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CD177 / CD177 antigen · IHC design guide
Design Immunohistochemistry for CD177
Use bone marrow hematopoietic cells or colonic enterocyte microvilli as positive IHC references (HPA tissue IHC). This guide covers expected staining patterns and a catalog antibody starting dilution of 1:100 (HPA tissue IHC; datasheet: 1:100).
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 membrane (HPA tissue IHC) | |
| Staining pattern | Marrow/spleen subsets: cytoplasmic; enterocyte microvilli: 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 formalin 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 CXCL8 increase surface CD177 (UniProt) | |
| Isoform / epitope | 3 isoforms; check the mature ectodomain epitope (UniProt) |
Section 1
Recommended CD177 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by four published CD177 IHC protocols (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); optimize against the published conditions for your sample.
Section 2
What Is the Expected CD177 Staining Pattern?
CD177 is a GPI-anchored surface protein with no transmembrane segment; it also occurs in neutrophil granule membranes and can be shed (UniProt Q8N6Q3 topology, subcellular location). In IHC, expect staining in a subset of marrow hematopoietic cells and at intestinal enterocyte microvilli; HPA also reports cytoplasmic staining in a subset of splenic cells (HPA: Enhanced tissue IHC).
| Distinct staining marks only some marrow hematopoietic cells; nearby cells remain pale. | A mixed result fits CD177 expression in a subset of mature neutrophils. Expression begins at the metamyelocyte stage, and the positive fraction varies between people; score the fraction and compartment rather than expecting every granulocyte to stain (UniProt Q8N6Q3 tissue specificity). |
| Colon or rectum shows a crisp apical enterocyte border. | This matches HPA's high microvillar staining and reported membranous expression in these tissues (HPA: colon and rectum tissue IHC). Read the epithelial border separately from leukocytes in the same section so their different expected patterns are not conflated (HPA: tissue IHC; UniProt Q8N6Q3 tissue specificity). |
| Staining is predominantly nuclear, without the expected cell surface or cytoplasmic pattern. | A nuclear pattern does not match the supplied CD177 locations and should prompt an artefact check (UniProt Q8N6Q3 subcellular location; HPA: tissue IHC). Review the counterstain and detection controls before calling those nuclei CD177 positive (standard IHC practice). |
| Strong signal appears in adipocytes or another cell type HPA lists as undetected. | That result conflicts with the specified negative cell population, though it does not by itself prove cross-reactivity (HPA: adipose tissue, adipocytes, not detected). Check antibody specificity and endogenous detection activity with appropriate controls; assess each named cell population rather than declaring an entire tissue negative (standard IHC practice; HPA: tissue IHC). |
| Diffuse colour covers tissue and blank spaces, or a known-positive marrow section has no specific signal. | Diffuse deposition obscures compartment scoring and suggests background; an absent signal in a known-positive section leaves the run uninterpretable (standard IHC practice). Marrow hematopoietic cells provide an HPA high-staining comparator, while their subset pattern means a small sampled field can miss positive cells (HPA: bone marrow tissue IHC; UniProt Q8N6Q3 tissue specificity). |
💡Expected CD177 appearanceCall a result positive when discrete CD177 staining marks a subset of marrow hematopoietic cells or the apical microvillar border of colon or rectal enterocytes, consistent with HPA's high staining; isolated nuclear colour or diffuse deposit is suspect (HPA: tissue IHC; UniProt Q8N6Q3 subcellular location; standard IHC practice).
| Cell mixture and maturation | CD177 appears from the metamyelocyte stage onward and labels only a variable subset of mature neutrophils. Field selection and the proportion of those cells therefore affect the apparent positive fraction (UniProt Q8N6Q3 tissue specificity). |
| Compartment and processing | CD177 has a signal peptide, a processed mature chain and a GPI-anchor annotation. Neutrophils store it mainly in secondary and tertiary granules; surface expression can rise after stimulation, and CD177 can be shed (UniProt Q8N6Q3 processing, keywords, subcellular location). These facts support more than one plausible cellular pattern, without predicting how paraffin processing changes it. |
| Tissue-specific appearance | HPA reports cytoplasmic expression in subsets of marrow and splenic cells, but membranous expression in colon and rectum. Its listed high signal in enterocyte microvilli gives a separate epithelial pattern to recognize (HPA: Enhanced tissue IHC). |
| Antibody evidence | HPA marks tissue IHC reliability Enhanced and lists HPA041820, HPA046601 and HPA077640 as IHC Enhanced (HPA: tissue IHC and antibody validation). That supports the observed tissue pattern; it does not supply target-specific antigen retrieval, fixation sensitivity or IF performance. |
| Antigen retrieval and detection controls | Retrieval conditions, blocking and chromogenic detection should be assessed with positive and negative controls for the chosen IHC assay (standard IHC practice). The supplied HPA and UniProt records do not specify a CD177 retrieval condition or show that fixation changes CD177 signal. |
| IF/ICC Q: Is an image-backed localisation available? | A: HPA lists no main subcellular location and no cell lines with ICC-IF images; its listed antibodies have no ICC validation entry (HPA: subcellular record and antibody validation). Treat the UniProt locations as biological context, not an IF-validated staining pattern (UniProt Q8N6Q3 subcellular location). |
| Situation | Likely cause | Next action |
|---|---|---|
| No specific staining in a marrow positive control | The run may have failed, or the examined field may contain too few CD177-positive cells (standard IHC practice; UniProt Q8N6Q3 tissue specificity). | Check that hematopoietic cells and multiple fields were examined, then review the assay's retrieval, antibody and detection steps against its own validated conditions (HPA: bone marrow high; standard IHC practice). |
| Uniform nuclear staining dominates | Nuclear localisation is unsupported by the supplied CD177 patterns; counterstain or nonspecific detection may be contributing (UniProt Q8N6Q3 subcellular location; HPA: tissue IHC; standard IHC practice). | Compare the nuclear colour with the counterstain and a no-primary control, and require the expected cellular compartment before scoring positive (standard IHC practice; HPA: tissue IHC). |
| Diffuse chromogen obscures cell borders | Background can arise from detection chemistry or insufficient blocking and washing (standard IHC practice). | Inspect a no-primary control, review blocking and wash steps, and score only resolved cellular staining after background is controlled (standard IHC practice). |
| Adipocytes stain strongly | HPA lists adipocytes in adipose tissue as not detected, so that cell-specific result warrants a specificity or endogenous-activity check (HPA: adipose tissue IHC; standard IHC practice). | Confirm cell identity, compare a no-primary control and the IHC-validated antibody's pattern, then avoid assigning CD177 positivity until the unexpected signal is resolved (standard IHC practice; HPA: antibody validation). |
| Every marrow granulocyte appears positive | That uniform pattern conflicts with the reported variable positive subset; cell identity, staining threshold or background may be misread (UniProt Q8N6Q3 tissue specificity; standard IHC practice). | Recheck morphology and controls, and record the positive fraction with its scoring threshold across several fields (standard IHC practice; UniProt Q8N6Q3 tissue specificity). |
| Colon or rectum lacks an apical border despite tissue colour | Diffuse colour does not reproduce HPA's enterocyte-microvillar pattern and cannot establish epithelial CD177 staining (HPA: colon and rectum tissue IHC). | Inspect border preservation and a no-primary control; compare the compartment with an HPA high-staining reference pattern before calling the section positive (HPA: tissue IHC; standard IHC practice). |
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 and look for staining at enterocyte microvilli (HPA: High in colon enterocytes–microvilli). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the colon slide, cells outside the enterocyte microvilli should lack that specific staining pattern, although infiltrating neutrophils may stain (UniProt Q8N6Q3 tissue specificity).
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, a host- and immunoglobulin-class-matched isotype control, and CD177-knockout material if available as a biological negative (standard IHC practice). For chromogenic colon IHC, quench endogenous peroxidase and block endogenous biotin if using avidin–biotin detection; for IF, assess tissue autofluorescence (standard IHC/IF practice).
⚠️Feasibility: The selected A03958 paraffin-section colon caption reports a 1:100 dilution but does not report the fixative (catalog caption: human-colon paraffin section, 1:100). A target-specific fixation window, fixation effect, and antigen-retrieval dependency are unreported in the supplied evidence; optimize retrieval with the IHC-validated antibody rather than infer processing from the HPA staining pattern (catalog caption; HPA: High in colon enterocytes–microvilli). The evidence does not establish whether frozen sections or IF are easier; when scoring colon, distinguish microvillar staining from possible staining of infiltrating neutrophils (HPA: High in colon enterocytes–microvilli; UniProt Q8N6Q3 tissue specificity).
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 in paraffin sections by checking retrieval, cell identity, staining compartment and controls before scoring chromogenic signal.
What retrieval should I try first when CD177 staining is weak?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting). Apply the same cooling and wash sequence to test and control sections, then compare signal within matched cell compartments (standard IHC practice). Check hematopoietic cells in bone marrow or enterocyte microvilli in colon as positive reference patterns (HPA tissue IHC). If staining remains weak, trial another retrieval buffer only on matched sections while holding antibody concentration and detection constant (standard IHC practice). Score any gain against background and tissue morphology, since retrieval changes can alter both (standard IHC practice).
How should I troubleshoot CD177 loss after tissue fixation?
CD177-specific sensitivity to fixation is unknown from the supplied evidence; the selected colon image reports paraffin embedding and 1:100 antibody dilution but no fixative (A03958 caption). Record fixative, fixation duration, tissue thickness and processing history for each specimen before comparing staining (standard IHC practice). Run specimens with differing processing histories in one staining batch, using the same retrieval and detection conditions (standard IHC practice). Include a section with an expected CD177 pattern, such as bone marrow hematopoietic cells or colon enterocyte microvilli, to assess assay performance (HPA tissue IHC). Treat differences between processing groups as observational until fixation is varied under controlled conditions (standard IHC practice).
Where should convincing CD177 signal appear in a paraffin section?
Assess membranous staining at colon and rectal enterocyte microvilli, and cytoplasmic staining in subsets of bone marrow and spleen cells (HPA tissue IHC). In neutrophils, CD177 occurs at the cell surface and predominantly in secondary and tertiary granules, so compartment can vary with cell state (UniProt Q8N6Q3 localisation). Surface expression increases after TNF, fMLP or CXCL8 stimulation, and CD177 can be shed (UniProt Q8N6Q3 localisation). Use morphology and a matched positive control to decide whether apparent diffuse signal follows the expected cells (standard IHC practice; HPA tissue IHC). Do not classify nuclear-only staining as the expected CD177 pattern (UniProt Q8N6Q3 localisation).
Could epitope location explain discordant CD177 staining?
CD177 has 3 listed isoforms and a processed chain spanning residues 22–408; its precursor includes a signal peptide and propeptide (UniProt Q8N6Q3 processing and isoforms). It has no transmembrane segment, carries a GPI-anchor annotation, and has a glycosylation site at residue 189 (UniProt Q8N6Q3 topology and keywords). Determine the catalog antibody’s documented immunogen or epitope before comparing results across antibodies; that information is absent here (supplied evidence; standard IHC practice). Compare staining on adjacent sections with matched retrieval and detection, then check whether differences track cell type or compartment (standard IHC practice). Do not assign an isoform-specific signal without epitope and isoform validation (standard IHC practice).
How can I compare multiplex IF with chromogenic CD177 IHC?
Use IF as a separate validation experiment: the supplied HPA subcellular record has no ICC/IF images or assigned main location (HPA subcellular). Multiplex CD177 with a validated marker of the expected cell type, then assess overlap and morphology against bone marrow hematopoietic cells or colon enterocytes (HPA tissue IHC; standard IF practice). Choose fluorophores after measuring tissue autofluorescence in unstained sections, and include single-stain controls for channel separation (standard IF practice). For an extracellular epitope on GPI-anchored CD177, test staining without permeabilisation; use mild permeabilisation only when testing access to granule-associated or intracellular antigen (UniProt Q8N6Q3 topology and localisation; standard IF practice). Keep IF conclusions separate from the paraffin-section IHC result (standard assay validation practice).
How do I distinguish CD177 staining from chromogenic background?
Inspect a no-primary control and a known positive section in the same run to locate detection background and confirm staining performance (standard IHC practice). Block endogenous peroxidase before an HRP/DAB reaction, and review nonspecific signal after changing antibody concentration or blocking conditions (standard chromogenic IHC practice). The selected paraffin-embedded colon image used 1:100, which is a documented image condition rather than a universal working dilution (A03958 caption). Compare diffuse precipitate with the expected membranous enterocyte microvilli and subset-restricted marrow staining (HPA tissue IHC). If background persists, change one variable at a time and retain identical exposure to chromogen across controls (standard IHC practice).
What should I count when quantifying CD177-positive tissue? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because CD177 appears in subsets of marrow and spleen cells and at colon and rectal enterocyte membranes (HPA tissue IHC). Report the percentage of positive cells within the chosen population; an H-score can additionally capture intensity across that same denominator (standard IHC quantification practice). For infiltrating cells, report positive-cell density per mm² of viable tissue and state the sampled area (standard IHC quantification practice). Keep segmentation, staining threshold and chromogen development consistent across slides, with blinded review of borderline cells (standard IHC quantification practice). Record cell identity separately from total tissue signal because CD177-positive neutrophils form a variable subset (UniProt Q8N6Q3 tissue specificity).
When is an apparent CD177-positive cell likely an artefact?
Favor signal in the expected cell and compartment: subset-restricted hematopoietic staining or enterocyte microvillar membrane staining (HPA tissue IHC). Check suspicious nuclear-only or widespread uniform staining against CD177’s documented surface, granule-membrane and secreted locations (UniProt Q8N6Q3 localisation). Review section edges, folds and necrotic areas for uneven deposition, and compare each pattern with a no-primary control (standard IHC practice). Endogenous peroxidase can complicate HRP/DAB interpretation, so verify that its block worked in the relevant tissue (standard chromogenic IHC practice). A negative neutrophil does not alone invalidate the assay, since only a subset of mature neutrophils expresses CD177 (UniProt Q8N6Q3 tissue specificity).
Boster reagents
Best CD177 / CD177 antigen IHC Antibodies
A03958 has a human paraffin-section IHC image (IHC image caption: human colon) and is listed for IF and for human and mouse reactivity (catalog: applications and reactivity).
Real IHC data
Anti-CD177 antigen CD177 Antibody
Cat # A03958
A03958 will render with a human colon paraffin-section IHC image at 1:100 (IHC image caption). The catalog lists A03958 for IHC and IF in human and mouse, but provides no IF image (catalog: applications, reactivity, image alts).
Which to pick: Choose A03958 for tissue IHC when a human paraffin-section example is useful; its own caption documents human colon at 1:100, with the fixative unreported (IHC image caption). A03958 is also the listed IF option, though the catalog provides no IF/ICC image (catalog: applications, image alts). For mouse work, A03958 is the only listed choice because its reactivity includes mouse; it is a rabbit polyclonal antibody, while its IHC image shows human tissue only (catalog: reactivity and host; dilution_raw: polyclonal; IHC 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 membrane (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 formalin 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 the mature ectodomain epitope (UniProt); Regulation: TNF, fMLP or CXCL8 increase 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.