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CRYAA / Alpha-crystallin A chain · IHC design guide
Design Immunohistochemistry for CRYAA
Plan chromogenic CRYAA IHC in paraffin sections using lens fiber cells as a positive reference (HPA tissue IHC). This guide covers the catalog antibody’s IHC-P workflow (datasheet: PB9940) and interpretation of cytoplasmic lens staining (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CRYAA 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 | Lens cytoplasm (HPA tissue IHC) | |
| Staining pattern | Medium cytoplasmic staining in lens fiber cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet PB9940) | |
| Positive control | Eye | |
| 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 | Several sampled non-lens tissues had no detectable staining (HPA tissue IHC) | |
| Regulation | Heat shock shifts CRYAA to nuclei (UniProt) | |
| Isoform / epitope | 0 isoforms; chains end at residues 162–173, so C-terminal epitopes may vary (UniProt) |
Section 1
Recommended CRYAA IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet) with the published CRYAA staining protocol for human lens capsules (PMC6334984).
| Sample | Paraffin-embedded mouse spleen tissues; fixative not specified (datasheet PB9940) |
| Fixation | Image fixative and duration unreported (datasheet PB9940); 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 PB9940) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9940) |
| Primary antibody | Rabbit anti-CRYAA, 0.5-1μg/ml (datasheet PB9940) |
| Primary incubation | Overnight at 4 °C (datasheet PB9940) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9940) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CRYAA-positive staining in lens fiber cells of eye (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in lens. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate retrieval at pH 6 (PB9940 datasheet); the published lens-capsule excerpt does not report retrieval conditions (PMC6334984 methods).
Section 2
What Is the Expected CRYAA Staining Pattern?
CRYAA is a soluble protein found in the cytoplasm and, under heat stress, the nucleus (UniProt P02489 localization and topology). In tissue IHC, expect cytoplasmic staining in lens fiber cells (HPA: cytoplasmic expression in lens; lens fiber cells, Medium). HPA rates its tissue staining Supported, with external characterization but no internal RNA data for correlation (HPA: tissue IHC reliability).
| Lens fiber cells show cytoplasmic chromogenic staining at roughly medium intensity. | This matches the reported positive tissue and compartment (HPA: lens fiber cells, Medium; cytoplasmic expression in lens). Assess the fiber cell area against adjacent tissue and controls; intensity is an HPA observation, not a required score for every section. |
| Staining appears mainly along cell borders or in another membrane-like pattern. | A membrane-dominant pattern does not match the reported cytoplasmic IHC pattern (HPA: cytoplasmic expression in lens) or the absence of a transmembrane segment (UniProt P02489 topology). Check morphology, counterstain and background before interpreting it as CRYAA. |
| Adipocytes, respiratory epithelial cells or another listed HPA-negative population stain strongly. | Those specific cell populations were reported as Not detected (HPA: adipose adipocytes; bronchus respiratory epithelial cells). Cross-reactivity or endogenous detection activity is possible; a positive elsewhere is not automatically false because the HPA list is limited. |
| Color spreads across the section without a clear cellular or cytoplasmic pattern. | Diffuse staining cannot establish the reported lens fiber cell pattern (HPA: lens fiber cells, Medium; cytoplasmic expression in lens). Review a no-primary control and the detection steps; nonspecific reagent binding or residual endogenous activity can create background in chromogenic IHC. |
| No staining is visible in an intact lens fiber cell area. | That conflicts with the reported positive reference (HPA: lens fiber cells, Medium), but one negative section cannot identify the cause. Confirm that the expected cells are present, then assess antibody performance, retrieval and detection with same-run controls; target-specific fixation sensitivity is unreported. |
💡Expected CRYAA appearanceCall a result concordant when lens fiber cells show predominantly cytoplasmic, approximately medium staining (HPA: lens fiber cells, Medium; cytoplasmic expression in lens); membrane-dominant color or strong staining in listed HPA-negative cells warrants control review.
| Tissue selection and evidence strength | Lens fiber cells provide the documented positive IHC reference (HPA: Eye, lens fiber cells, Medium). HPA calls tissue staining Supported through external characterization, without internal RNA correlation (HPA: reliability description); its Kidney and Retina group-enriched RNA annotation does not establish an IHC-positive cell population (HPA: RNA specificity). |
| Compartment and topology | Use cytoplasmic lens staining as the tissue benchmark (HPA: tissue IHC profile). CRYAA has no transmembrane segment and may move to nuclear speckles during heat shock (UniProt P02489 topology and localization). Nuclear signal alone therefore needs context; it is not the routine lens IHC pattern documented here. |
| Antibody validation by application | The HPA IHC-supported antibody is HPA038430; the ICC-supported antibody is HPA037737 (HPA: antibody validation). These are application-specific records, so the ICC result does not validate the IHC antibody for IF, and the IHC result does not validate the ICC antibody for paraffin IHC. |
| IF/ICC Q&A: where should signal appear? | Mainly in the cytosol, with additional nucleoplasmic localization (HPA: subcellular ICC-IF, both Supported). UniProt reports nuclear translocation during heat shock (UniProt P02489 localization). This is an interpretation reference for IF/ICC; assess that application on its own guide page. |
| Situation | Likely cause | Next action |
|---|---|---|
| Lens fiber cells are negative on the IHC slide. | The positive population may be absent or poorly preserved, or an assay step may have failed; HPA reports Medium lens fiber cell staining (HPA: tissue IHC). | Verify lens fiber cells on the counterstain and compare a same-run positive section. Review antibody application, retrieval and chromogenic detection as general IHC checks; no CRYAA-specific fixation or retrieval sensitivity is established here. |
| The whole section has diffuse brown background. | Nonspecific binding, incomplete washing or endogenous detection activity can cause broad chromogenic background (general IHC practice). | Compare a no-primary control, inspect wash and blocking steps, and address endogenous enzyme activity when relevant. Score CRYAA only where a cellular pattern can be distinguished from background (HPA: cytoplasmic lens profile). |
| Signal is mainly nuclear in lens cells. | UniProt describes heat-shock-associated nuclear translocation, while HPA reports cytoplasmic lens IHC staining (UniProt P02489 localization; HPA: tissue IHC profile). Nuclear predominance alone does not identify the condition. | Confirm nuclear boundaries with the counterstain and review controls and tissue handling. Record the nuclear pattern separately from the expected lens IHC pattern; avoid assigning heat shock without independent evidence. |
| Color outlines membranes more than cytoplasm. | The pattern conflicts with cytoplasmic lens staining (HPA: tissue IHC profile) and CRYAA's lack of a transmembrane segment (UniProt P02489 topology). | Recheck focus and cellular boundaries, then compare the no-primary control and detection background. Treat membrane-dominant staining as inconclusive until a cytoplasmic lens pattern is resolved. |
| A listed negative cell population stains strongly. | Cross-reactivity or endogenous detection activity may explain staining in a population reported Not detected (HPA: adipose adipocytes, adrenal glandular cells or bronchus respiratory epithelial cells). | Identify the stained cells on the counterstain, compare a no-primary control and the same-run lens reference, and report the discrepancy by cell type. HPA's negative observations do not cover every tissue or condition. |
Experiment evidence
IHC & IF images showing CRYAA staining
PB9940 · IHCIHC analysis of CRYAA using anti-CRYAA antibody (PB9940). CRYAA was detected in paraffin-embedded section of mouse spleen tissues. Heat medi
PB9940 · IHCIHC analysis of CRYAA using anti-CRYAA antibody (PB9940). CRYAA was detected in paraffin-embedded section of rat spleen tissues. Heat mediat
M01900-2 · IFIF analysis of Alpha A Crystallin using anti-Alpha A Crystallin antibody (M01900-2). Alpha A Crystallin was detected in an immunocytochemica
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000160202-CRYAA/tissue/cerebral+cortex#imid_20203877
HPA · EyeHuman Protein Atlas IHC, Eye. Source: https://www.proteinatlas.org/ENSG00000160202-CRYAA/tissue/retina#imid_943
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000160202-CRYAA/tissue/colon#imid_20203803
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000160202-CRYAA/tissue/liver#imid_20203804
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA037737; green = CRYAA, blue = nucleus, red = microtubules.
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA037737; green = CRYAA, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CRYAA IHC & IF
🧪Run eye first: lens fiber cells should stain at the HPA-reported Medium level (HPA: Eye, lens fiber cells, Medium). Use adipose tissue as a negative, focusing on adipocytes (HPA: Adipose tissue, adipocytes, Not detected); no internal negative cell type is identified for the eye slide in the supplied HPA rows, so do not assign one by appearance alone (HPA: supplied positive row).
Positive control tissue: Eye (Lens fiber cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CRYAA in Hep-G2, U-251MG, U2OS, with annotated localisation: Cytosol (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and concentration-matched rabbit IgG isotype controls, matching the catalog primary’s clonality where known, plus CRYAA-knockout tissue as a biological negative (caption: rabbit primary; standard IHC controls). For the biotin-based DAB method, block endogenous peroxidase and assess endogenous biotin background (caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected PB9940 paraffin-section caption does not state a fixative (caption: fixative not stated). That caption uses citrate retrieval at pH 6 for 20 minutes; it documents one working approach, without establishing that retrieval is required (caption: citrate retrieval). The supplied evidence does not establish whether frozen sections or IF are easier; lens autofluorescence can complicate IF interpretation, so include an unstained control (standard IF practice).
HPA tissue IHC evidence for CRYAA
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — External characterization data supports antibody staining but no internal RNA data available for correlation.). 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 |
|---|---|---|---|---|
| Eye | Lens fiber 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 | Glandular 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 CRYAA IHC Tips
Use lens staining and compartment controls to troubleshoot CRYAA in paraffin section chromogenic IHC; assess IF separately.
How should I retrieve CRYAA when staining paraffin sections?
Start with heat mediated citrate retrieval at pH 6 for 20 minutes (datasheet PB9940). Keep heating, cooling, section thickness and staining conditions consistent between the positive control and test sections so a retrieval change can be judged fairly (standard IHC practice). If staining remains weak, adjust heating time in small steps while checking whether tissue structure and background remain acceptable; the supplied caption documents only the 20 minute condition (datasheet PB9940). Include a lens positive control, where cytoplasmic lens fiber cell staining is reported, before interpreting a weak result in another tissue (HPA: Medium in eye lens fiber cells).
Could fixation explain weak or uneven CRYAA staining?
The selected paraffin section caption does not state a fixative, so CRYAA specific sensitivity to fixation is unknown for PB9940 (datasheet PB9940: fixative not stated). Record the actual fixative, fixation duration and processing history for each specimen, and compare matched sections stained in the same run (standard IHC practice). If signal differs across batches, examine tissue preservation and retrieval consistency before attributing the difference to CRYAA abundance (standard IHC practice). Use a lens control to check whether the run can show the expected cytoplasmic staining, but do not use its result to infer a preferred fixative (HPA: cytoplasmic expression in lens).
Should CRYAA staining be cytoplasmic or nuclear?
Expect cytoplasmic lens staining as the clearest tissue reference, with medium staining reported in lens fiber cells (HPA: cytoplasmic expression in lens; Medium in lens fiber cells). CRYAA is also annotated in the nucleus and can move there during heat shock, including to nuclear splicing speckles (UniProt P02489: subcellular location). Score cytoplasmic and nuclear chromogen separately, using the counterstain to define nuclei and avoiding a single combined intensity score (standard IHC practice). A nuclear pattern outside the lens needs appropriate biological context and controls; location alone cannot establish a stress response in a paraffin section (UniProt P02489: heat shock translocation; standard IHC practice).
Could processing or modification alter the CRYAA epitope?
The record lists no isoforms, but it does list chains ending at residues 173, 172, 168 and 162 (UniProt P02489: isoforms and processing). It also lists a glycosylation site at residue 162 and modifications including phosphorylation at residue 45 (UniProt P02489: glycosylation and modified residues). If staining varies, check the antibody's documented epitope before linking a negative region to a particular chain or modification; no epitope location is supplied here (record: epitope unspecified). Compare retrieval conditions and matched tissue controls first, because altered accessibility in processed sections can also change staining (standard IHC practice).
How can IF help assess a questionable CRYAA pattern?
For IF, multiplex CRYAA with an independently validated lens fiber cell marker to test cell identity; lens fiber cells are the reported positive tissue population (HPA: Medium in eye lens fiber cells). Choose fluorophores and imaging channels after checking unstained tissue for autofluorescence, and include single stain controls when assessing overlap (standard IF practice). CRYAA has no transmembrane segment and is reported in cytosol and nucleoplasm, so permeabilise the plasma membrane and ensure nuclear access if nuclear signal is being assessed (UniProt P02489: topology; HPA: subcellular location). Optimise IF staining independently; the supplied PB9940 tissue caption documents chromogenic paraffin section IHC conditions (datasheet PB9940).
What should I check when the DAB background is high?
Check whether background tracks section edges, damaged areas or the whole tissue rather than the expected lens cell pattern (HPA: cytoplasmic expression in lens; standard IHC practice). The selected caption used 10% goat serum, 1 µg/ml primary antibody overnight at 4°C, and a biotin based DAB detection workflow (datasheet PB9940). Compare a no primary control, review endogenous peroxidase blocking, and assess endogenous biotin when using that detection chemistry (standard IHC practice). If background remains high, titrate primary antibody and detection exposure together, retaining a lens positive control so reduced background is not mistaken for lost sensitivity (standard IHC practice; HPA: lens fiber cells positive).
How should I score CRYAA across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring: lens fiber cells provide a reported positive reference, and the reported lens pattern is cytoplasmic (HPA: Medium in lens fiber cells; cytoplasmic expression in lens). Within comparable regions, record the percentage of positive cells and an intensity based H-score, or positive area density per mm² when individual cells cannot be counted reliably (standard IHC practice). Normalise to evaluable tissue or cell number and use the same threshold, imaging conditions and background rule across sections (standard IHC practice). Report nuclear signal separately because CRYAA also has a documented nuclear location (UniProt P02489: subcellular location).
When is apparent CRYAA positivity likely to be artefactual?
Give greatest weight to a reproducible cytoplasmic pattern in lens fiber cells, the reported positive population, while checking an appropriate negative control section in the same run (HPA: Medium in lens fiber cells; standard IHC practice). Diffuse staining confined to cut edges, necrotic areas or tissue folds warrants review as a processing or staining artefact (standard IHC practice). Brown signal in a no primary control points toward detection background, including possible endogenous enzyme activity in a DAB workflow (standard IHC practice). Nuclear staining is biologically possible during heat shock, but an unexpected compartment or cell population requires corroboration before assigning it to CRYAA (UniProt P02489: heat shock translocation; HPA: lens pattern).
Boster reagents
Best CRYAA / Alpha-crystallin A chain IHC Antibodies
The catalog shows CRYAA IHC in paraffin-embedded mouse and rat spleen (PB9940 IHC captions) and IF/ICC in human HepG2 cells (M01900-2 IF caption).
Real IHC data
Anti-Alpha A Crystallin/CRYAA Antibody ®
Cat # PB9940
Real IF data
Anti-Alpha A Crystallin Antibody ® (monoclonal, 10B9)
Cat # M01900-2
PB9940 will render with a mouse spleen IHC figure; its catalog also documents rat spleen IHC and lists Human, Mouse, and Rat reactivity (PB9940 IHC captions; catalog: PB9940 reactivity). M01900-2 will render with a human HepG2 IF/ICC figure and lists Human, Mouse, and Rat reactivity (M01900-2 IF caption; catalog: M01900-2 reactivity).
Which to pick: Choose PB9940 for tissue IHC: its own captions document paraffin-embedded mouse and rat spleen, and its IHC dilution is 0.5–1 μg/ml; the fixative is unreported (PB9940 IHC captions; catalog: PB9940 IHC dilution). Choose M01900-2 for IF/ICC when a mouse monoclonal antibody is preferred: clone 10B9 has an IF figure in human HepG2 cells, with 5 μg/ml used in that example (catalog: M01900-2 host and clone; M01900-2 IF caption). For cross-species tissue IHC, PB9940 lists Human, Mouse, and Rat reactivity, with figure evidence for mouse and rat paraffin sections (catalog: PB9940 applications and reactivity; PB9940 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P02489 (CRYAA_HUMAN, Alpha-crystallin A chain).
- Human Protein Atlas. CRYAA tissue IHC expression (reliability: Supported).
- Human Protein Atlas. CRYAA subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the nucleoplasm..
- Human Protein Atlas. CRYAA antibody validation summary (2 antibodies).
- Novel protein constituents of pathological ocular pseudoexfoliation syndrome deposits identified with mass spectrometry. Molecular vision 2018 — PMC6334984.
- Epigenetic regulation of αA-crystallin in high myopia-induced dark nuclear cataract. PloS one 2013 — PMC3849391.
- PubMed PMID:817940 — UniProt-cited evidence.
- PubMed PMID:8587135 — UniProt-cited evidence.
- PubMed PMID:8943244 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CRYAA (Alpha-crystallin A chain). Expected localisation Lens cytoplasm (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet PB9940). Validated IHC antibody: PB9940 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Eye (Lens fiber cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded mouse spleen tissues; fixative not specified (datasheet PB9940); 2. Fixation: Image fixative and duration unreported (datasheet PB9940); 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 PB9940); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9940); 8. Primary antibody: Rabbit anti-CRYAA, 0.5-1μg/ml (datasheet PB9940); 9. Primary incubation: Overnight at 4 °C (datasheet PB9940). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Several sampled non-lens tissues had no detectable staining (HPA tissue IHC); Isoform / epitope: 0 isoforms; chains end at residues 162–173 , so C-terminal epitopes may vary (UniProt); Regulation: Heat shock shifts CRYAA to nuclei (UniProt). Evidence: UniProt P02489, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the…, Datasheet PB9940, PMC6334984. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CRYAA/immunohistochemistry-ihc-guide.