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CSTA / Cystatin-A · IHC design guide
Design Immunohistochemistry for CSTA
Plan CSTA IHC in paraffin sections using the PA2105 protocol and esophageal squamous cells as a high-staining reference (datasheet: PA2105 IHC-P; HPA tissue IHC). Score cytoplasmic and nuclear staining by cell type, using adipocytes as a not-detected comparator (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CSTA 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 tissue staining (HPA tissue IHC) | |
| Staining pattern | Squamous and lymphoid cells: cytoplasmic and nuclear signal (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet PA2105) | |
| 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 sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet PA2105); verify before use. | |
| Caveat | Lymphoid cells can stain alongside squamous epithelium (HPA tissue IHC) | |
| Regulation | RNA tissue enhanced: cervix, esophagus, vagina (HPA tissue RNA) | |
| Isoform / epitope | No isoforms; chains start at residues 1 or 2 (UniProt) |
Section 1
Recommended CSTA IHC & IF Protocols
The catalog antibody protocol (datasheet: PA2105) is followed by three published CSTA IHC protocols (PMC5908574; PMC5649615; PMC1626467).
| Sample | Paraffin-embedded human tonsil tissues; fixative not specified (datasheet PA2105) |
| Fixation | Image fixative and duration unreported (datasheet PA2105); 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 PA2105) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PA2105) |
| Primary antibody | Rabbit anti-CSTA, 0.5-1μg/ml (datasheet PA2105) |
| Primary incubation | Overnight at 4 °C (datasheet PA2105) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PA2105) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CSTA-positive staining in germinal center cells of appendix (HPA tissue IHC: High). HPA tissue profile: Selective cytoplasmic and nuclear expression in squamous epithelia, lymphoid cells and a few types of glandular epithelium. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate pH 6 retrieval (datasheet: PA2105); published CSTA protocols use citrate pH 6 with an autoclave or microwave (PMC5908574; PMC5649615).
Section 2
What Is the Expected CSTA Staining Pattern?
CSTA is cytoplasmic by UniProt and has no transmembrane segment (UniProt P01040: subcellular location and topology). HPA tissue IHC shows selective cytoplasmic and nuclear staining in squamous epithelia, lymphoid cells and some glandular epithelia (HPA: tissue profile). HPA rates the tissue profile Enhanced, while noting medium consistency between antibody staining and RNA expression (HPA: reliability). Interpret each compartment in its tissue and cell context.
| Squamous epithelial cells stain in esophagus; cells in the skin corneal layer stain strongly. | These are expected positive populations (HPA: High in esophageal squamous cells and skin corneal-layer cells). Cytoplasmic staining fits UniProt localisation; nuclear staining can also fit the HPA tissue profile (UniProt P01040: cytoplasm; HPA: tissue profile). |
| Cytoplasmic and nuclear signal appears within an expected positive cell population. | Both compartments are compatible with HPA tissue IHC; HPA ICC-IF separately places CSTA in the cytosol and nucleoplasm (HPA: tissue profile; HPA: approved ICC-IF locations). Judge the IHC slide by its cell pattern as well as its compartment. |
| Signal is confined to cell borders, extracellular material or an unrelated structure. | A purely membrane or extracellular pattern does not fit the reported intracellular locations or absence of a transmembrane segment (UniProt P01040: cytoplasm and topology; HPA: tissue profile). Treat it as suspect and check staining controls before scoring it as CSTA. |
| Adipocytes or adrenal glandular cells stain as strongly as the selected positive cells. | Those cell populations are listed as not detected (HPA: adipose adipocytes; HPA: adrenal glandular cells). Unexpected signal can reflect antibody cross-reactivity or endogenous chromogenic detection activity; verify with appropriate negative and detection controls (general IHC practice). |
| A known-positive compartment is blank, or color covers many cell types without clear boundaries. | A blank esophageal squamous population conflicts with its High HPA level (HPA: esophagus). Widespread haze prevents a reliable cell-level call. Check the positive-control run, detection controls and background before interpreting either appearance (general IHC practice). |
💡Expected CSTA appearanceCall the IHC result positive when expected cells show discernible cytoplasmic staining, with possible nuclear staining and strong signal in HPA High populations; isolated extracellular, border-only or broadly diffuse color is suspect (HPA: tissue profile and High populations; UniProt P01040: cytoplasm and topology).
| Tissue and cell choice | Use a defined positive population such as esophageal squamous cells or skin corneal-layer cells (HPA: High in both). Adipocytes provide a population listed as not detected (HPA: adipose tissue). Score the specified cells, because an HPA tissue entry does not assign one level to every cell in that tissue. |
| Compartment and assay | UniProt lists cytoplasm, while HPA tissue IHC reports cytoplasmic and nuclear expression (UniProt P01040: location; HPA: tissue profile). HPA ICC-IF approves cytosol and nucleoplasm (HPA: subcellular summary). That IF observation supports interpretation here; it is not an IF protocol option. |
| Antibody validation | The overall HPA tissue profile is Enhanced, with medium staining–RNA consistency (HPA: reliability). Individual IHC ratings differ: CAB000469 is Enhanced; HPA001031 and CAB047315 are Supported (HPA: antibody validation). Keep that distinction when comparing slides made with different antibodies. |
| Processing and annotated variants | UniProt lists full-length residues 1–98 and an N-terminally processed 2–98 chain, with no annotated isoforms or glycosylation sites (UniProt P01040: processing, isoforms and glycosylation). These annotations do not identify the antibody epitope or establish different staining between the two chains. |
| Chromogenic detection background | Endogenous tissue activity or nonspecific detection can create color unrelated to primary-antibody binding (general IHC practice). Evaluate the relevant detection control alongside cell morphology. Neither HPA tissue levels nor UniProt annotations identify a CSTA-specific source of such background. |
| Situation | Likely cause | Next action |
|---|---|---|
| Esophageal squamous cells show no convincing signal. | A High reference population is expected to stain (HPA: esophageal squamous cells). A failed run, weak detection or an unsuitable antibody condition can leave it blank (general IHC practice). | Confirm that the positive-control section and detection reagents worked; then review the antibody's IHC-P instructions for retrieval and dilution (general IHC practice). Do not infer CSTA-specific retrieval sensitivity from HPA. |
| Only nuclei stain in the expected positive cells. | Nuclear staining can be compatible with HPA tissue IHC and approved nucleoplasmic ICC-IF localisation (HPA: tissue profile and ICC-IF locations). Its absence from cytoplasm alone does not prove artefact. | Check whether the signal follows the expected cell distribution, then compare background and antibody controls (general IHC practice). Record nuclear and cytoplasmic staining separately rather than discarding a plausible nuclear result. |
| Strong color appears in adipocytes or adrenal glandular cells. | Both populations are reported as not detected (HPA: adipose adipocytes; HPA: adrenal glandular cells). Cross-reactivity or endogenous chromogenic activity is possible (general IHC practice). | Check a control omitting the primary antibody and review the blocking and detection steps (general IHC practice). Interpret any residual staining against the HPA cell-specific references. |
| Color is diffuse across tissue or obscures cell outlines. | Poorly resolved color cannot demonstrate the selective cell and compartment pattern reported by HPA (HPA: tissue profile). Excess background may arise during blocking, antibody incubation or detection (general IHC practice). | Compare the negative detection control, review wash and blocking steps, and adjust incubation conditions using the antibody's IHC-P instructions (general IHC practice). Reassess only when individual cells can be scored. |
| Signal is restricted to extracellular deposits or cell borders. | That distribution conflicts with intracellular localisation and the absence of a transmembrane segment (UniProt P01040: cytoplasm and topology; HPA: tissue profile). | Inspect morphology and detection controls, then compare a positive reference population on the same run (general IHC practice; HPA: High esophageal squamous cells). Do not count border-only color as the expected pattern. |
| Two antibodies give different staining patterns. | HPA assigns different IHC validation ratings to the listed antibodies (HPA: CAB000469 Enhanced; HPA001031 and CAB047315 Supported). Ratings alone do not identify the cause of a particular disagreement. | Compare the same cell populations and compartments, review each antibody's IHC-P conditions, and retain run controls (general IHC practice). Report the discordance rather than combining unlike patterns into one score. |
Experiment evidence
IHC & IF images showing CSTA staining
PA2105 · IHCIHC analysis of Cystatin A/CSTA using anti-Cystatin A/CSTA antibody (PA2105). Cystatin A/CSTA was detected in paraffin-embedded section of h
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000121552-CSTA/tissue/cerebral+cortex#imid_389150
HPA · EsophagusHuman Protein Atlas IHC, Esophagus. Source: https://www.proteinatlas.org/ENSG00000121552-CSTA/tissue/esophagus#imid_389122
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000121552-CSTA/tissue/colon#imid_389076
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000121552-CSTA/tissue/liver#imid_389069
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001031; green = CSTA, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001031; green = CSTA, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CSTA IHC & IF
🧪Run esophagus first; its squamous epithelial cells should stain (HPA: High in squamous epithelial cells). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); on the esophagus slide, assess adjacent stromal cells for background staining without assuming they are CSTA-negative.
Positive control tissue: Appendix (Germinal center cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CSTA in A-431, U2OS, hTCEpi, with annotated localisation: Nucleoplasm (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; nonimmune rabbit IgG matched to the primary antibody’s clonality; and, if available, CSTA-knockout tissue or a validated peptide-block control (caption: rabbit primary antibody; standard IHC practice). Block endogenous peroxidase and, for the caption’s biotin-based detection, endogenous biotin; inspect the esophageal surface for nonspecific chromogen deposition (caption: biotinylated secondary and DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected PA2105 paraffin-section caption does not state a fixative (caption: fixative not stated). The caption uses citrate retrieval at pH 6 for 20 minutes, but does not establish that retrieval is required (caption: citrate retrieval conditions). The supplied evidence does not establish that frozen sections or IF are easier; check keratinized esophageal surface material for nonspecific staining (standard IHC practice).
HPA tissue IHC evidence for CSTA
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 |
|---|---|---|---|---|
| Appendix | Germinal center cells | High | Protein (IHC) | HPA → |
| Endometrium | Ciliated epithelial cells | High | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Skin | Cells in corneal layer | High | Protein (IHC) | HPA → |
| Lung | Macrophages | 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 → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CSTA IHC Tips
Troubleshoot CSTA staining in paraffin sections by checking retrieval, cell type, compartment, detection background and scoring against documented expression patterns.
How should I adjust retrieval when CSTA staining is weak in paraffin sections?
Start with heat-mediated citrate antigen retrieval at pH 6 for 20 minutes (datasheet PA2105). The selected paraffin-section image used that treatment before an overnight incubation with 1 μg/ml catalog antibody at 4°C (datasheet PA2105). If signal is weak, verify that the section reached the intended retrieval temperature, then vary heating or cooling time on serial sections while holding antibody concentration and detection constant (standard IHC practice). Include a known positive compartment, such as esophageal squamous epithelium, in the comparison (HPA: High in esophageal squamous epithelial cells). Record tissue integrity alongside signal, because excessive heating can damage morphology (standard IHC practice).
Could fixation explain inconsistent CSTA staining between paraffin blocks?
The selected paraffin-section caption does not state a fixative, and target-specific CSTA sensitivity to fixation is unknown (datasheet PA2105). Record each block’s fixative and fixation duration before comparing staining, because these variables can change antigen accessibility in paraffin-section IHC (standard IHC practice). Process serial sections with the documented citrate pH 6, 20-minute retrieval and identical antibody incubation to isolate differences associated with specimen preparation (datasheet PA2105; standard IHC practice). Compare preservation and staining in the same expected cell population, rather than treating a weak field alone as fixation damage (HPA: High in esophageal squamous epithelial cells; standard IHC practice).
Should nuclear staining be rejected when evaluating CSTA IHC?
Evaluate cytoplasmic staining first because UniProt assigns CSTA to the cytoplasm and describes it as an intracellular thiol proteinase inhibitor (UniProt P01040). Nuclear signal also warrants review: HPA reports selective cytoplasmic and nuclear tissue staining and approved nucleoplasm and cytosol locations in subcellular images (HPA: tissue IHC profile; HPA: subcellular). Compare nuclear and cytoplasmic staining separately within the expected cell types, including squamous epithelial cells, rather than combining both compartments into one positive score (HPA: esophagus; standard IHC practice). Check the no-primary control and neighbouring cells before accepting isolated nuclear colour, since chromogenic background can imitate compartment-specific staining (standard IHC practice).
Could CSTA processing or an unknown epitope alter the IHC pattern?
The record lists no alternative isoforms, so an apparent change in paraffin-section staining should not automatically be assigned to isoform switching (UniProt P01040: isoforms 0). It lists chains spanning residues 1–98 and 2–98, plus N-terminal acetylmethionine, but supplies no mapped antibody epitope (UniProt P01040: processing and modified residues; datasheet PA2105). Compare serial sections under identical retrieval and detection conditions before proposing that N-terminal processing changes antibody recognition (standard IHC practice). CSTA has no annotated transmembrane segment or glycosylation sites, so describe unusual membrane-dominant staining as a finding requiring validation, rather than a demonstrated glycoform or membrane isoform (UniProt P01040: topology, glycosylation and isoforms).
How can IF help resolve ambiguous chromogenic CSTA staining?
Use IF as a separate localisation check when the paraffin-section DAB pattern leaves cell identity or compartment uncertain (standard IHC/IF practice). Multiplex CSTA with a validated marker for the expected squamous epithelial population, then assess overlap cell by cell; HPA reports high CSTA staining in esophageal squamous epithelial cells (HPA: esophagus). Choose a spectrally separated fluorophore in a channel with low measured tissue autofluorescence, and include single-colour and no-primary controls (standard IF practice). For this intracellular protein, assess whether permeabilisation permits access to cytosolic and nuclear epitopes, without assuming the antibody epitope has been mapped (UniProt P01040: cytoplasm; HPA: nucleoplasm and cytosol; standard IF practice).
What should I check when CSTA DAB staining is widespread?
Run a no-primary control through the same secondary and chromogen steps to locate detection-system background (standard IHC practice). The selected image used a biotinylated goat anti-rabbit secondary, a streptavidin–biotin complex and DAB, so test endogenous biotin and peroxidase contributions when colour appears outside expected cells (datasheet PA2105; standard IHC practice). Apply an appropriate peroxidase block and compare with a biotin-independent detection system if the background pattern persists (standard IHC practice). Review the documented 10% goat-serum block and 1 μg/ml primary concentration before changing them one variable at a time (datasheet PA2105; standard IHC practice).
How should I quantify CSTA staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and compartment before analysis; HPA reports selective cytoplasmic and nuclear staining across several cell types (HPA: tissue IHC profile). For a defined population, record the percentage of positive cells and, when intensity is reproducible, an H-score using fixed intensity categories across all sections (standard IHC practice). If the question concerns scattered positive cells, report their density per mm² of evaluable tissue or within a prespecified region (standard IHC practice). Normalise to the number of eligible cells or evaluable tissue area, exclude folds and necrosis, and keep retrieval, exposure and threshold settings consistent across the comparison (standard IHC practice).
How can I distinguish genuine CSTA signal from artefact?
Treat staining as more convincing when it follows a defined cell population and shows cytoplasmic signal, with nuclear signal assessed separately (UniProt P01040: cytoplasm; HPA: tissue IHC profile). Compare expected high-staining esophageal squamous epithelial cells with an appropriate low-expression or undetected population, such as adipocytes, under the same run conditions (HPA: esophagus High; HPA: adipose adipocytes Not detected; standard IHC practice). Question isolated membrane-dominant colour because CSTA has no transmembrane segment, and inspect section edges or necrotic areas for nonspecific accumulation (UniProt P01040: topology; standard IHC practice). A no-primary control helps identify endogenous enzyme or detection background before positive cells are counted (standard IHC practice).
Boster reagents
Best CSTA / Cystatin-A IHC Antibodies
Anti-CSTA antibodies have image data for human tonsil paraffin sections (PA2105 and PB9564 IHC captions) and A431 cells by IF (PB9564 IF caption).
Real IHC data
Anti-Cystatin A/CSTA Antibody ®
Cat # PA2105
PA2105 will render with a human tonsil paraffin-section IHC image (PA2105 IHC caption; catalog: Human, IHC). PB9564 will render with its own human tonsil paraffin-section IHC image; its IF image shows A431 cells (PB9564 IHC and IF captions).
Which to pick: Choose PA2105 for tissue IHC because IHC is listed and its own image shows human tonsil paraffin sections (PA2105 catalog and IHC caption). Choose PB9564 for IF/ICC because those applications are listed and its IF image shows A431 cells; its catalog also lists Human, Mouse and Rat reactivity, though the supplied IF image shows A431 cells only (PB9564 catalog and IF caption). PB9564 has a paraffin-section IHC caption but IHC is absent from its application list; neither IHC caption reports the fixative (PB9564 catalog and IHC caption; PA2105 IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P01040 (CYTA_HUMAN, Cystatin-A).
- Human Protein Atlas. CSTA tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CSTA subcellular location (ICC-IF): Localized to the nucleoplasm and cytosol..
- Human Protein Atlas. CSTA antibody validation summary (3 antibodies).
- Clinicopathological significance of cystatin A expression in progression of esophageal squamous cell carcinoma. Medicine 2018 — PMC5908574.
- Particular gene upregulation and p53 heterogeneous expression in TP53-mutated maxillary carcinoma. Oncology letters 2017 — PMC5649615.
- Gene expressions and copy numbers associated with metastatic phenotypes of uterine cervical cancer. BMC genomics 2006 — PMC1626467.
- Identification and Validation of Three-Gene Signature in Lung Squamous Cell Carcinoma by Integrated Transcriptome and Methylation Analysis. Journal of oncology 2022 — PMC9525794.
- PubMed PMID:6689312 — UniProt-cited evidence.
- PubMed PMID:2768224 — UniProt-cited evidence.
- PubMed PMID:2442723 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CSTA (Cystatin-A). Expected localisation Cytoplasmic and nuclear tissue staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet PA2105). Validated IHC antibody: PA2105 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Germinal center cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissues; fixative not specified (datasheet PA2105); 2. Fixation: Image fixative and duration unreported (datasheet PA2105); 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 PA2105); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PA2105); 8. Primary antibody: Rabbit anti-CSTA, 0.5-1μg/ml (datasheet PA2105); 9. Primary incubation: Overnight at 4 °C (datasheet PA2105). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet PA2105); verify before use.; Caveat: Lymphoid cells can stain alongside squamous epithelium (HPA tissue IHC); Isoform / epitope: No isoforms; chains start at residues 1 or 2 (UniProt); Regulation: RNA tissue enhanced: cervix, esophagus, vagina (HPA tissue RNA). Evidence: UniProt P01040, HPA tissue IHC · Enhanced, HPA ICC-IF · nucleoplasm and cytosol., Datasheet PA2105, PMC5908574, PMC5649615. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CSTA/immunohistochemistry-ihc-guide.