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CSTB / Cystatin-B · IHC design guide
Design Immunohistochemistry for CSTB
Plan CSTB IHC in paraffin sections using the cytoplasmic and nuclear staining seen in squamous epithelia as a reference (HPA tissue IHC). This guide covers fixation, chromogenic detection and scoring; the catalog antibody M02794 lists 0.5–1 μg/ml for IHC in paraffin sections (datasheet M02794).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CSTB 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 and nuclear staining in squamous epithelia (HPA tissue IHC) | |
| Staining pattern | Squamous and urothelial cells show cytoplasmic and nuclear staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet M02794) | |
| Positive control | Esophagus+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) | |
| Caveat | Antibody staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Tissue enriched in esophagus at the RNA level (HPA RNA) | |
| Isoform / epitope | One 1–98 chain; no isoforms annotated (UniProt) |
Section 1
Recommended CSTB IHC & IF Protocols
The catalog antibody protocol uses citrate pH 6 retrieval (datasheet). The published IHC protocols below cover ovarian tumors (PMC3977810), cholangiocarcinoma (PMC11854580), and cervical cancer (PMC10913530).
| Sample | Paraffin-embedded human tonsil tissue; fixative not specified (datasheet M02794) |
| Fixation | Image fixative and duration unreported (datasheet M02794); 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 M02794) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M02794) |
| Primary antibody | Mouse monoclonal (clone 2B6) anti-CSTB, 0.5-1μg/ml recommended; image 2μg/ml (datasheet M02794) |
| Primary incubation | Overnight at 4 °C (datasheet M02794) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet M02794) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CSTB-positive staining in squamous epithelial cells of esophagus (HPA tissue IHC: High). HPA tissue profile: Selective cytoplasmic and nuclear expression in squamous epithelia and urothelium. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate pH 6 retrieval (datasheet); the ovarian tumor protocol also specifies citrate pH 6.0 (PMC3977810).
Section 2
What Is the Expected CSTB Staining Pattern?
CSTB is intracellular, with cytoplasmic and nuclear localization (UniProt P04080: subcellular location; no transmembrane segment). In paraffin-section IHC, expect selective staining of squamous epithelium and urothelium, strongest in esophageal squamous epithelial cells (HPA tissue IHC: selective profile; High in esophagus). HPA rates the tissue pattern Enhanced, while reporting medium consistency between antibody staining and RNA expression (HPA tissue IHC: reliability).
| Esophageal squamous epithelial cells show clear cytoplasmic and nuclear chromogen. | This fits the strongest listed positive tissue and the reported compartments (HPA tissue IHC: High in esophageal squamous epithelial cells; UniProt P04080: cytoplasm and nucleus). Assess the epithelial cells themselves, since staining elsewhere on the section does not establish the expected cell-specific pattern (HPA tissue IHC: selective profile). |
| A crisp membrane rim, extracellular deposit, or exclusively surface-directed signal dominates. | Treat this as a possible staining artefact and review the assay before assigning it to CSTB: the reported pattern is intracellular, and CSTB has no transmembrane segment (UniProt P04080: location and topology). Nuclear signal alone is not a wrong compartment (UniProt P04080: nucleus). |
| Strong signal appears in a cell type listed as undetected, such as adipocytes. | Investigate cross-reactivity or endogenous detection activity, especially if the expected epithelial pattern is absent (HPA tissue IHC: adipocytes Not detected; esophageal squamous epithelial cells High). A listed negative is a comparator for that cell type, not proof that every cell in its tissue must be blank (HPA tissue IHC: cell-level entries). |
| Chromogen coats tissue broadly or fills areas without recognizable cellular boundaries. | Diffuse background cannot establish cytoplasmic or nuclear localization. Check the no-primary control and detection reagents, then reassess recognizable epithelial cells (general IHC practice; HPA tissue IHC: selective cytoplasmic and nuclear profile). |
| Esophageal squamous epithelium is blank while the slide and controls are readable. | The result conflicts with the listed High signal in that cell type (HPA tissue IHC: esophagus). Review antibody suitability and the validated IHC-P workflow, including retrieval and detection settings, before interpreting the blank as absent CSTB (general IHC practice). |
💡Expected CSTB appearanceA convincing positive is High cytoplasmic and nuclear staining in esophageal squamous epithelial cells (HPA tissue IHC: esophagus and selective profile); a dominant membrane rim or acellular deposit is suspect (UniProt P04080: intracellular location; no transmembrane segment).
| Positive tissue choice | Esophageal squamous epithelial cells are the strongest listed benchmark; oral mucosa, tonsil, vagina, and urothelium are listed at Medium (HPA tissue IHC: positive entries). Compare the named cell types when judging a section. |
| Negative comparator choice | HPA lists adipocytes and several other specified cell types as Not detected (HPA tissue IHC: negative entries). Use the named cells as comparators rather than assuming a whole tissue has no CSTB staining. |
| Localization and molecular form | CSTB is cytoplasmic and nuclear, with no transmembrane segment, signal peptide, or annotated propeptide (UniProt P04080: location, topology, processing). These annotations support an intracellular readout; they do not predict fixation sensitivity. |
| Antibody evidence | HPA assigns Enhanced IHC status to HPA017380, HPA058557, and CAB047320 (HPA antibodies: IHC validation). The tissue profile also has medium staining–RNA consistency (HPA tissue IHC: reliability description), so examine morphology and controls with the intensity call. |
| IF/ICC Q&A: where should CSTB appear? | HPA reports supported nucleolar and cytosolic localization in ICC-IF (HPA subcellular: main location). This is an IF/ICC interpretation cue; the paraffin-section IHC expectation remains cytoplasmic and nuclear staining in selected epithelia (HPA tissue IHC: profile). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in esophageal squamous epithelium. | The result misses an HPA-listed High cell type; the cause is not determined by that observation (HPA tissue IHC: esophagus). | Confirm that squamous epithelium is present and review the catalog antibody's IHC-P instructions, retrieval, detection, and run controls (general IHC practice). |
| Broad brown haze obscures cells. | Background prevents a cell-level localization call (general IHC practice; HPA tissue IHC: selective profile). | Inspect the no-primary control and review blocking, washing, and detection conditions; score only interpretable cellular staining (general IHC practice). |
| A membrane rim dominates the positive area. | A surface-dominant pattern conflicts with intracellular CSTB localization and its lack of a transmembrane segment (UniProt P04080: location and topology). | Compare with expected cytoplasmic and nuclear staining and inspect the no-primary control before accepting specificity (HPA tissue IHC: profile; general IHC practice). |
| Adipocytes stain strongly. | That cell-level result conflicts with the HPA Not detected entry; cross-reactivity or detection background is possible (HPA tissue IHC: adipocytes; general IHC practice). | Verify the cell identity and check the no-primary control alongside an esophageal epithelial positive comparator (HPA tissue IHC: esophagus; general IHC practice). |
| Only faint epithelial staining appears in oral mucosa or tonsil. | Both are listed at Medium, below esophageal High; faint staining alone cannot identify a technical cause (HPA tissue IHC: positive entries). | Compare the same run with esophageal squamous epithelium and judge signal within the listed cell type (HPA tissue IHC: positive entries; general IHC practice). |
| Nuclear staining is dismissed as nonspecific. | Nuclear localization is reported for CSTB, so compartment alone does not justify rejection (UniProt P04080: nucleus; HPA tissue IHC: nuclear expression). | Judge nuclear signal together with cytoplasmic staining, epithelial cell identity, morphology, and controls (HPA tissue IHC: selective profile; general IHC practice). |
Experiment evidence
IHC & IF images showing CSTB staining
M02794 · IHCIHC analysis of Stefin B using anti-Stefin B antibody (M02794). Stefin B was detected in paraffin-embedded section of human tonsil tissue. H
PB9565 · IHCIHC analysis of Stefin B using anti-Stefin B antibody (PB9565). Stefin B was detected in a paraffin-embedded section of human prostatic canc
PB9565 · IFIF analysis of Stefin B using anti-Stefin B antibody (PB9565). Stefin B was detected in an immunocytochemical section of A431 cells. Enzyme
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000160213-CSTB/tissue/cerebral+cortex#imid_12543337
HPA · EsophagusHuman Protein Atlas IHC, Esophagus. Source: https://www.proteinatlas.org/ENSG00000160213-CSTB/tissue/esophagus#imid_12543301
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000160213-CSTB/tissue/colon#imid_12543263
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000160213-CSTB/tissue/liver#imid_12543264
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA017380; green = CSTB, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA017380; green = CSTB, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CSTB IHC & IF
🧪Run esophagus first; its squamous epithelial cells should stain (HPA: High in esophageal squamous epithelial cells). Use adipose tissue adipocytes as the negative comparator (HPA: Not detected in adipocytes); on the esophagus slide, treat cells outside the squamous epithelium as internal background comparators only where they remain unstained, since their CSTB status is not specified by the supplied HPA row.
Positive control tissue: Esophagus (Squamous epithelial cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CSTB in A-431, U-251MG, KOLF2.1J, with annotated localisation: Nucleoli (supported), Cytosol (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host-species, immunoglobulin-class-matched isotype controls, plus CSTB-knockout tissue or a validated peptide-block control (standard IHC controls). For chromogenic staining, quench endogenous peroxidase and check endogenous biotin background when using the caption’s streptavidin–biotin detection system (caption: SABC with DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected M02794 tissue-IHC caption does not state the fixative (caption: paraffin-embedded tonsil section; fixative not stated). The caption reports citrate pH 6 heat retrieval for 20 minutes in tonsil, but does not establish whether retrieval is required for CSTB (caption: M02794 tonsil IHC). Frozen sections cannot be judged easier from these data; IF/ICC images support evaluating cytosolic and nucleolar signal, while squamous epithelium should be assessed for nonspecific chromogenic background in IHC (HPA: supported cytosol and nucleoli; HPA: High in esophageal squamous epithelial cells; standard IHC practice).
HPA tissue IHC evidence for CSTB
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 |
|---|---|---|---|---|
| Esophagus | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Tonsil | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Urinary bladder | Urothelial cells | Medium | Protein (IHC) | HPA → |
| Vagina | Squamous epithelial cells | Medium | 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 → |
| Appendix | Endocrine cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CSTB IHC Tips
Troubleshoot CSTB staining in paraffin sections by checking retrieval, compartment, cell type and detection controls against the evidence available for this IHC workflow.
How should I optimize retrieval when CSTB staining is weak?
Start with heat-mediated citrate retrieval at pH 6 for 20 minutes (datasheet M02794). Run the catalog antibody on a human tonsil section with its documented overnight incubation at 4°C, keeping detection conditions consistent while assessing retrieval (M02794 tissue-IHC caption). Compare sections retrieved together and inspect both staining intensity and tissue morphology; uneven heating or damaged sections can mislead an IHC comparison (standard IHC practice). If staining remains weak, test a different retrieval condition on adjacent sections as a fallback, but record the change separately because only citrate pH 6 is documented for this antibody (datasheet M02794).
Could fixation explain inconsistent CSTB staining between paraffin blocks?
The selected paraffin-section caption does not state a fixative, so CSTB-specific fixation sensitivity is unknown for this antibody (M02794 tissue-IHC caption). Record the fixative, fixation duration, processing history and section age for each block before attributing a staining difference to biology (standard IHC practice). Compare adjacent sections under the same citrate pH 6, 20-minute retrieval and antibody conditions to isolate differences associated with specimen handling (datasheet M02794; standard IHC practice). Include a concurrently stained positive control, and do not infer fixation tolerance from CSTB’s cytoplasmic and nuclear annotation or from tissue staining patterns (UniProt P04080; HPA tissue IHC).
Which staining compartments and cell types should count as plausible CSTB signal?
Assess cytoplasmic and nuclear staining in the appropriate cells, since CSTB is annotated in both compartments (UniProt P04080). HPA describes selective cytoplasmic and nuclear expression in squamous epithelia and urothelium, with high staining in esophageal squamous epithelial cells and medium staining in tonsillar squamous epithelial cells (HPA tissue IHC). Nucleolar staining can also be plausible: supported nucleolar and cytosolic locations come from subcellular imaging, although that evidence is separate from this IHC assay (HPA subcellular). Score compartments separately, compare them with section morphology, and investigate strong staining outside the expected cell population using controls before calling it CSTB (standard IHC practice; HPA tissue IHC).
How can epitope placement affect interpretation of CSTB IHC?
CSTB is annotated as one 98-amino-acid chain with no annotated isoforms, signal peptide, propeptide or transmembrane segment (UniProt P04080). Its record lists no glycosylation sites and identifies N-terminal acetylmethionine at residue 1, but these annotations do not establish which epitope the catalog antibody recognizes (UniProt P04080; M02794 tissue-IHC caption). Check the antibody’s stated immunogen or epitope, if available, before interpreting a negative section as loss of CSTB (standard IHC practice). If epitope information remains unavailable, retain the documented citrate pH 6 retrieval and compare staining with a positive control under matched conditions (datasheet M02794; standard IHC practice).
How should I adapt the CSTB question to multiplex IF?
Treat IF/ICC as a separate assay and validate its antibody and controls before comparing fluorescent signal with the chromogenic paraffin-section result (standard IF practice; M02794 tissue-IHC caption). Multiplex with a marker identifying the expected squamous epithelial or urothelial cells, and examine CSTB in the cytosol and nucleus, including nucleoli where resolved (HPA tissue IHC; HPA subcellular). Choose a fluorophore channel with low tissue autofluorescence and include single-stain controls to assess bleed-through (standard IF practice). Because CSTB is intracellular and has no transmembrane segment, optimize permeabilisation to expose cytosolic and nuclear epitopes while preserving morphology; verify that access experimentally (UniProt P04080; standard IF practice).
What should I check when DAB staining appears diffuse or widespread?
Compare the stained section with a no-primary control to identify signal arising from the chromogenic detection workflow (standard IHC practice). The documented tonsil assay used 10% goat serum, a biotinylated secondary antibody, a streptavidin–biotin complex and DAB, so check blocking and detection reagents when background rises (M02794 tissue-IHC caption). Apply a peroxidase block and assess endogenous biotin where relevant to this detection system; these are general IHC safeguards, not demonstrated CSTB-specific requirements (standard IHC practice). Reassess washing, antibody concentration and development time on matched sections, retaining the documented 2 µg/mL primary condition as the comparison point (M02794 tissue-IHC caption; standard IHC practice).
How should I quantify CSTB staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because the reported tissue pattern includes cytoplasmic and nuclear staining in squamous epithelia and urothelium (HPA tissue IHC). For chromogenic IHC, report the percentage of positive cells and an H-score using intensity grades 0–3 multiplied by each grade’s percentage, yielding 0–300 (standard IHC practice). When counting discrete positive cells, express density per mm² of viable, evaluable tissue and exclude folds or necrotic areas (standard IHC practice). Normalize comparisons to the same cell type, compartment, section-selection rules and staining batch, with a concurrently processed control to monitor technical variation (standard IHC practice).
How can I distinguish genuine CSTB staining from artefact?
Look for reproducible cytoplasmic or nuclear signal in the expected epithelial cells, using the reported squamous epithelial and urothelial pattern as a biological check (UniProt P04080; HPA tissue IHC). Nucleolar signal is plausible, but its supporting evidence comes from subcellular imaging and should be assessed alongside the IHC section’s morphology and controls (HPA subcellular; standard IHC practice). Treat strong signal in an unexpected cell type or compartment, staining confined to section edges, and staining over necrotic areas as reasons to investigate artefact (standard IHC practice; HPA tissue IHC). Compare no-primary and peroxidase-control sections when DAB persists, because endogenous enzyme activity can imitate positive chromogenic staining (standard IHC practice).
Boster reagents
Best CSTB / Cystatin-B IHC Antibodies
Anti-CSTB antibodies have IHC images from human tonsil and prostatic cancer paraffin sections, plus an IF image from A431 cells (catalog image captions).
Real IHC data
Anti-Stefin B CSTB Antibody ® (monoclonal, 2B6)
Cat # M02794
Real IHC data
Anti-Stefin B/CSTB Antibody ®
Cat # PB9565
M02794 has an IHC image from a human tonsil paraffin section and lists human reactivity (M02794 IHC image caption; catalog reactivity). PB9565 has an IHC image from a human prostatic cancer paraffin section and an IF image from A431 cells (PB9565 image captions).
Which to pick: For tissue IHC, choose M02794 for human tonsil or PB9565 for human prostatic cancer paraffin sections (M02794 and PB9565 IHC image captions); both captions leave the fixative unreported (M02794 and PB9565 IHC image captions). For IF/ICC, choose PB9565: both applications are listed, and its IF image shows A431 cells (catalog applications; PB9565 IF image caption). For rat tissue IHC, M02794-1 lists rat reactivity and IHC, but has no IHC image in the payload (catalog reactivity, applications and image alts).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P04080 (CYTB_HUMAN, Cystatin-B).
- Human Protein Atlas. CSTB tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CSTB subcellular location (ICC-IF): Localized to the nucleoli and cytosol..
- Human Protein Atlas. CSTB antibody validation summary (3 antibodies).
- Cystatin B is a progression marker of human epithelial ovarian tumors mediated by the TGF-β signaling pathway. International journal of oncology 2014 — PMC3977810.
- Cystatin B Promotes the Proliferation, Migration, and Invasion of Intrahepatic Cholangiocarcinoma. Current oncology (Toronto, Ont.) 2025 — PMC11854580.
- Brain inflammation is accompanied by peripheral inflammation in Cstb (-/-) mice, a model for progressive myoclonus epilepsy. Journal of neuroinflammation 2016 — PMC5127053.
- Biomarker cystatin B expression correlates with pathogenesis in cervical cancer. The Journal of international medical research 2024 — PMC10913530.
- PubMed PMID:3902020 — UniProt-cited evidence.
- PubMed PMID:8596935 — UniProt-cited evidence.
- PubMed PMID:10830953 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CSTB (Cystatin-B). Expected localisation Cytoplasmic and nuclear staining in squamous epithelia (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet M02794). Validated IHC antibody: M02794 at 0.5-1μg/ml recommended; image 2μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Esophagus (Squamous epithelial cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissue; fixative not specified (datasheet M02794); 2. Fixation: Image fixative and duration unreported (datasheet M02794); 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 M02794); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M02794); 8. Primary antibody: Mouse monoclonal (clone 2B6) anti-CSTB, 0.5-1μg/ml recommended; image 2μg/ml (datasheet M02794); 9. Primary incubation: Overnight at 4 °C (datasheet M02794). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Antibody staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: One 1–98 chain; no isoforms annotated (UniProt); Regulation: Tissue enriched in esophagus at the RNA level (HPA RNA). Evidence: UniProt P04080, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoli and cytosol., Datasheet M02794, PMC3977810, PMC11854580. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CSTB/immunohistochemistry-ihc-guide.