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ALCAM / CD166 antigen · IHC design guide
Design Immunohistochemistry for ALCAM
Plan chromogenic ALCAM IHC on paraffin sections with the catalog antibody at 2–5 μg/mL (datasheet A01788-1). Use the membranous tissue profile to select controls and assess staining by cell population (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ALCAM 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 | Membranous tissue staining (HPA tissue IHC) | |
| Staining pattern | Membranous staining in most tissues, including parathyroid (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A01788-1) | |
| Positive control | Bronchus+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 sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01788-1) | |
| Caveat | A secreted variant may complicate protein–RNA concordance (HPA tissue IHC) | |
| Regulation | Activated T and B cells express ALCAM (UniProt) | |
| Isoform / epitope | Map the epitope across 4 isoforms and extracellular versus cytoplasmic regions (UniProt) |
Section 1
Recommended ALCAM IHC & IF Protocols
Start with the catalog antibody’s IHC-P protocol (datasheet A01788-1); the published options below cover tPVAT (PMC8698607), medulloblastoma (PMC7714159), and melanoma (PMC4489046).
| Sample | Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A01788-1) |
| Fixation | Image fixative and duration unreported (datasheet A01788-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A01788-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01788-1) |
| Primary antibody | Rabbit anti-ALCAM, 2-5μg/ml (datasheet A01788-1) |
| Primary incubation | Overnight at 4 °C (datasheet A01788-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A01788-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ALCAM-positive staining in respiratory epithelial cells of bronchus (HPA tissue IHC: High). HPA tissue profile: Membranous expression in most tissues, including parathyroid gland. No signal in the no-primary control. |
💡Decision noteTry heat-mediated EDTA at pH 8.0 first for the catalog antibody (datasheet A01788-1). The citrate method used a different ALCAM clone (PMC7714159).
Section 2
What Is the Expected ALCAM Staining Pattern?
In paraffin-section IHC, expect ALCAM chiefly at cell membranes, with strong staining reported in bronchial respiratory epithelium and several glandular cell populations (HPA: tissue IHC). Its extracellular region spans residues 28–527, followed by one transmembrane segment and a short cytoplasmic region (UniProt Q13740 topology). HPA rates tissue IHC reliability Enhanced but reports only medium consistency with RNA expression, partly because a protein variant is secreted (HPA: reliability description).
| Distinct cell borders in bronchial respiratory epithelial cells or cervical glandular cells (HPA: High in both). | This fits the expected membrane pattern (HPA: membranous expression in most tissues; UniProt Q13740: cell membrane). Judge the named cell population rather than overall tissue color; adjacent unstained structures do not by themselves invalidate a positive result (general IHC interpretation). |
| Predominantly nuclear staining, with little or no cell-border staining. | Treat this as a suspect compartment pattern: the supplied localisation records place ALCAM at the cell membrane, projections and in a secreted form, without a nuclear assignment (UniProt Q13740: subcellular location). Review controls and detection before scoring it as ALCAM (general IHC practice). |
| Strong stain in cells outside the annotated positive population, without convincing borders. | For example, liver's reported high signal is in cholangiocytes (HPA: High in liver cholangiocytes). Unexpected staining elsewhere warrants a check for cross-reactivity or endogenous detection activity; HPA's cell-specific annotation alone cannot prove that every other liver cell is negative (general IHC interpretation). |
| Diffuse brown color across tissue and empty spaces, obscuring cell boundaries. | A spread-out deposit cannot establish the reported membranous pattern (HPA: tissue IHC profile). Compare with a no-primary control and inspect blocking, washes and chromogen development; diffuse background can arise from nonspecific binding or detection chemistry (general chromogenic IHC practice). |
| No staining in bronchial respiratory epithelium that should be strongly positive. | HPA reports High staining in that cell population, so a blank result calls for checking tissue identity, control performance, retrieval and detection conditions (HPA: High in bronchus; general IHC practice). A single negative section does not establish absence of ALCAM expression. |
💡Expected ALCAM appearanceCall a result positive when strong, discernible membrane staining appears in an HPA High cell population such as bronchial respiratory epithelium (HPA: High; HPA: membranous profile); isolated nuclear color or diffuse deposit is suspect (UniProt Q13740: subcellular location; general IHC interpretation).
| Epitope location | ALCAM has a large extracellular region (residues 28–527) and a cytoplasmic tail (550–583) (UniProt Q13740 topology). Interpret staining against the antibody's documented epitope if available; the supplied records do not specify one. |
| Secreted variant and RNA comparison | At least one protein variant is secreted, so tissue RNA and protein location can differ (HPA: reliability description). Do not reject cell-specific IHC solely because its RNA map looks different (HPA: medium RNA–staining consistency). |
| Cell-specific reference intensity | HPA reports High in kidney Bowman's capsule and liver cholangiocytes, Low in colon endothelial cells, and Not detected in adipocytes (HPA: tissue IHC). These are distinct cell-level references, not uniform expectations for their whole tissues. |
| IHC antibody evidence | HPA010926 and CAB002148 each have Enhanced IHC validation (HPA: antibody validation). That supports the reported IHC pattern, while the tissue IHC reliability description still flags medium consistency with RNA data (HPA: reliability description). |
| IF/ICC: should vesicles be expected? | HPA lists vesicles as an uncertain ICC-IF location and rates HPA010926 ICC Uncertain (HPA: subcellular; HPA: antibody validation). Use the separate IF/ICC guide for that application; this observation does not replace the membranous tissue-IHC expectation (HPA: tissue IHC profile). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive bronchial epithelium is blank (HPA: High in respiratory epithelial cells). | The run may have inadequate retrieval, primary-antibody exposure or chromogenic detection (general IHC practice); the supplied sources identify no ALCAM-specific retrieval or fixation sensitivity. | Verify tissue identity and a working positive control, then optimize retrieval and detection using the antibody's IHC instructions (general IHC practice). |
| A test tissue is negative while the positive control stains. | The sampled cell type may have little detectable ALCAM: HPA reports Not detected in adipocytes and Low in colon endothelial cells (HPA: tissue IHC). | Check the exact cell population against the HPA tissue annotation and score the control separately; do not equate an HPA tissue label with uniform staining throughout that tissue (HPA: tissue IHC). |
| Brown signal covers the section without clear cell borders. | Nonspecific binding or excess chromogen can hide a membranous pattern (general chromogenic IHC practice; HPA: membranous tissue profile). | Compare a no-primary control, inspect washes and blocking, and adjust detection development according to the assay instructions (general IHC practice). |
| Unexpected staining appears outside the annotated cell population. | Cross-reactivity or endogenous detection activity is possible (general IHC practice); HPA's High designation applies to named cells, such as liver cholangiocytes (HPA: tissue IHC). | Review cellular morphology and a no-primary control; if enzyme detection is used, check its endogenous-activity block before assigning the stain to ALCAM (general IHC practice). |
| Signal is mainly nuclear rather than along cell borders. | That compartment conflicts with the supplied ALCAM localisation, which includes membrane, projections and secreted protein (UniProt Q13740: subcellular location; HPA: membranous tissue profile). | Recheck controls and detection specificity, then score only a reproducible, cell-specific pattern consistent with the membrane reference (general IHC interpretation; HPA: tissue IHC). |
| IHC and an ICC-IF image appear to show different compartments. | HPA's vesicle assignment for ICC-IF is uncertain, while its tissue-IHC profile is membranous (HPA: subcellular; HPA: tissue IHC). | Interpret each application against its own validation record: the supplied antibody ratings include Enhanced IHC and Uncertain ICC for HPA010926 (HPA: antibody validation). |
Experiment evidence
IHC & IF images showing ALCAM staining
A01788-1 · IHCIHC analysis of CD166/ALCAM using anti-CD166/ALCAM antibody (A01788-1). CD166/ALCAM was detected in a paraffin-embedded section of human bre
A01788-1 · IHCIHC analysis of CD166/ALCAM using anti-CD166/ALCAM antibody (A01788-1). CD166/ALCAM was detected in a paraffin-embedded section of human lun
A01788-1 · IFIF analysis of CD166/ALCAM using anti-CD166/ALCAM antibody (A01788-1). CD166/ALCAM was detected in a paraffin-embedded section of human live
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000170017-ALCAM/tissue/cerebral+cortex#imid_2989452
HPA · StomachHuman Protein Atlas IHC, Stomach. Source: https://www.proteinatlas.org/ENSG00000170017-ALCAM/tissue/stomach+1#imid_2989368
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000170017-ALCAM/tissue/colon#imid_2989394
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000170017-ALCAM/tissue/liver#imid_2989395
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA010926; green = ALCAM, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA010926; green = ALCAM, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ALCAM IHC & IF
🧪Run bronchus first and expect high staining in respiratory epithelial cells (HPA: High in bronchus respiratory epithelial cells). Use adipose tissue as the negative tissue, focusing on adipocytes (HPA: Not detected in adipose adipocytes). On the bronchus slide, compare stained epithelium with any morphologically distinct unstained cells; the supplied HPA row does not identify a specific internal negative cell type (HPA: bronchus respiratory epithelial cells only).
Positive control tissue: Bronchus (Respiratory epithelial cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ALCAM in A-431, U-251MG, U2OS, with annotated localisation: Vesicles (uncertain) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host-matched rabbit IgG isotype controls, plus ALCAM-knockout tissue or cells as a biological negative (caption: rabbit primary antibody; standard IHC practice). Check endogenous peroxidase before HRP/DAB detection in bronchus; for IF, check tissue autofluorescence with a no-primary control (caption: HRP/DAB detection; standard IHC/IF practice).
⚠️Feasibility: The selected A01788-1 paraffin-section caption reports heat retrieval in EDTA, pH 8.0, but its fixative is unreported; no target-specific fixation window or fixation effect is supplied (A01788-1 tissue-IHC caption). Treat retrieval as a condition to validate for the catalog antibody, rather than a proven requirement for all ALCAM antibodies (A01788-1 tissue-IHC caption). The supplied evidence does not establish whether frozen sections or IF are easier; for bronchus, assess background from endogenous peroxidase in chromogenic IHC or autofluorescence in IF (standard IHC/IF practice).
HPA tissue IHC evidence for ALCAM
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 |
|---|---|---|---|---|
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Processes in granular layer | High | Protein (IHC) | HPA → |
| Cervix | Glandular cells | High | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular 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 → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ALCAM IHC Tips
Troubleshoot ALCAM staining in paraffin sections by checking retrieval, cell compartment, controls, and scoring before interpreting chromogenic signal.
How should I optimize retrieval when ALCAM staining is weak in paraffin sections?
Start with heat-mediated retrieval in EDTA pH 8.0 for this catalog antibody (datasheet A01788-1). Its paraffin-section example used that retrieval before an overnight primary incubation at 4°C, so first check heating consistency and section handling while keeping the documented conditions fixed (datasheet A01788-1; standard IHC practice). Compare a positive control processed alongside the weak section, because ALCAM staining can vary by cell population within a tissue (HPA tissue IHC; standard IHC practice). If signal remains weak, vary retrieval time cautiously across matched sections and monitor tissue damage and background; a longer treatment is an optimization trial, not a documented ALCAM requirement (standard IHC practice).
Could fixation explain weak or uneven ALCAM staining?
The selected paraffin-section caption does not state a fixative, so this antibody’s target-specific sensitivity to fixation is unknown (datasheet A01788-1). Record the fixative, fixation duration, processing history, and section age for each specimen before comparing staining intensity (standard IHC practice). Run a control section through the same processing and staining batch, then assess whether weak signal follows a specimen or a batch (standard IHC practice). If processing histories differ, interpret intensity differences cautiously and optimize on matched material; neither the reported tissue staining pattern nor ALCAM topology establishes a fixation effect for this antibody (HPA tissue IHC; UniProt Q13740 topology; standard IHC practice).
Where should convincing ALCAM signal appear in chromogenic IHC?
Prioritize staining along cell boundaries: ALCAM is a membrane protein with an extracellular region at residues 28–527 and a transmembrane segment at 528–549 (UniProt Q13740 topology). Membranous expression across many tissues supports that expectation, although a secreted variant can complicate agreement between tissue protein and RNA patterns (HPA tissue IHC). Score membrane staining separately from diffuse cytoplasmic or extracellular DAB, and inspect neighboring cells at contacts rather than treating every brown deposit as equivalent (standard IHC practice; UniProt Q13740 function). The reported vesicular IF location is uncertain and does not, by itself, establish that punctate chromogenic tissue staining is specific (HPA subcellular).
Can isoforms or epitope position change how I interpret ALCAM staining?
ALCAM has 4 reported isoforms, but the supplied antibody caption does not identify its epitope or establish which isoforms it detects (UniProt Q13740 isoforms; datasheet A01788-1). The canonical sequence places 5 Ig-like domains in the extracellular region and a short cytoplasmic region at residues 550–583 (UniProt Q13740 domains and topology). It also lists 9 glycosylation sites, so epitope accessibility may depend on the antibody’s binding site; no such dependence has been shown here (UniProt Q13740 glycosylation; standard IHC practice). Avoid assigning a staining difference to a particular isoform or glycan without epitope mapping or an independent validation assay (standard IHC practice).
How can IF help investigate an ambiguous ALCAM IHC pattern?
Use IF as a separate validation experiment, pairing ALCAM with a marker for the suspected cell population so signal can be assigned to the right cells (standard IF practice). Choose fluorophores after checking tissue autofluorescence in unstained controls, and include single-color controls when assessing overlap (standard IF practice). If the antibody recognizes an extracellular epitope, test minimal permeabilisation; if it recognizes the cytoplasmic region, permeabilisation may be needed, but the supplied caption does not map this antibody’s epitope or validate it for IF (UniProt Q13740 topology; datasheet A01788-1; standard IF practice). The reported vesicular IF location is uncertain, so interpret puncta alongside membrane staining and controls (HPA subcellular).
What should I check when DAB background obscures ALCAM staining?
First compare a no-primary control with the test section to distinguish detection-system signal from antibody-associated staining (standard IHC practice). The documented example 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 A01788-1). Check peroxidase blocking, wash quality, and DAB development time on matched sections; these are general chromogenic IHC controls, not demonstrated ALCAM-specific requirements (standard IHC practice). If background persists, titrate the primary around the documented concentration and compare membrane contrast with the no-primary control before changing several steps together (datasheet A01788-1; standard IHC practice).
How should I quantify ALCAM staining across heterogeneous sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because ALCAM is predominantly membranous in tissue IHC and expression varies among cell types (HPA tissue IHC). Report the percentage of positive cells and membrane intensity using a prespecified scale; an H-score can combine intensity with the percentage at each intensity (standard IHC practice). For spatial comparisons, count positive cells per mm² of viable, annotated tissue and record the sampled area (standard IHC practice). Normalize comparisons to the same cell population, compartment, staining batch, and exposure to DAB development, while excluding necrotic regions and recording how boundaries and positivity thresholds were set (standard IHC practice).
How do I distinguish true ALCAM staining from tissue or detection artefacts?
Look for reproducible cell-boundary staining in plausible cells, consistent with ALCAM’s membrane location and the HPA tissue profile (UniProt Q13740 subcellular location; HPA tissue IHC). For example, HPA reports high staining in respiratory epithelial cells of bronchus and no detection in adipocytes of adipose tissue; apply those observations to the specified cells rather than every cell in either tissue (HPA tissue IHC). Treat staining limited to section edges, necrotic material, or a no-primary control as suspect, and investigate endogenous peroxidase when DAB appears without primary antibody (standard IHC practice). Diffuse signal outside expected cells needs corroboration, especially because at least one ALCAM variant is secreted (HPA tissue IHC).
Boster reagents
Best ALCAM / CD166 antigen IHC Antibodies
The IHC-validated antibody A01788-1 has illustrated paraffin-section IHC in human breast, lung and liver cancer and mouse lung, plus IF in human liver cancer (catalog image captions).
Real IHC data
Anti-CD166/ALCAM Antibody ®
Cat # A01788-1
A01788-1 has illustrated IHC in human breast, lung and liver cancer and mouse lung paraffin sections, plus IF in human liver cancer paraffin sections (catalog image captions). M01788 lists IHC and ICC/IF applications and human, mouse and rat reactivity, but has no supplied IHC or IF image captions (catalog applications, reactivity and image fields).
Which to pick: For tissue IHC, pick A01788-1 when its illustrated paraffin-section examples match your sample; its captions do not report the fixative (A01788-1 IHC image captions). For IF on paraffin sections, A01788-1 has an illustrated human liver cancer example; for ICC/IF, M01788 lists that application and is a rabbit monoclonal, but has no supplied image example (A01788-1 IF image caption; M01788 catalog applications, clone and image fields). Both list human, mouse and rat reactivity, while the supplied IHC images document human and mouse samples only (catalog reactivity; A01788-1 IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q13740 (CD166_HUMAN, CD166 antigen).
- Human Protein Atlas. ALCAM tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. ALCAM subcellular location (ICC-IF): Localized to vesicles..
- Human Protein Atlas. ALCAM antibody validation summary (2 antibodies).
- Comparative Proteomic Analysis of tPVAT during Ang II Infusion. Biomedicines 2021 — PMC8698607.
- Activated leukocyte cell adhesion molecule expression correlates with the WNT subgroup in medulloblastoma and is involved in regulating tumor cell proliferation and invasion. PloS one 2020 — PMC7714159.
- ALCAM (CD166) expression and serum levels in pancreatic cancer. PloS one 2012 — PMC3380038.
- Prognostic significance of ALCAM (CD166/MEMD) expression in cutaneous melanoma patients. Diagnostic pathology 2015 — PMC4489046.
- PubMed PMID:7760007 — UniProt-cited evidence.
- PubMed PMID:15496415 — UniProt-cited evidence.
- PubMed PMID:16641997 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ALCAM (CD166 antigen). Expected localisation Membranous tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A01788-1). Validated IHC antibody: A01788-1 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bronchus (Respiratory epithelial cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human breast cancer tissue; fixative not specified (datasheet A01788-1); 2. Fixation: Image fixative and duration unreported (datasheet A01788-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: EDTA pH 8.0 (datasheet A01788-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01788-1); 8. Primary antibody: Rabbit anti-ALCAM, 2-5μg/ml (datasheet A01788-1); 9. Primary incubation: Overnight at 4 °C (datasheet A01788-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01788-1); Caveat: A secreted variant may complicate protein–RNA concordance (HPA tissue IHC); Isoform / epitope: Map the epitope across 4 isoforms and extracellular versus cytoplasmic regions (UniProt); Regulation: Activated T and B cells express ALCAM (UniProt). Evidence: UniProt Q13740, HPA tissue IHC · Enhanced, HPA ICC-IF · vesicles., Datasheet A01788-1, PMC8698607, PMC7714159. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ALCAM/immunohistochemistry-ihc-guide.