigning an isoform to an IHC pattern (standard IHC practice).
How can I check PAM localisation with multiplex immunofluorescence?
Treat IF as a separate antibody validation, since the supplied catalog image documents chromogenic tissue IHC rather than IF for this antibody (datasheet A00332-2). Pair PAM with a marker for the expected cell population; HPA reports high staining in appendix and colon endocrine cells, among other cell types (HPA tissue IHC). Choose spectrally separated fluorophores and inspect unstained tissue for autofluorescence before assigning a faint channel to PAM (standard IF practice). If the epitope is intragranular, establish permeabilisation that gives antibody access across the vesicle membrane; a cytoplasmic-tail epitope has different access requirements (UniProt P19021 topology; standard IF practice). Because this antibody’s epitope is unspecified, test permeabilisation with appropriate single-stain and secondary-only controls (datasheet A00332-2; standard IF practice).
How can I reduce diffuse brown staining without losing PAM signal?
Begin by checking a no-primary control and blocking endogenous peroxidase before HRP and DAB development (standard chromogenic IHC practice). The catalog image used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and a peroxidase-conjugated secondary for 30 minutes at 37°C (datasheet A00332-2). Keep those reported conditions as a reference while varying one blocking, washing or detection step at a time (datasheet A00332-2; standard IHC practice). Assess background in compartments and cells expected to be low or unstained, such as HPA-listed adipocytes in adipose tissue (HPA tissue IHC). A widespread precipitate or section-edge signal should prompt a check of detection conditions before it is scored as PAM (standard IHC practice).
How should I score PAM staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and intracellular compartment before scoring, since HPA describes cytoplasmic expression in neuroendocrine cells, islets of Langerhans and heart muscle (HPA tissue IHC). For a cell-based readout, report the percentage of positive viable target cells and, when intensity categories are reproducible, an H-score from 0–300 (standard IHC scoring practice). For a spatial readout, report positive-cell density per mm² of viable tissue rather than total section area (standard IHC scoring practice). Apply the same retrieval, staining batch, exposure and scoring thresholds to compared sections (standard IHC practice). Record compartment-specific scores separately when intracellular staining and extracellular signal coexist, because a PAM variant may be secreted (HPA tissue IHC).
When is a PAM-positive DAB signal biologically credible?
Give greatest weight to reproducible intracellular staining in an expected cell population, with signal above matched negative controls (UniProt P19021 localisation; HPA tissue IHC; standard IHC practice). HPA reports high staining in appendix and colon endocrine cells and heart cardiomyocytes, providing cell-specific reference patterns rather than a universal tissue threshold (HPA tissue IHC). Predominantly nuclear staining conflicts with the annotated vesicle, Golgi and cytosol locations and warrants further validation (UniProt P19021 localisation; HPA subcellular). Exclude section edges, necrotic regions and signal retained in a no-primary control before interpreting brown deposits as PAM (standard IHC practice). Check endogenous peroxidase blocking when DAB persists in controls, and avoid treating secreted signal alone as proof of its producing cell (standard IHC practice; HPA tissue IHC).
Boster reagents
Best PAM / Peptidyl-glycine alpha-amidating monooxygenase IHC Antibodies
A00332-2 has IHC images from paraffin-embedded human prostate cancer and heart, mouse heart, and rat heart sections (A00332-2 IHC captions). No IF image is supplied (catalog payload).
Real IHC data
Anti-PAM Antibody ®
Cat # A00332-2
A00332-2 is listed for IHC in human, mouse, and rat (catalog applications and reactivity). Its IHC captions show paraffin-embedded human prostate cancer and heart, mouse heart, and rat heart sections (A00332-2 IHC captions).
Which to pick: For tissue IHC, choose A00332-2: its rabbit antibody was shown on paraffin-embedded sections at 2 μg/ml after EDTA pH 8.0 heat retrieval (A00332-2 IHC captions); the fixative is unreported (A00332-2 IHC captions). For cross-species IHC, the same SKU has human, mouse, and rat tissue images (A00332-2 IHC captions). No IF/ICC application or image is supplied for A00332-2, so this payload supports no IF/ICC pick (catalog applications and images).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P19021 (AMD_HUMAN, Peptidyl-glycine alpha-amidating monooxygenase).
- Human Protein Atlas. PAM tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. PAM subcellular location (ICC-IF): Localized to the Golgi apparatus and cytosol..
- Human Protein Atlas. PAM antibody validation summary (2 antibodies).
- PAM staining intensity of primary neuroendocrine neoplasms is a potential prognostic biomarker. Scientific reports 2020 — PMC7331689.
- Retinal safety evaluation of photoacoustic microscopy. Experimental eye research 2021 — PMC8018531.
- Effects of pamidronate disodium on the loss of osteoarthritic subchondral bone and the expression of cartilaginous and subchondral osteoprotegerin and RANKL in rabbits. BMC musculoskeletal disorders 2014 — PMC4240862.
- Type 2 diabetes risk alleles in PAM impact insulin release from human pancreatic β-cells. Nature genetics 2018 — PMC6237273.
- PubMed PMID:2357221 — UniProt-cited evidence.
- PubMed PMID:7999037 — UniProt-cited evidence.
- PubMed PMID:12699694 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for PAM (Peptidyl-glycine alpha-amidating monooxygenase). Expected localisation Observed cytoplasmic (HPA tissue IHC); secretory granule membrane expected (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A00332-2). Validated IHC antibody: A00332-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human prostate cancer tissue; fixative not specified (datasheet A00332-2); 2. Fixation: Image fixative and duration unreported (datasheet A00332-2); 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: EDTA pH 8.0 (datasheet A00332-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A00332-2); 8. Primary antibody: Rabbit anti-PAM, 2-5 μg/ml (datasheet A00332-2); 9. Primary incubation: Overnight at 4 °C (datasheet A00332-2). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: A secreted variant may weaken RNA–protein concordance (HPA tissue IHC); Isoform / epitope: 6 isoforms; map the epitope against processing and TM sites (UniProt); Regulation: No expression regulator specified in the record (UniProt). Evidence: UniProt P19021, HPA tissue IHC · Enhanced, HPA ICC-IF · Golgi apparatus and cytosol., Datasheet A00332-2, PMC7331689. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PAM/immunohistochemistry-ihc-guide.