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CYGB / Cytoglobin · IHC design guide
Design Immunohistochemistry for CYGB
Plan chromogenic CYGB IHC on paraffin sections with the catalog antibody at 0.5–1 μg/mL (datasheet A01197). Assess cytoplasmic staining by cell type, using high staining in skeletal myocytes and the prominent immune-cell subset as tissue-pattern references (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CYGB 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 | Cytoplasmic in tissue (HPA tissue IHC); nucleus annotated (UniProt) | |
| Staining pattern | Cytoplasmic; most abundant in an immune-cell subset (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A01197) | |
| Positive control | Skeletal muscle+4 more · see all | |
| Negative control | Bone marrow+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01197) | |
| Caveat | Immune-cell staining may dominate mixed tissue (HPA tissue IHC) | |
| Regulation | Intensity varies by tissue and cell type (HPA tissue IHC) | |
| Isoform / epitope | One 1–190 chain; no isoforms or signal peptide (UniProt) |
Section 1
Recommended CYGB IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by published CYGB staining protocols for human liver biopsies and glioma sections (PMC5849339; PMC3663650).
| Sample | Paraffin-embedded mouse intestine tissues; fixative not specified (datasheet A01197) |
| Fixation | Image fixative and duration unreported (datasheet A01197); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet A01197) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01197) |
| Primary antibody | Rabbit anti-CYGB, 0.5-1μg/ml (datasheet A01197) |
| Primary incubation | Overnight at 4 °C (datasheet A01197) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A01197) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CYGB-positive staining in myocytes of skeletal muscle (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression, most abundant in a subset of immune cells. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 heat retrieval for the catalog antibody (datasheet A01197); the glioma protocol also specifies citrate pH 6 (PMC3663650).
Section 2
What Is the Expected CYGB Staining Pattern?
CYGB is a soluble cytoplasmic and nuclear protein with no transmembrane segment (UniProt Q8WWM9). In paraffin-section IHC, expect mainly cytoplasmic staining, most abundant in a subset of immune cells (HPA tissue IHC). Skeletal-muscle myocytes stain strongly, while several glandular and epithelial populations stain moderately (HPA tissue IHC). HPA rates its tissue IHC reliability Approved, with medium consistency between staining and RNA expression (HPA tissue IHC).
| Strong cytoplasmic staining in skeletal-muscle myocytes; moderate staining in colon glands (HPA tissue IHC). | This fits the reported High myocyte and Medium glandular-cell patterns (HPA tissue IHC). Judge the named cell populations, not the entire section: HPA also describes CYGB as most abundant in a subset of immune cells (HPA tissue IHC). A matched positive control helps anchor the intensity call (general IHC practice). |
| Predominantly crisp membrane staining, with little cytoplasmic signal. | A membrane-dominant pattern conflicts with CYGB's cytoplasmic and nuclear locations and lack of a transmembrane segment (UniProt Q8WWM9). Treat it as suspect, then compare another validated positive tissue and a detection-only control before assigning it to CYGB (general IHC practice). Nuclear signal alone needs separate assessment because a nuclear location is documented (UniProt Q8WWM9). |
| Strong staining in skin fibroblasts or bone-marrow hematopoietic cells. | Those specific populations were not detected in HPA tissue IHC (HPA tissue IHC). Unexpected signal raises possible antibody cross-reactivity or endogenous detection activity; it does not prove either cause (general IHC interpretation). Check whether signal persists when primary antibody is omitted, and compare cell identity and morphology with the HPA reference (general IHC practice). |
| Diffuse chromogen over cells, stroma and empty areas, obscuring cell boundaries. | This distribution is difficult to score against HPA's cell-specific cytoplasmic profile (HPA tissue IHC). Nonspecific antibody binding, incomplete blocking or residual detection activity are possible workflow causes (general IHC practice). Because background can mimic weak positive cells, compare a primary-omission control before changing the biological interpretation (general IHC practice). |
| No staining in skeletal-muscle myocytes on the test run. | HPA reports High staining in these cells, so an entirely blank result warrants a technical check (HPA tissue IHC). Confirm the tissue and myocyte morphology, then review the antibody's IHC-P instructions, retrieval, dilution and detection controls (general IHC practice). One failed section does not establish that CYGB is absent from all skeletal muscle. |
💡Expected CYGB appearanceCall a convincing positive when skeletal-muscle myocytes show strong, mainly cytoplasmic staining (HPA tissue IHC); isolated strong membrane staining or broad signal in HPA-negative cell populations is suspect (UniProt Q8WWM9; HPA tissue IHC).
| Compartment and topology | CYGB is cytoplasmic and nuclear, with no transmembrane segment or signal peptide (UniProt Q8WWM9). Use cytoplasmic staining as the principal tissue-IHC reference (HPA tissue IHC); do not classify every nuclear signal as artefact, since UniProt also lists the nucleus (UniProt Q8WWM9). |
| Choice of comparison cells | Skeletal-muscle myocytes are High; bronchial respiratory epithelium and colon glands are Medium (HPA tissue IHC). Skin fibroblasts and bone-marrow hematopoietic cells are listed as not detected (HPA tissue IHC). Compare the specified cells rather than treating an entire tissue as uniformly positive or negative. |
| Antibody evidence | HPA017757 has IHC status Approved; HPA061642 has no listed IHC status (HPA antibodies). HPA's tissue assessment reports medium agreement between antibody staining and RNA expression (HPA tissue IHC). This supports cautious pattern comparison, while the catalog antibody's own IHC-P instructions govern its working conditions (general IHC practice). |
| Processing and variants | UniProt lists one CYGB chain spanning residues 1–190, no signal peptide or propeptide, and no listed isoforms or glycosylation sites (UniProt Q8WWM9). These annotations offer no basis for predicting a shed extracellular staining pattern or an isoform-specific tissue pattern; they do not establish an antigen-retrieval requirement. |
| IF/ICC Q&A | What localisation might IF/ICC show? HPA reports supported nuclear speckles plus cytosol in its ICC-IF assessment (HPA subcellular). That cell-imaging observation can inform interpretation, but it does not make punctate nuclei a required paraffin-section IHC pattern (HPA subcellular; HPA tissue IHC). Use the separate IF/ICC guide for that application. |
| Situation | Likely cause | Next action |
|---|---|---|
| Skeletal-muscle myocytes are blank. | The result conflicts with HPA's High myocyte staining; the failing step is unknown (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. |
| A presumed negative control stains strongly. | The chosen cell population may differ from the HPA-negative population, or staining may be nonspecific (HPA tissue IHC; general IHC interpretation). | Identify the cells before scoring: HPA calls skin fibroblasts, specifically, not detected (HPA tissue IHC). Run a primary-omission control and reassess signal confined to the intended cell type (general IHC practice). |
| Staining is exclusively nuclear. | Nuclear CYGB is plausible (UniProt Q8WWM9), but HPA's tissue-IHC summary emphasizes cytoplasm (HPA tissue IHC). | Compare a reference positive tissue and review the primary-omission control before rejecting the signal (general IHC practice). HPA's supported nuclear-speckle finding comes from ICC-IF and should not be imposed as an IHC scoring rule (HPA subcellular). |
| Broad brown background masks cell outlines. | Residual endogenous detection activity or nonspecific binding could obscure the cell-specific pattern (general IHC practice; HPA tissue IHC). | Inspect primary-omission and detection controls; review blocking, washes and chromogen development for the chosen system (general IHC practice). Score CYGB only after discrete cells can be distinguished from background. |
| Cell surfaces stain more strongly than cytoplasm. | A dominant surface pattern conflicts with CYGB's soluble locations and lack of a transmembrane segment (UniProt Q8WWM9). | Compare staining in HPA-positive myocytes with a primary-omission control, and verify the catalog antibody's IHC-P validation before calling surface signal specific (HPA tissue IHC; general IHC practice). |
| Moderate tissue staining seems weaker than expected. | HPA grades colon glandular cells Medium, versus High for skeletal-muscle myocytes (HPA tissue IHC). | Check the named cell population and compare sections processed in the same run (general IHC practice). Do not require a moderate population to match the myocyte intensity; investigate a technical failure if the positive control is also absent (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing CYGB staining
A01197 · IHCIHC analysis of Cytoglobin using anti-Cytoglobin antibody (A01197). Cytoglobin was detected in paraffin-embedded section of mouse intestine
A01197 · IHCIHC analysis of Cytoglobin using anti-Cytoglobin antibody (A01197). Cytoglobin was detected in paraffin-embedded section of mouse cardiac mu
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000161544-CYGB/tissue/cerebral+cortex#imid_4658304
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000161544-CYGB/tissue/colon#imid_4658246
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000161544-CYGB/tissue/liver#imid_4658247
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000161544-CYGB/tissue/kidney#imid_4658240
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA017757; green = CYGB, blue = nucleus, red = microtubules.
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA017757; green = CYGB, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CYGB IHC & IF
🧪Run skeletal muscle first and look for staining in myocytes (HPA: High in skeletal-muscle myocytes). Use bone marrow hematopoietic cells as the negative tissue (HPA: Not detected); on the positive slide, adjacent non-myocyte areas should provide a low-background reference, although their CYGB status is not specified by HPA.
Positive control tissue: Skeletal muscle (Myocytes, HPA High)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CYGB in Hep-G2, SH-SY5Y, U-251MG, with annotated localisation: Nuclear speckles (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and rabbit immunoglobulin controls matched to the primary’s clonality (caption: rabbit anti-Cytoglobin); a CYGB knockout specimen is a stronger biological negative where available. Quench endogenous peroxidase and control endogenous biotin before interpreting muscle DAB staining (caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected A01197 tissue-IHC caption does not state a fixative. The caption uses citrate retrieval at pH 6 for 20 minutes in paraffin-embedded mouse intestine, but does not establish retrieval dependence in skeletal muscle; it also gives no basis to call frozen sections or IF easier (caption: mouse intestine IHC). Assess muscle DAB background with the process controls; for IF/ICC, HPA reports nuclear speckles and cytosol in its imaged cell lines, which requires separate interpretation (HPA: supported subcellular locations).
HPA tissue IHC evidence for CYGB
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium 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 |
|---|---|---|---|---|
| Skeletal muscle | Myocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Cerebellum | Cells in molecular layer | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CYGB IHC Tips
Use compartment and cell type controls alongside the catalog antibody’s paraffin-section IHC conditions to assess CYGB staining (UniProt Q8WWM9; datasheet A01197; HPA tissue IHC).
How should I optimize retrieval when CYGB staining is weak in paraffin sections?
Start with heat-mediated retrieval in citrate buffer at pH 6 (datasheet A01197). The catalog antibody’s mouse intestine image used 20 minutes of retrieval before chromogenic detection, providing a directly documented starting condition for this antibody (datasheet A01197). If staining remains weak, compare a modestly longer heating period on matched sections while keeping buffer, antibody concentration, and detection constant (general IHC practice). Include a section processed without primary antibody to detect retrieval-associated background (general IHC practice). Judge improvement by staining in plausible cells and compartments, since CYGB occurs in cytoplasm and nucleus and HPA reports predominantly cytoplasmic tissue staining (UniProt Q8WWM9; HPA tissue IHC).
Could fixation explain inconsistent CYGB staining between paraffin-section samples?
Target-specific fixation sensitivity is unknown: the catalog mouse intestine caption identifies a paraffin section but does not report its fixative (datasheet A01197). Record each sample’s fixative, fixation duration, section thickness, and storage history before attributing differences to CYGB biology (general IHC practice). Compare sections processed together using citrate pH 6 retrieval for 20 minutes and the same antibody and detection conditions (datasheet A01197; general IHC practice). Overfixation and underfixation can change antigen accessibility or tissue preservation in IHC generally; neither effect has been established specifically for CYGB here (general IHC practice). Interpret differences only after checking morphology and matched negative controls (general IHC practice).
Should CYGB appear in the cytoplasm, nucleus, or both?
Assess cytoplasmic and nuclear staining separately because both locations are listed for CYGB (UniProt Q8WWM9). HPA tissue IHC describes general cytoplasmic expression, while HPA cell imaging supports nuclear speckles as the main location and cytosol as an additional location (HPA tissue IHC; HPA subcellular). In chromogenic sections, inspect nuclear staining against the counterstain and compare it with the surrounding cytoplasm at the same magnification (general IHC practice). The catalog mouse intestine image establishes a paraffin-section staining workflow, but its caption does not assign signal to either compartment (datasheet A01197). Use a no-primary control when a compartment appears diffusely colored (general IHC practice).
How can I troubleshoot a suspected epitope-access problem without assuming another CYGB isoform?
The supplied UniProt record lists 0 isoforms and a single 1–190 CYGB chain, so it supplies no alternative isoform to explain a changed IHC pattern (UniProt Q8WWM9). It also lists no signal peptide, transmembrane segment, glycosylation sites, or modified residues; those annotations do not locate this antibody’s epitope (UniProt Q8WWM9). Begin with the catalog antibody’s citrate pH 6, 20-minute retrieval condition and hold detection settings constant while comparing sections (datasheet A01197; general IHC practice). If signal varies, check tissue preservation, retrieval uniformity, and staining controls before assigning an epitope mechanism (general IHC practice). Request an epitope map if that distinction is essential (general IHC practice).
How should I use IF to investigate an ambiguous CYGB IHC pattern?
Use IF as an orthogonal localization check, with a marker for the expected cell population: HPA reports high staining in skeletal-muscle myocytes and medium staining in several glandular cell populations (HPA tissue IHC). Choose fluorophores after inspecting unstained tissue for autofluorescence, and use single-stain controls to assess channel overlap (general IF practice). Because CYGB is listed in cytoplasm and nucleus and has no transmembrane segment, permeabilize to give antibodies access to intracellular epitopes, then compare cytosolic and nuclear signals (UniProt Q8WWM9; general IF practice). HPA cell images support nuclear speckles and cytosol, but their localization does not establish a paraffin-section IF protocol (HPA subcellular). Keep conclusions tied to the cell types and compartments resolved by each method (general microscopy practice).
What should I check when DAB staining obscures CYGB-positive cells?
First examine a no-primary section and the tissue edges for staining unrelated to the primary antibody (general IHC practice). The catalog mouse intestine workflow used 10% goat serum blocking, a biotinylated secondary antibody, streptavidin–biotin detection, and DAB; these are documented conditions for that image, not proof that every brown deposit is CYGB (datasheet A01197). Check endogenous peroxidase blocking and, for biotin-based detection, an appropriate endogenous biotin control when background is widespread (general IHC practice). Compare the documented 1 μg/ml primary concentration with a lower concentration on matched sections if diffuse staining persists (datasheet A01197; general IHC practice). Score only cells with convincing morphology and compartmental signal (general IHC practice).
How should I quantify CYGB staining across heterogeneous tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, because HPA reports different staining levels among myocytes, glandular cells, and other populations (HPA tissue IHC). For chromogenic IHC, report the percentage of positive cells and an H-score based on staining intensity; count positive cells per mm² if cell density is the biological question (general IHC practice). Normalize counts to viable tissue area or the number of evaluable cells in the same annotated population, and exclude folds, edges, and necrosis (general IHC practice). Keep retrieval, DAB development, imaging, and positivity thresholds consistent across samples (general IHC practice). Report cytoplasmic and nuclear scores separately when both are visible (UniProt Q8WWM9; general IHC practice).
When is a CYGB-positive chromogenic pattern convincing rather than an artefact?
A convincing pattern has interpretable cells and compartments: CYGB is annotated in cytoplasm and nucleus, and HPA tissue IHC describes predominantly cytoplasmic staining (UniProt Q8WWM9; HPA tissue IHC). Compare candidate signal with the expected population, such as skeletal-muscle myocytes, while recognizing that HPA rates agreement between staining and RNA as medium (HPA tissue IHC). Treat staining limited to section edges, folds, or necrotic areas as suspect, and check no-primary and endogenous-enzyme controls for DAB signal (general IHC practice). HPA reports CYGB as not detected in bone-marrow hematopoietic cells and liver cholangiocytes; these observations provide comparison points, not absolute exclusion rules (HPA tissue IHC). Reassess surprising nuclear-only or cell-type-restricted staining with matched controls (general IHC practice).
Boster reagents
Best CYGB / Cytoglobin IHC Antibodies
A01197 has IHC images from paraffin sections of mouse intestine and cardiac muscle, rat intestine, and human intestinal cancer (catalog: A01197 IHC image captions). No IF data are listed (catalog: A01197 IF images).
Real IHC data
Anti-Cytoglobin/CYGB Antibody ®
Cat # A01197
A01197 is listed for IHC in human, mouse, and rat (catalog: A01197 applications/reactivity). Its IHC captions show paraffin sections of mouse intestine and cardiac muscle, rat intestine, and human intestinal cancer (catalog: A01197 IHC image captions).
Which to pick: Choose A01197 for tissue IHC across human, mouse, and rat; it is a rabbit antibody with no clone reported (catalog: A01197 applications/reactivity/host/clone). Its paraffin-section captions use citrate retrieval at pH 6 for 20 minutes and 1 μg/ml primary antibody; the fixative is unreported (catalog: A01197 IHC image captions). No IF/ICC SKU is supported here: A01197 has no listed IF application or IF image (catalog: A01197 applications/IF images).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q8WWM9 (CYGB_HUMAN, Cytoglobin).
- Human Protein Atlas. CYGB tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CYGB subcellular location (ICC-IF): Mainly localized to the nuclear speckles. In addition localized to the cytosol..
- Human Protein Atlas. CYGB antibody validation summary (2 antibodies).
- IRP1/ARID3A complex promotes pancreatic cancer chemoresistance by suppressing CYGB-related ferroptosis. Genes & diseases 2026 — PMC13254595.
- Stagnation of histopathological improvement is a predictor of hepatocellular carcinoma development after hepatitis C virus eradication. PloS one 2018 — PMC5849339.
- The expression of cytoglobin as a prognostic factor in gliomas: a retrospective analysis of 88 patients. BMC cancer 2013 — PMC3663650.
- Cytoglobin deficiency potentiates Crb1-mediated retinal degeneration in rd8 mice. Developmental biology 2020 — PMC6995765.
- PubMed PMID:11919282 — UniProt-cited evidence.
- PubMed PMID:12359339 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CYGB (Cytoglobin). Expected localisation Cytoplasmic in tissue (HPA tissue IHC); nucleus annotated (UniProt). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A01197). Validated IHC antibody: A01197 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Skeletal muscle (Myocytes); negative Bone marrow. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded mouse intestine tissues; fixative not specified (datasheet A01197); 2. Fixation: Image fixative and duration unreported (datasheet A01197); 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: Citrate pH 6 , 20 min (datasheet A01197); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01197); 8. Primary antibody: Rabbit anti-CYGB, 0.5-1μg/ml (datasheet A01197); 9. Primary incubation: Overnight at 4 °C (datasheet A01197). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01197); Caveat: Immune-cell staining may dominate mixed tissue (HPA tissue IHC); Isoform / epitope: One 1–190 chain; no isoforms or signal peptide (UniProt); Regulation: Intensity varies by tissue and cell type (HPA tissue IHC). Evidence: UniProt Q8WWM9, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nuclear speckles. In addition localized to…, Datasheet A01197, PMC5849339, PMC3663650. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CYGB/immunohistochemistry-ihc-guide.