CSTA / Cystatin-A · IHC design guide

Design Immunohistochemistry for CSTA

Plan CSTA staining in paraffin sections using observed cytoplasmic and nuclear patterns in selected tissues (HPA tissue IHC). This guide covers fixation, controls and interpretation for chromogenic IHC.

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for CSTA (IHC for CSTA): expected localisation Cytoplasmic and nuclear staining in select cells (HPA tissue IHC), antibody PA2105, validated IHC image, and IHC protocol steps — Printable **CSTA** IHC protocol sheet — expected localisation Cytoplasmic and nuclear staining in select cells (HPA tissue IHC), antibody PA2105, controls and protocol steps. Open the full CSTA IHC guide →

Navigate to: Protocols Staining pattern Controls Antibodies

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 staining in select cells (HPA tissue IHC)
	Staining pattern	Squamous, lymphoid and some glandular cells stain in cytoplasm and nuclei (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)
	Caveat	Lymphoid cells may stain alongside squamous epithelium (HPA tissue IHC)
	Regulation	Regulation of expression is unreported (UniProt)
	Isoform / epitope	0 isoforms; N-terminal processing may affect N-terminal epitopes (UniProt)

Section 1

Recommended CSTA IHC & IF Protocols

The catalog antibody protocol (datasheet: PA2105) is accompanied by four published CSTA paraffin IHC protocols (PMC5908574; PMC5649615; PMC1626467; PMC9525794).

Recommended immunohistochemistry (IHC-P) protocol parameters
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 retrieval at pH 6 (datasheet: PA2105); two published protocols also use citrate at pH 6 (PMC5908574; PMC5649615).

Section 2

What Is the Expected CSTA Staining Pattern?

CSTA is an intracellular protein without a transmembrane segment (UniProt P01040: cytoplasm, topology). In paraffin-section IHC, expect cytoplasmic staining in squamous epithelial cells, especially in esophagus, and in cells of the skin’s corneal layer (HPA: High in both). Nuclear staining can also occur in selected cells (HPA tissue IHC: cytoplasmic and nuclear profile). HPA rates the tissue pattern Enhanced, while reporting medium consistency between staining and RNA data (HPA tissue IHC).

What am I looking at on my slide?
Cytoplasmic staining in esophageal squamous epithelial cells or cells in the skin’s corneal layer.	This fits the strongest listed tissue observations (HPA: High in both cell populations). Assess the named cells within each section; a tissue label alone does not establish that the expected cells stained (HPA tissue IHC).
Nuclear staining accompanies cytoplasmic staining in a plausible positive cell population.	Nuclear signal is compatible with the reported tissue profile (HPA tissue IHC: selective cytoplasmic and nuclear expression). Nuclear staining alone needs comparison with cell identity and controls; cytoplasm remains the annotated UniProt location (UniProt P01040: cytoplasm).
Signal appears chiefly at cell borders, on extracellular material, or in empty spaces.	That distribution does not match the annotated intracellular location or the observed tissue profile (UniProt P01040: cytoplasm; HPA tissue IHC: cytoplasmic and nuclear). Review morphology and controls before assigning it to CSTA; deposits or detection artefact are possible (general IHC practice).
Strong stain appears in adipocytes while the expected epithelial cells are weak or unstained.	Adipocytes in the listed adipose-tissue and breast examples were not detected (HPA tissue IHC). The mismatch raises concern for nonspecific binding, endogenous detection activity, or tissue-identification error; it does not prove which occurred (general IHC practice).
No staining appears in correctly identified esophageal squamous epithelium.	This conflicts with the listed High observation (HPA: esophageal squamous epithelial cells). First confirm that the expected cells are present and intact, then review the staining run and controls (general IHC practice). A negative result alone cannot establish absent CSTA.

💡Expected CSTA appearanceCall a section positive when staining is predominantly intracellular in the expected cells, with clear signal in esophageal squamous epithelial cells or skin corneal-layer cells (HPA: High); cytoplasmic signal is expected and nuclear signal is permissible (UniProt P01040: cytoplasm; HPA tissue IHC: cytoplasmic and nuclear), whereas dominant border, extracellular, or adipocyte staining warrants investigation (UniProt P01040: topology; HPA: adipocytes Not detected).

How each factor affects the staining
Cell population and tissue context	Intensity varies across the supplied examples: esophageal squamous cells and skin corneal-layer cells are High, oral-mucosal squamous cells and lung macrophages are Medium, and adipocytes in the listed examples are Not detected (HPA tissue IHC). Compare like cell populations when scoring.
Intracellular location and processing	CSTA has no signal peptide or transmembrane segment, and UniProt lists full-length and N-terminally processed chains (UniProt P01040: topology, processing). Those entries support intracellular interpretation but do not establish an extracellular staining pattern or a processing-specific IHC difference.
Antibody evidence	The overall tissue-IHC reliability is Enhanced despite medium staining-to-RNA consistency (HPA tissue IHC). Antibody status differs: CAB000469 is IHC Enhanced, while HPA001031 and CAB047315 are IHC Supported (HPA antibodies). Interpret discrepancies with the antibody used in mind.
IF/ICC Q&A: Can nuclear CSTA staining be expected?	Yes. Nucleoplasm and cytosol are approved ICC-IF locations (HPA subcellular). This supports considering nuclear signal alongside cytosolic signal; it does not supply an IF protocol or make every nuclear-positive cell a confirmed CSTA-positive cell.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
A known-positive esophageal section has little or no signal.	The result differs from the reported High squamous-cell staining (HPA tissue IHC); the failed step cannot be identified from the image alone.	Verify squamous cells are present; inspect run controls, antibody dilution, retrieval conditions, and detection steps using the assay’s documented settings (general IHC practice).
Only the section edge or broad areas outside cells stain.	The distribution lacks the selective intracellular pattern reported for CSTA (HPA tissue IHC; UniProt P01040: cytoplasm). Background from staining or detection is possible (general IHC practice).	Compare with a no-primary control; review blocking, washes, chromogen development, and section handling (general IHC practice).
Adipocytes stain strongly.	That conflicts with Not detected adipocytes in the listed adipose-tissue and breast examples (HPA tissue IHC). Cross-reactivity or endogenous detection activity is possible (general IHC practice).	Confirm cell identity and compare a no-primary control; review blocking of endogenous activity appropriate to the detection system (general IHC practice).
Nuclear signal is judged automatically as an artefact.	The tissue profile includes nuclear expression, and ICC-IF places CSTA in the nucleoplasm (HPA tissue IHC; HPA subcellular).	Reassess the stained cell population, accompanying cytoplasmic signal, and controls before excluding nuclear staining (HPA tissue IHC; general IHC practice).
A medium or low-expression sample looks weaker than esophagus.	HPA reports Medium oral-mucosal squamous cells and Low respiratory epithelial cells in nasopharynx and bronchus, versus High esophageal squamous cells (HPA tissue IHC).	Score each named cell population against its reported level; use an expected High tissue as a run control when available (HPA tissue IHC; general IHC practice).
An IF/ICC image and an IHC slide appear to disagree on compartment.	ICC-IF records approved nucleoplasm and cytosol locations, while tissue IHC reports selective cytoplasmic and nuclear staining (HPA subcellular; HPA tissue IHC). Sample context can differ (general microscopy practice).	Check cell identity, image channels or counterstain, and controls within each assay before calling a conflict; do not infer an IF protocol from the IHC result (general microscopy practice).

Source: UniProt P01040 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing CSTA staining

IHC analysis of Cystatin A/CSTA using anti-Cystatin A/CSTA antibody (PA2105). Cystatin A/CSTA was detected in paraffin-embedded section of human tonsil tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-Cystatin A/CSTA Antibody (PA2105) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

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.

← 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 and look for staining in squamous epithelial cells (HPA: High in esophageal squamous epithelial cells). Use adipose tissue as the negative tissue, focusing on adipocytes (HPA: Not detected in adipocytes); on the esophagus slide, use adjacent nonsquamous cells as an internal background comparator and call them negative only if they remain unstained.

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 no-primary (secondary-only) and host- and isotype-matched nonimmune rabbit IgG controls (selected PA2105 caption: rabbit primary antibody), plus CSTA-knockout material as a biological negative if available. Quench endogenous peroxidase and check for endogenous biotin when using the caption’s biotin-based DAB detection (selected PA2105 caption: biotinylated secondary, SABC and DAB).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected PA2105 tissue-IHC caption does not state a fixative (selected PA2105 caption: fixative not stated). Citrate retrieval at pH 6 for 20 minutes is a documented starting condition, but retrieval dependency is unreported (selected PA2105 caption: citrate retrieval); no supplied evidence shows frozen sections or IF to be easier than paraffin IHC. In esophagus, inspect surface keratin for retained chromogen before scoring epithelial staining.

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 identity, subcellular pattern and matched controls before comparing signal intensity.

What should I change if CSTA staining is weak after antigen retrieval?

Start with heat-mediated retrieval in citrate buffer at pH 6 for 20 minutes (datasheet PA2105). The selected paraffin-section example then used 10% goat serum and 1 μg/ml primary antibody overnight at 4°C (datasheet PA2105). If staining remains weak, compare a modestly longer heat exposure on adjacent sections while keeping antibody concentration and detection constant (standard IHC practice). Include a section with expected staining in squamous epithelial cells to distinguish inadequate retrieval from a low-expressing specimen (HPA: High in esophageal squamous epithelial cells; standard IHC practice). Record tissue morphology alongside signal, because excessive heating can make an apparent gain difficult to interpret (standard IHC practice).

How can I troubleshoot fixation-related loss of CSTA staining?

The selected paraffin-section caption does not state a fixative, so CSTA-specific fixation sensitivity is unknown (datasheet PA2105). Record fixative, fixation duration and tissue thickness for each case, then compare sections processed under documented conditions before changing retrieval or antibody concentration (standard IHC practice). Use the reported citrate pH 6, 20-minute heat retrieval as the initial staining condition for this antibody (datasheet PA2105). Keep a known positive squamous epithelial section in each run to help separate specimen processing from staining-run variation (HPA: High in esophageal squamous epithelial cells; standard IHC practice). Do not infer fixation tolerance from CSTA’s cytoplasmic annotation or tissue staining profile (UniProt P01040 subcellular location; HPA tissue IHC profile).

Should CSTA stain nuclei, cytoplasm or cell membranes in tissue sections?

Expect a substantial cytoplasmic component because CSTA is annotated as cytoplasmic and lacks a transmembrane segment (UniProt P01040 subcellular location and topology). Nuclear signal is also plausible in some tissue cells: the tissue IHC profile describes selective cytoplasmic and nuclear expression, while cell imaging places CSTA in the nucleoplasm and cytosol (HPA tissue IHC profile; HPA subcellular). Assess localisation within morphologically identified cells rather than calling a stained boundary membrane localisation (standard IHC practice; UniProt P01040 topology). Compare epithelial and lymphoid compartments separately, since both occur in the reported tissue staining profile (HPA tissue IHC profile). If nuclear staining appears alone or only at section edges, review counterstain, retrieval and negative controls before interpreting it (standard IHC practice).

Could CSTA processing or an epitope difference explain inconsistent staining?

UniProt lists no annotated CSTA isoforms, so an isoform-specific explanation is unsupported by this record (UniProt P01040 isoforms). It lists chains spanning residues 1–98 and 2–98, plus N-terminal acetylmethionine at residue 1 (UniProt P01040 processing and modified residues). If the antibody epitope is known, check whether it includes the extreme N-terminus before attributing a change to processing; the supplied caption does not identify an epitope (UniProt P01040 processing; datasheet PA2105 caption). CSTA has no annotated glycosylation sites or transmembrane segment, so neither feature supplies an epitope explanation here (UniProt P01040 glycosylation and topology). Compare matched sections with a common positive control before assigning a biological cause to discordant staining (standard IHC practice).

How should I verify CSTA localisation when moving from chromogenic IHC to IF?

Treat IF as a separate optimisation: the selected antibody example documents paraffin-section chromogenic IHC, with no IF conditions supplied (datasheet PA2105). Multiplex CSTA with a validated marker for the expected cell type, and inspect each channel separately before interpreting overlap (HPA: High in esophageal squamous epithelial cells; standard IF practice). Choose fluorophores in channels with low specimen autofluorescence, checking an unstained section and single-label controls under the same exposure settings (standard IF practice). Because CSTA is intracellular and has no transmembrane segment, optimise permeabilisation for access to cytosolic and, where assessed, nuclear signal (UniProt P01040 subcellular location and topology; HPA subcellular; standard IF practice). Keep compartment boundaries visible with an appropriate counterstain (standard IF practice).

What controls distinguish CSTA staining from chromogenic background?

The selected example used a biotinylated secondary, a streptavidin–biotin detection complex and DAB, so evaluate background within that detection sequence (datasheet PA2105). Include a no-primary control and inspect whether signal follows tissue edges, folds or damaged areas rather than intact cells (standard IHC practice). Apply a peroxidase block before DAB development and use an appropriate biotin-blocking control if the detection system produces tissue-dependent signal (standard IHC practice; datasheet PA2105 detection method). Compare signal in expected squamous epithelial cells with adipocytes, which are reported as undetected, while matching section processing (HPA: High in esophageal squamous epithelial cells; HPA: Not detected in adipocytes of adipose tissue; standard IHC practice). Titrate primary antibody from the reported 1 μg/ml condition if background obscures cellular detail (datasheet PA2105; standard IHC practice).

How should I score CSTA staining across heterogeneous tissue compartments? ⚠ ANSWER MARKED FOR VERIFICATION

Define the scored cell population before measuring CSTA, because reported staining differs across squamous epithelia, lymphoid cells and selected glandular cells (HPA tissue IHC profile). For chromogenic sections, score the percentage of positive cells and intensity within each defined population, or calculate an H-score using the same thresholds for every case (standard IHC practice). Normalise positive-cell counts to the number of eligible cells, or express positive-cell density per mm² of viable, analysable tissue (standard IHC practice). Keep cytoplasmic and nuclear scores separate when both are present, since both patterns are reported (HPA tissue IHC profile; standard IHC practice). Exclude folds, necrosis and section edges using prespecified rules, and include a common control section across staining runs (standard IHC practice).

When is an unexpected CSTA-positive cell a credible biological finding?

Check whether staining lies in intact cytoplasm, with or without nuclear signal, and whether the cell identity fits the reported tissue pattern (UniProt P01040 subcellular location; HPA tissue IHC profile). Squamous epithelial cells can show strong signal, while macrophages and some other cell populations have reported staining at different levels (HPA: High in esophageal squamous epithelial cells; HPA: Medium in lung macrophages). A crisp membrane-only pattern conflicts with CSTA’s lack of a transmembrane segment and warrants review of morphology and controls (UniProt P01040 topology; standard IHC practice). Discount signal confined to necrosis, folds or cut edges, and compare with a no-primary control for endogenous enzyme or detection-system background (standard IHC practice). Repeat a surprising result on another well-preserved section before assigning biological meaning (standard IHC practice).

Boster reagents

Best CSTA / Cystatin-A IHC Antibodies

Anti-CSTA antibodies have IHC data from human paraffin-embedded tonsil sections and IF data from A431 cells (catalog image captions); the catalog lists human, mouse and rat reactivity (catalog applications/reactivity).

Real IHC data

Anti-Cystatin A/CSTA Antibody ®

Cat # PA2105

View product →

PA2105 will render with IHC staining of human paraffin-embedded tonsil sections (PA2105 IHC image caption). PB9564 is listed for human IF/ICC and has an IF image from A431 cells; its catalog also lists mouse and rat reactivity (PB9564 applications/reactivity and IF image caption).

Which to pick: Choose PA2105 for human tissue IHC because IHC is a listed application and its own image shows staining in paraffin-embedded tonsil sections (PA2105 applications/reactivity and IHC image caption). Choose PB9564 for human IF/ICC, or to investigate mouse or rat reactivity, because IF/ICC and those species are listed; its IF image documents A431 cells (PB9564 applications/reactivity and IF image caption). Both antibodies are rabbit-hosted with clonality unreported, and neither IHC caption reports a fixative (catalog host/clone fields and IHC image captions).

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.