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CD177 / CD177 antigen · IHC design guide
Design Immunohistochemistry for CD177
Plan chromogenic CD177 IHC on paraffin sections with bone marrow or colon as positive tissues and the catalog antibody at 1:100 (HPA tissue IHC; datasheet: 1:100). Compare cytoplasmic staining in marrow cells with membranous staining in colon enterocytes, and score the positive cell fraction (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CD177 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 | Marrow/spleen cytoplasm; colon/rectum membranes (HPA tissue IHC) | |
| Staining pattern | Marrow/spleen cell subset: cytoplasmic; colon/rectum enterocytes: membranous (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Appendix+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 | Only a variable subset of mature neutrophils expresses CD177 (UniProt) | |
| Regulation | TNF, fMLP or IL-8 raises surface CD177 (UniProt) | |
| Isoform / epitope | 3 isoforms; check ectodomain epitope coverage (UniProt) |
Section 1
Recommended CD177 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by four published CD177 IHC protocols covering gastric cancer, lung adenocarcinoma, mouse gastric mucosa, and mouse mammary tissue (PMC3734037; PMC10941067; PMC6476150; PMC10641157).
| Sample | Paraffin-embedded human colon tissue; fixative not specified (datasheet A03958) |
| Fixation | Image fixative and duration unreported (datasheet A03958); 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-CD177, 1:100 (datasheet A03958) |
| 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 | CD177-positive staining in enterocytes - Microvilli of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in a subset of cells in bone marrow and spleen. Membranous expression in colon and rectum. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval); the published protocols show tissue-specific alternatives (PMC3734037; PMC10941067; PMC6476150).
Section 2
What Is the Expected CD177 Staining Pattern?
CD177 is a GPI-anchored protein associated with the cell surface, membrane rafts and neutrophil granules; it has no transmembrane segment (UniProt Q8N6Q3 topology, subcellular location, keywords). Expect staining in a subset of maturing and mature neutrophils, alongside the tissue patterns HPA reports in marrow, spleen and intestinal epithelium (UniProt Q8N6Q3 tissue specificity; HPA tissue IHC). HPA rates its tissue IHC profile Enhanced, citing agreement between staining and RNA expression (HPA tissue IHC).
| A subset of marrow hematopoietic cells stains strongly; some cells show cytoplasmic signal (HPA tissue IHC: High in bone marrow; cytoplasmic subset). | This fits the reported marrow pattern. CD177 appears from the metamyelocyte stage onward, and only a subset of mature neutrophils expresses it; a mixed positive and negative population is expected (UniProt Q8N6Q3 tissue specificity). |
| Colon or rectum shows strong staining at enterocyte microvilli, with a membranous pattern (HPA tissue IHC: High; membranous expression). | This is an observed epithelial IHC pattern, despite CD177's well-described neutrophil expression (HPA tissue IHC; UniProt Q8N6Q3 tissue specificity). Evaluate the epithelial edge separately from any stained inflammatory cells. |
| Nuclei dominate the stain, or broad cytoplasmic staining obscures the expected cell boundaries (UniProt Q8N6Q3 subcellular location; HPA tissue IHC). | Treat nuclear dominance as a compartment mismatch: the supplied localization record lists membranes and granules, with no nuclear location (UniProt Q8N6Q3 subcellular location). Diffuse cytoplasm needs comparison with HPA's restricted marrow and spleen pattern (HPA tissue IHC). |
| Strong staining appears in an HPA-listed negative cell population, such as adipocytes or adrenal glandular cells (HPA tissue IHC: Not detected). | Question specificity or endogenous chromogenic activity before assigning CD177 positivity. HPA's negative calls apply to those listed cells; they do not exclude staining in a different cell population within the section (HPA tissue IHC; standard IHC control practice). |
| No signal appears in marrow hematopoietic cells or colon enterocyte microvilli (HPA tissue IHC: High in both populations). | Check the assay and specimen before calling the sample negative. Marrow positivity is confined to a subset, so inspect enough cells; colon offers a distinct reported epithelial pattern (HPA tissue IHC; UniProt Q8N6Q3 tissue specificity). |
💡Expected CD177 appearanceCall a result positive when it shows strong, cell-restricted staining in a subset of marrow hematopoietic cells or at colon or rectum enterocyte microvilli; widespread nuclear stain or color across HPA-listed negative cells is suspect (HPA tissue IHC: High and Not detected; UniProt Q8N6Q3 subcellular location; standard IHC control practice).
| Cellular distribution (UniProt Q8N6Q3 tissue specificity) | Expression begins at the metamyelocyte stage and persists through later neutrophil stages, but the CD177-positive fraction of mature neutrophils varies. Score the fraction of stained cells as well as intensity (UniProt Q8N6Q3 tissue specificity). |
| Surface and granule pools (UniProt Q8N6Q3 subcellular location) | CD177 occurs at the neutrophil surface and predominantly in secondary and tertiary granules; stimulation increases surface expression. Membrane and intracellular signals can therefore differ with cell state (UniProt Q8N6Q3 subcellular location). |
| Processing and shedding (UniProt Q8N6Q3 processing, subcellular location) | The record lists a signal peptide, a 22–408 mature chain, a removed C-terminal propeptide and shedding from the surface. Without the antibody epitope, these facts cannot predict which pool its IHC stain detects (UniProt Q8N6Q3 processing, subcellular location). |
| Epithelial patterns (HPA tissue IHC) | HPA reports High staining in appendix, colon and rectum enterocyte microvilli and in cervix and oral mucosa squamous cells. Do not classify every epithelial signal as cross-reactivity; compare its cell type and location with the specific HPA entry (HPA tissue IHC). |
| Antibody evidence (HPA antibodies; HPA tissue IHC) | Three listed rabbit polyclonal antibodies carry Enhanced IHC status; the tissue profile is also Enhanced for staining and RNA agreement. Those labels support the reported patterns, but do not identify the epitope or establish performance for another antibody (HPA antibodies; HPA tissue IHC). |
| Fixation and retrieval evidence (UniProt Q8N6Q3; HPA tissue IHC) | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. |
| Situation | Likely cause | Next action |
|---|---|---|
| Marrow looks negative despite an expected positive control (HPA tissue IHC: High in marrow hematopoietic cells). | The positive population may be sparse, or assay sensitivity may be insufficient; CD177 marks a variable subset of neutrophils (UniProt Q8N6Q3 tissue specificity; standard IHC practice). | Inspect more hematopoietic cells and compare a confirmed positive section processed in the same run; then assess retrieval, antibody concentration and detection as general IHC variables (HPA tissue IHC; standard IHC practice). |
| The whole section has brown haze, including HPA-listed negative cells (HPA tissue IHC: Not detected in specified cells). | Nonspecific binding or endogenous detection activity can produce diffuse chromogenic background (standard IHC practice). | Compare a no-primary control, review blocking and washes, and check endogenous peroxidase suppression if using a peroxidase system (standard IHC practice). |
| Nuclear staining is prominent across unrelated cell types (UniProt Q8N6Q3 subcellular location). | This conflicts with the listed membrane, granule and lamellipodial locations and raises concern for nonspecific signal (UniProt Q8N6Q3 subcellular location; standard IHC practice). | Review control sections and the antibody's IHC validation; score only a pattern consistent with the relevant tissue and cell population (HPA tissue IHC; standard IHC practice). |
| Colon signal appears broadly cytoplasmic instead of concentrated at the enterocyte edge (HPA tissue IHC: membranous colon pattern). | Excess background or a different stained cell population may obscure the reported microvillar pattern (HPA tissue IHC; standard IHC practice). | Inspect enterocyte borders at higher magnification and compare controls before assigning epithelial CD177 positivity (HPA tissue IHC; standard IHC practice). |
| Only some morphologically similar neutrophils stain (UniProt Q8N6Q3 tissue specificity). | A variable CD177-positive subset is a documented feature of mature neutrophils; stimulation can also increase surface expression (UniProt Q8N6Q3 tissue specificity, subcellular location). | Record the positive fraction and staining compartment. Do not require uniform neutrophil positivity to accept a run with an appropriate positive control (UniProt Q8N6Q3 tissue specificity; standard IHC practice). |
| IF/ICC Q: Is there a verified CD177 image pattern to use here? | HPA provides no main ICC-IF location and lists no cell lines with ICC-IF images (HPA subcellular ICC-IF). | A: Use the membrane and granule locations only as biological context; do not treat this IHC pattern as an ICC-IF validation or protocol (UniProt Q8N6Q3 subcellular location; HPA subcellular ICC-IF). |
Experiment evidence
IHC & IF images showing CD177 staining
A03958 · IHCImmunohistochemical analysis of paraffin-embedded human-colon, antibody was diluted at 1:100
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000204936-CD177/tissue/cerebral+cortex#imid_13375439
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000204936-CD177/tissue/colon#imid_13375653
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000204936-CD177/tissue/liver#imid_13375646
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000204936-CD177/tissue/kidney#imid_13375639
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CD177 IHC & IF
🧪Run colon first: enterocyte microvilli should stain strongly (HPA: High in colon enterocyte microvilli). Run adipose tissue as the negative, where adipocytes are listed as not detected (HPA: Not detected in adipocytes); on the colon slide, use adjacent non-enterocyte cells as candidate internal negatives, while recognizing that some neutrophils may stain (UniProt Q8N6Q3: expression in a subset of mature neutrophils).
Positive control tissue: Appendix (Enterocytes - Microvilli, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for CD177; derive a cell-line control from the positive tissue's cell type (Enterocytes - Microvilli) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and clonality; use CD177 knockout material or a validated immunogen-peptide block as a biological specificity control (standard IHC practice). For chromogenic colon IHC, quench endogenous peroxidase and check for staining unrelated to the primary antibody; for IF, assess tissue autofluorescence (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window and antigen-retrieval dependency are unreported in the supplied evidence; the selected A03958 paraffin-colon IHC caption gives 1:100 but does not report a fixative (selected A03958 tissue-IHC caption). The evidence does not establish whether frozen sections or IF are easier for CD177, and HPA supplies no ICC-IF images for this gene (HPA subcellular). In colon, interpret staining by cell type because enterocyte microvilli are HPA-positive and a subset of neutrophils can also express CD177 (HPA: High in colon enterocyte microvilli; UniProt Q8N6Q3: subset of mature neutrophils).
HPA tissue IHC evidence for CD177
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data.). 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 |
|---|---|---|---|---|
| Appendix | Enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Colon | Enterocytes - Microvilli | High | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CD177 IHC Tips
Troubleshoot CD177 staining by comparing compartment, cell type and section controls before changing detection conditions.
What retrieval should I start with for CD177 in paraffin sections?
Start with citrate buffer at pH 6.0 for heat induced retrieval at 95–98 °C for 20 minutes (page retrieval specification). Allow sections to cool in buffer, then keep heating, cooling and wash conditions consistent across the staining run (standard IHC practice). Compare the catalog antibody’s paraffin embedded human colon image, stained at 1:100, with a matched control section when judging recovery of signal (caption A03958). Evaluate membrane staining in colon alongside positive cells in bone marrow, since their reported staining patterns differ (HPA tissue IHC). If signal remains weak, titrate primary antibody and check tissue preservation before changing retrieval conditions (standard IHC practice).
Could fixation explain weak or patchy CD177 staining?
Target specific fixation sensitivity is unknown from the supplied evidence; the catalog image identifies paraffin embedded human colon but does not state its fixative (caption A03958). Record the fixative, fixation duration and tissue processing for each specimen before comparing CD177 intensity across cases (standard IHC practice). Include sections processed together when assessing staining differences, because fixation and processing can change antigen accessibility in paraffin IHC (standard IHC practice). Compare both tissue morphology and the expected membrane pattern in colon to decide whether weak staining tracks a processing problem (HPA tissue IHC; standard IHC practice). Do not assign a CD177 specific fixation effect without a controlled comparison (supplied fixation evidence).
Which compartments should show convincing CD177 staining?
Interpret a membrane outline in colon or rectum against the reported membranous tissue pattern, while allowing cytoplasmic staining in a subset of bone marrow and spleen cells (HPA tissue IHC). CD177 is associated with the cell surface, membrane rafts and cytoplasmic granule membranes, and it has no transmembrane segment (UniProt Q8N6Q3 localisation and topology). Neutrophil staining can vary with activation because surface expression increases after TNF, fMLP or CXCL8 stimulation (UniProt Q8N6Q3 localisation). Examine high magnification fields for the location of signal within intact cells rather than relying on whole section intensity (standard IHC practice). Treat a diffuse pattern that ignores cell boundaries as a reason to inspect controls and morphology (standard IHC practice).
How could CD177 processing or isoforms affect an antibody result?
Check the antibody’s stated immunogen or epitope against the mature CD177 chain, residues 22–408, before interpreting absent staining (UniProt Q8N6Q3 processing; standard IHC practice). The precursor also contains a signal sequence at residues 1–21 and a propeptide at 409–437 (UniProt Q8N6Q3 processing). Three isoforms and one recorded glycosylation site at residue 189 make epitope coverage and accessibility reasonable questions, but these records do not establish an effect on this antibody (UniProt Q8N6Q3 isoforms and glycosylation). Ask whether the supplier maps the antibody epitope, and compare a second epitope only if its identity is documented (standard IHC practice). Avoid calling a negative section CD177 deficient on one antibody result alone (standard IHC practice).
How should I investigate a CD177 result by multiplex IF?
Use IF as a separate assay and pair CD177 with a validated neutrophil marker when testing the neutrophil population reported to express it (UniProt Q8N6Q3 tissue specificity; standard IF practice). Select fluorophores after inspecting unstained tissue for autofluorescence, and place the weaker signal in a spectrally cleaner channel where possible (standard IF practice). If the antibody recognizes an extracellular CD177 epitope, first assess staining without permeabilisation; test mild permeabilisation separately when investigating granule associated signal (UniProt Q8N6Q3 topology and localisation; standard IF practice). The supplied subcellular record contains no ICC/IF images, so it cannot validate an IF pattern for this antibody (HPA subcellular record). Keep single stain and secondary only controls for multiplex interpretation (standard IF practice).
What should I check when CD177 chromogenic staining is widespread?
First compare stained sections with a no primary control to identify signal from the detection system or tissue rather than the primary antibody (standard IHC practice). For peroxidase based chromogenic IHC, include an endogenous peroxidase block and inspect the control before attributing brown DAB deposits to CD177 (standard IHC practice). Titrate the primary antibody around the catalog image’s 1:100 dilution using the same paraffin section workflow; that caption alone does not establish an optimum for every specimen (caption A03958; standard IHC practice). Assess whether background is concentrated at cut edges, folds or damaged tissue, where staining is less reliable (standard IHC practice). Judge any residual signal against the expected cell and compartment patterns (HPA tissue IHC; UniProt Q8N6Q3 localisation).
How should I score heterogeneous CD177 staining in tissue? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, since HPA reports membranous colon and rectum staining but cytoplasmic staining in subsets of bone marrow and spleen cells (HPA tissue IHC). For epithelial regions, report the percentage of positive cells and, when intensity is reproducible, an H-score using the same thresholds across slides (standard IHC practice). For infiltrating cells, report CD177 positive cell density per mm² of viable tissue and the percentage among the relevant counted cells (standard IHC practice). Exclude folds, necrosis and section edges using prespecified rules, and normalise counts to viable area or the appropriate cell denominator (standard IHC practice). Record region selection because CD177 is expressed by only a subset of mature neutrophils (UniProt Q8N6Q3 tissue specificity).
How can I distinguish genuine CD177 staining from an artefact?
Look for signal in intact cells with the expected compartment pattern: membranous staining in colon or rectum and cytoplasmic staining in subsets of bone marrow or spleen cells (HPA tissue IHC). CD177 also occurs on a subset of mature neutrophils, so patchy neutrophil positivity can be biologically plausible (UniProt Q8N6Q3 tissue specificity). Compare suspicious staining with morphology and a no primary control, particularly around necrosis, section edges and areas prone to endogenous enzyme signal (standard IHC practice). A diffuse deposit spanning unrelated compartments or damaged cells warrants review before scoring it positive (standard IHC practice). Interpret a negative field cautiously because the proportion of CD177 positive neutrophils varies across the population (UniProt Q8N6Q3 tissue specificity).
Boster reagents
Best CD177 / CD177 antigen IHC Antibodies
A03958 has a human-colon paraffin-section IHC image (catalog image caption), lists IF (catalog applications), and lists human and mouse reactivity (catalog reactivity); no IF image is supplied (catalog image payload).
Real IHC data
Anti-CD177 antigen CD177 Antibody
Cat # A03958
A03958 will render with its human-colon paraffin-section IHC image at 1:100 (catalog image caption). It lists IHC and IF applications and human and mouse reactivity (catalog applications/reactivity).
Which to pick: For tissue IHC, choose A03958: its own image shows paraffin-embedded human colon at 1:100 (catalog image caption); the fixative is unreported (catalog image caption). For IF/ICC, A03958 lists IF at 1:50 (catalog applications/dilution), but ICC validation and an IF image are unreported (catalog payload). For cross-species work, A03958 is a rabbit polyclonal antibody with listed human and mouse reactivity (catalog host/clonality/reactivity); its IHC image documents human colon only (catalog image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8N6Q3 (CD177_HUMAN, CD177 antigen).
- Human Protein Atlas. CD177 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CD177 subcellular location (ICC-IF): Highest expression in EFO-21: 4.4 nTPM.
- Human Protein Atlas. CD177 antibody validation summary (3 antibodies).
- Gene expression analysis of a Helicobacter pylori-infected and high-salt diet-treated mouse gastric tumor model: identification of CD177 as a novel prognostic factor in patients with gastric cancer. BMC gastroenterology 2013 — PMC3734037.
- Prognostic value of tumor‑associated CD177(+) neutrophils in lung adenocarcinoma. Oncology letters 2024 — PMC10941067.
- CD177 Expression and Inflammation Grade in Helicobacter pylori-Infected Wild-Type and CD177(-/-) C57BL/6 Mice. Analytical cellular pathology (Amsterdam) 2019 — PMC6476150.
- Network pharmacology and in vivo and in vitro experiments to determine the mechanism behind the effects of Jiawei Yanghe decoction via TLR4/Myd88/NF-κB against mastitis. Heliyon 2023 — PMC10641157.
- PubMed PMID:10753836 — UniProt-cited evidence.
- PubMed PMID:11465086 — UniProt-cited evidence.
- PubMed PMID:12010833 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CD177 (CD177 antigen). Expected localisation Marrow/spleen cytoplasm; colon/rectum membranes (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A03958 at 1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Enterocytes - Microvilli); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon tissue; fixative not specified (datasheet A03958); 2. Fixation: Image fixative and duration unreported (datasheet A03958); 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-CD177, 1:100 (datasheet A03958); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Only a variable subset of mature neutrophils expresses CD177 (UniProt); Isoform / epitope: 3 isoforms; check ectodomain epitope coverage (UniProt); Regulation: TNF, fMLP or IL-8 raises surface CD177 (UniProt). Evidence: UniProt Q8N6Q3, HPA tissue IHC · Enhanced, HPA cell-line RNA · Highest expression in EFO-21: 4.4 nTPM, Datasheet A03958, PMC3734037, PMC10941067. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CD177/immunohistochemistry-ihc-guide.