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CRYAB / Alpha-crystallin B chain · IHC design guide
Design Immunohistochemistry for CRYAB
Plan chromogenic CRYAB IHC in paraffin sections using cytoplasmic staining as the expected tissue pattern (HPA tissue IHC). Compare heart muscle cardiomyocytes with adrenal gland glandular cells, which have no detected staining (HPA tissue IHC), and start catalog antibody A03496-2 at 2–5 μg/mL (datasheet A03496-2).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CRYAB 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 | Observed cytoplasmic staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in glia and cardiomyocytes (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03496-2) | |
| Positive control | Caudate+4 more · see all | |
| Negative control | Adrenal gland+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 | Heat shock can shift CRYAB to nuclear speckles (UniProt) | |
| Regulation | Expression varies by tissue (HPA tissue IHC) | |
| Isoform / epitope | No listed isoforms; one 1–175 chain (UniProt) |
Section 1
Recommended CRYAB IHC & IF Protocols
The catalog antibody uses EDTA pH 8.0 retrieval (datasheet A03496-2). The published IHC methods below cover ovarian paraffin sections (PMC6579977) and frozen cerebral contusions (PMC8963874).
| Sample | Paraffin-embedded rat brain tissue; fixative not specified (datasheet A03496-2) |
| Fixation | Image fixative and duration unreported (datasheet A03496-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A03496-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03496-2) |
| Primary antibody | Rabbit anti-CRYAB, 2-5μg/ml (datasheet A03496-2) |
| Primary incubation | Overnight at 4 °C (datasheet A03496-2) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A03496-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CRYAB-positive staining in glial cells of caudate (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in a subset of tissues including heart muscle, brain, thyroid gland, kidney and peripheral nerves. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 for the catalog antibody (datasheet A03496-2). Citrate pH 6.0 is a published alternative for ovarian paraffin sections (PMC6579977).
Section 2
What Is the Expected CRYAB Staining Pattern?
In paraffin IHC, expect CRYAB mainly in the cytoplasm of selected cells, including cardiomyocytes, lens fiber cells and glia (HPA: Enhanced tissue IHC; High in these cells). CRYAB has no transmembrane segment, so a membrane-only IHC pattern needs scrutiny (UniProt P02511: topology). Nuclear localisation can occur during heat shock (UniProt P02511: subcellular location).
| Strong cytoplasmic staining in cardiomyocytes or lens fiber cells, with identifiable cell boundaries. | This matches reported high staining in both cell types (HPA: High in heart cardiomyocytes and eye lens fiber cells). Assess the named cells rather than assigning one intensity to the whole section, because CRYAB expression is reported in a subset of tissues (HPA: tissue IHC profile). |
| Prominent membrane-only or nuclear-only staining replaces the expected cytoplasmic IHC pattern. | Check whether the compartment assignment is reliable before calling the result positive: tissue IHC is described as cytoplasmic (HPA: tissue IHC profile). A nuclear signal is possible during heat shock (UniProt P02511: subcellular location), while ICC-IF also reports plasma membrane localisation (HPA: subcellular). Neither observation alone validates an unexpected paraffin IHC pattern. |
| Strong signal appears in adrenal glandular cells, appendix glandular cells or marrow hematopoietic cells. | These named cell populations are reported as not detected (HPA: tissue IHC). Review whether the signal follows cellular structure; unexplained staining may reflect antibody cross-reactivity or endogenous detection activity (standard IHC practice). A negative reference is specific to the reported cell type, not every cell in that tissue. |
| Uniform colour covers unrelated cells, empty spaces or the section edge. | Treat this as diffuse background until a cell-specific pattern is visible (standard IHC practice). True tissue staining is reported in selected cytoplasmic cell populations (HPA: tissue IHC profile). Compare with a no-primary control and inspect section edges before scoring weak signal (standard IHC practice). |
| No signal appears in an intact heart muscle or eye section. | Check the assay before concluding CRYAB is absent: cardiomyocytes and lens fiber cells are reported as high (HPA: tissue IHC). Confirm that those cells are present, then review antibody dilution, antigen retrieval and detection controls as general IHC workflow checks (standard IHC practice). |
💡Expected CRYAB appearanceA convincing positive is strong, cell-associated cytoplasmic staining in an HPA-high population such as cardiomyocytes or lens fiber cells (HPA: tissue IHC); uniform background or equally strong staining in an HPA-not-detected cell population warrants a false-positive check (HPA: tissue IHC; standard IHC practice).
| Cell type within the section | Compare like cells: glia in caudate, cerebral cortex and hippocampus are high, as are cerebellar Bergmann glia (HPA: tissue IHC). Kidney staining is reported in Bowman's capsule (HPA: tissue IHC); an organ-wide score could hide this distribution. |
| Compartment and biological state | The tissue profile is cytoplasmic (HPA: tissue IHC). CRYAB can enter the nucleus during heat shock and localise to Z-bands and intercalated disks in cardiomyocytes (UniProt P02511: subcellular location). Interpret unusual structure-specific staining in its cellular context. |
| Antibody validation | HPA lists Enhanced IHC validation for HPA057100, CAB002053 and CAB040560 (HPA: antibodies). That supports the reported tissue pattern but does not establish the performance or working dilution of a different catalog antibody. |
| Detection chemistry | Endogenous enzyme activity or nonspecific detection reagents can create colour unrelated to the primary antibody (standard chromogenic IHC practice). Match blocking and a no-primary control to the chosen detection system (standard IHC practice). |
| IF/ICC question: should the same compartment be expected? | HPA reports enhanced plasma membrane and cytosol localisation in ICC-IF, with images from A-431, U-251MG and U2OS (HPA: subcellular). Use that as context for IF interpretation; the tissue IHC profile remains cytoplasmic (HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive cardiomyocytes remain blank. | The assay may have insufficient detectable signal; cardiomyocytes are reported as high (HPA: tissue IHC). | Verify that cardiomyocytes are present and the detection control works. Review the catalog antibody's IHC-P instructions for dilution and retrieval, then adjust those variables within that workflow (standard IHC practice). No CRYAB-specific retrieval condition is supplied here. |
| The whole section is weakly coloured, including spaces without cells. | Diffuse deposition is consistent with background rather than the selected cellular pattern (standard IHC practice; HPA: tissue IHC profile). | Compare a no-primary control, inspect wash quality and review blocking appropriate to the detection chemistry (standard IHC practice). Score CRYAB only where staining resolves to identifiable cells. |
| An HPA-not-detected cell population stains strongly. | Cross-reactivity or endogenous detection activity is possible (standard IHC practice); HPA reports no signal in the specified population (HPA: tissue IHC). | Confirm the exact cell identity, compare a no-primary control and assess a reported high population in the same run (standard IHC practice; HPA: tissue IHC). Do not extend the negative designation to other cells in that organ. |
| A membrane rim dominates the paraffin IHC slide. | The reported tissue IHC pattern is cytoplasmic, although plasma membrane localisation is reported by ICC-IF (HPA: tissue IHC profile; HPA: subcellular). | Check focus and cellular boundaries, then compare cytoplasmic signal in a high-staining reference population (standard IHC practice; HPA: tissue IHC). Record the rim separately if it persists. |
| Nuclear staining dominates without clear cytoplasmic staining. | CRYAB can translocate to the nucleus during heat shock, but that condition cannot be inferred from staining alone (UniProt P02511: subcellular location). | Record the nuclear pattern and sample context. Check for cytoplasmic staining in an HPA-high population and compare controls before interpreting nuclear signal as CRYAB (HPA: tissue IHC; standard IHC practice). |
| Kidney or brain appears mixed when scored as a whole organ. | High staining is assigned to particular cells: Bowman's capsule in kidney and specified glial populations in brain (HPA: tissue IHC). | Score the named cell populations separately and document their compartment and intensity (HPA: tissue IHC; standard IHC practice). Avoid treating unstained neighbouring cells as a failed positive control. |
Experiment evidence
IHC & IF images showing CRYAB staining
A03496-2 · IHCIHC analysis of Alpha B Crystallin/CRYAB using anti-Alpha B Crystallin/CRYAB antibody (A03496-2). Alpha B Crystallin/CRYAB was detected in p
A03496-2 · IHCIHC analysis of Alpha B Crystallin/CRYAB using anti-Alpha B Crystallin/CRYAB antibody (A03496-2). Alpha B Crystallin/CRYAB was detected in p
A03496-2 · IFIF analysis of Alpha B Crystallin/CRYAB using anti-Alpha B Crystallin/CRYAB antibody (A03496-2). Alpha B Crystallin/CRYAB was detected in im
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000109846-CRYAB/tissue/cerebral+cortex#imid_20694232
HPA · EyeHuman Protein Atlas IHC, Eye. Source: https://www.proteinatlas.org/ENSG00000109846-CRYAB/tissue/retina#imid_1338
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000109846-CRYAB/tissue/colon#imid_20696446
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000109846-CRYAB/tissue/liver#imid_20696463
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB002053; green = CRYAB, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB002053; green = CRYAB, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CRYAB IHC & IF
🧪Run cerebellum first: Bergmann glia should stain strongly (HPA: High in Bergmann glia), while nearby non-Bergmann cells should show only background signal if used as an internal background check; their CRYAB-negative status is not established by the supplied HPA row. Run adrenal gland as the negative tissue and assess its glandular cells (HPA: Not detected in adrenal gland glandular cells).
Positive control tissue: Caudate (Glial cells, HPA High)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CRYAB in A-431, U-251MG, U2OS, with annotated localisation: Plasma membrane (enhanced), Cytosol (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a concentration-matched rabbit IgG isotype control; and, if available, CRYAB-knockout tissue as a biological negative (caption: rabbit primary; standard IHC practice). Quench endogenous peroxidase and block endogenous biotin when using the caption’s biotin–streptavidin/DAB detection in cerebellum (caption: SABC and DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A03496-2 paraffin-section caption does not state a fixative (caption: fixative unreported). That rat brain example uses heat-mediated EDTA retrieval at pH 8.0, so retrieval is a documented starting condition, though its necessity for cerebellum is unreported (caption: EDTA pH 8.0; HPA: High in cerebellar Bergmann glia). The supplied evidence does not show frozen sections or IF/ICC to be easier; for this cerebellar IHC design, check background from the caption’s biotin-based detection (caption: SABC; standard IHC practice).
HPA tissue IHC evidence for CRYAB
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 |
|---|---|---|---|---|
| Caudate | Glial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Bergmann glia - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Cerebral cortex | Glial cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
| Eye | Lens fiber cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| 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 → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CRYAB IHC Tips
Use matched paraffin sections and cell-specific controls to troubleshoot CRYAB staining while keeping retrieval, detection, and scoring consistent.
What retrieval should I try first when CRYAB staining is weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A03496-2). Keep heating, cooling, and section thickness consistent across runs so differences in epitope exposure do not mimic biological variation (standard IHC practice). The selected rat brain section received 2 μg/ml primary antibody overnight at 4°C after retrieval, providing a tested starting point (datasheet A03496-2). If staining remains weak, compare adjusted heating times on identically processed positive sections while checking tissue integrity and background (standard IHC practice). Assess nuclear signal separately: CRYAB can enter nuclear speckles during heat shock (UniProt P02511 localisation).
Could fixation explain inconsistent CRYAB staining between paraffin specimens?
The selected paraffin-section caption does not report a fixative, so CRYAB-specific fixation sensitivity in this IHC workflow is unknown (datasheet A03496-2). Record each specimen's fixative, fixation duration, and processing conditions, then compare sections processed identically before changing retrieval or antibody concentration (standard IHC practice). If staining is unexpectedly weak, include a positive section from the same processing batch; heart cardiomyocytes are a supported positive cell population (HPA: High in cardiomyocytes). Compare signal alongside preserved morphology, and document use of EDTA retrieval at pH 8.0 without assigning any observed difference to a CRYAB-specific fixative effect (datasheet A03496-2; standard IHC practice).
Which staining compartments are plausible for CRYAB in tissue sections?
In chromogenic IHC, examine cytoplasmic signal within the relevant cells, including cardiomyocytes and brain glia (HPA: High in cardiomyocytes and glial cells). Cell imaging reports cytosolic and plasma-membrane localisation, while UniProt also lists nuclear, lysosomal, and secreted CRYAB (HPA subcellular; UniProt P02511 localisation). CRYAB has no transmembrane segment, so assess a sharp membrane outline against cell morphology and control staining before interpreting it (UniProt P02511 topology; standard IHC practice). Score cytoplasmic, membrane-edge, and nuclear patterns separately; nuclear speckle localisation has been reported during heat shock (UniProt P02511 localisation; standard IHC practice).
Can epitope accessibility or CRYAB modifications change the IHC pattern?
No CRYAB isoforms are listed in the supplied record, so an isoform-specific explanation requires evidence beyond this guide (UniProt P02511 isoforms). Reported modifications include phosphorylation at residues 19, 45, and 59, and glycosylation sites at 41 and 170 (UniProt P02511 modified residues and glycosylation). The small heat-shock-protein domain spans residues 56–164 (UniProt P02511 domains). The selected caption does not identify the antibody epitope, so those features alone cannot establish whether a modification masks staining (datasheet A03496-2). Compare retrieval conditions and a consistently processed positive control before attributing regional differences to epitope accessibility (standard IHC practice).
How should I investigate CRYAB localisation by multiplex IF?
For IF/ICC, pair CRYAB with a validated marker of the expected cell type, such as a glial marker when examining brain tissue (HPA: High in glial cells; standard IF practice). Choose spectrally separated fluorophores and evaluate a red or far-red channel against tissue autofluorescence using unstained and single-label controls (standard IF practice). Assess mild permeabilisation for an intracellular epitope, since CRYAB is reported in cytoplasm and nucleus and has no transmembrane segment; this antibody's epitope position is unreported (UniProt P02511 localisation and topology; datasheet A03496-2). Optimize IF fixation with matched controls because the selected IHC caption reports no fixative or IF/ICC fixation validation (datasheet A03496-2; standard IF practice).
How can I separate CRYAB signal from chromogenic background?
The selected paraffin IHC example used 10% goat serum blocking, a biotinylated secondary, streptavidin-biotin detection, and DAB (datasheet A03496-2). Include a no-primary control and assess endogenous peroxidase and endogenous biotin where relevant to this detection chemistry (standard IHC practice). Compare any widespread brown deposit with tissue morphology and the no-primary section before increasing primary-antibody concentration (standard IHC practice). The example used 2 μg/ml primary antibody overnight at 4°C, which offers a documented starting concentration and incubation (datasheet A03496-2). Use expected positive cardiomyocytes and cells reported as undetected, such as adrenal glandular cells, to judge staining specificity (HPA: High in cardiomyocytes; Not detected in adrenal glandular cells).
How should I quantify CRYAB staining across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring; CRYAB is reported in cardiomyocytes and glial cells, with cytoplasmic tissue expression (HPA: High in cardiomyocytes and glial cells; HPA tissue IHC profile). For each population, record percent-positive cells and an H-score using intensity grades 0–3, yielding a 0–300 scale (standard IHC practice). If counting positive cells per mm², divide by the measured viable tissue area of that same cell population (standard IHC practice). Normalize comparisons to viable target-cell counts or area, rather than total section size when cell composition differs (standard IHC practice). Apply one staining threshold and review matched controls across batches (standard IHC practice).
When is an unusual CRYAB staining pattern convincing?
Give greatest weight to reproducible signal in morphologically intact expected cells, including brain glia or cardiomyocytes (HPA: High in glial cells and cardiomyocytes; standard IHC practice). Compare suspect staining with a no-primary section and an appropriate negative population, such as adrenal glandular cells reported as undetected (HPA: Not detected in adrenal glandular cells; standard IHC practice). Edge staining, necrotic regions, and endogenous peroxidase activity can produce misleading chromogenic signal, so assess them against tissue morphology and controls (standard IHC practice). Do not dismiss every nuclear or membrane-associated pattern: nuclear translocation during heat shock and membrane localisation are reported (UniProt P02511 localisation; HPA subcellular). Require reproducibility and cell-specific context before assigning biological meaning to those patterns (standard IHC practice).
Boster reagents
Best CRYAB / Alpha-crystallin B chain IHC Antibodies
The IHC-validated antibody has paraffin-section images from rat brain and human cancer tissues, plus an IF image from U20S cells (A03496-2 image captions).
Real IHC data
Anti-Alpha B Crystallin/CRYAB Antibody ®
Cat # A03496-2
A03496-2 has IHC images from paraffin sections of rat brain and human lung, gastric, and skin cancer tissues, plus an IF image from U20S cells (A03496-2 image captions). M03496 lists IHC and ICC/IF applications with human and rat reactivity; no IHC or IF figure is supplied (M03496 catalog payload).
Which to pick: Choose A03496-2 for paraffin-section IHC because its own captions document those samples with 2 μg/ml primary antibody and EDTA retrieval at pH 8.0; the fixative is unreported (A03496-2 IHC image captions). Choose A03496-2 for IF/ICC when an imaged example matters: its U20S-cell IF caption uses 5 μg/ml; M03496 is a rabbit monoclonal listed for IHC and ICC/IF, but has no supplied figures (A03496-2 IF image caption; M03496 catalog payload). For broader listed species coverage, choose A03496-2, which lists human, monkey, mouse, and rat, versus human and rat for M03496; the imaged IHC examples cover human and rat (catalog reactivity; A03496-2 IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P02511 (CRYAB_HUMAN, Alpha-crystallin B chain).
- Human Protein Atlas. CRYAB tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CRYAB subcellular location (ICC-IF): Localized to the plasma membrane and cytosol..
- Human Protein Atlas. CRYAB antibody validation summary (3 antibodies).
- Expression of CRYAB with the angiogenesis and poor prognosis for human gastric cancer. Medicine 2019 — PMC6855521.
- High α B-crystallin and p53 co-expression is associated with poor prognosis in ovarian cancer. Bioscience reports 2019 — PMC6579977.
- Crystallin Alpha-B Overexpression as a Possible Marker of Reactive Astrogliosis in Human Cerebral Contusions. Frontiers in cellular neuroscience 2022 — PMC8963874.
- Cluster analyses of the TCGA and a TMA dataset using the coexpression of HSP27 and CRYAB improves alignment with clinical-pathological parameters of breast cancer and suggests different epichaperome influences for each sHSP. Cell stress & chaperones 2021 — PMC8943080.
- PubMed PMID:838078 — UniProt-cited evidence.
- PubMed PMID:2387586 — UniProt-cited evidence.
- PubMed PMID:1407707 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CRYAB (Alpha-crystallin B chain). Expected localisation Observed cytoplasmic staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03496-2). Validated IHC antibody: A03496-2 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Caudate (Glial cells); negative Adrenal gland. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded rat brain tissue; fixative not specified (datasheet A03496-2); 2. Fixation: Image fixative and duration unreported (datasheet A03496-2); 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: EDTA pH 8.0 (datasheet A03496-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03496-2); 8. Primary antibody: Rabbit anti-CRYAB, 2-5μg/ml (datasheet A03496-2); 9. Primary incubation: Overnight at 4 °C (datasheet A03496-2). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Heat shock can shift CRYAB to nuclear speckles (UniProt); Isoform / epitope: No listed isoforms; one 1–175 chain (UniProt); Regulation: Expression varies by tissue (HPA tissue IHC). Evidence: UniProt P02511, HPA tissue IHC · Enhanced, HPA ICC-IF · plasma membrane and cytosol., Datasheet A03496-2, PMC6579977, PMC8963874. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CRYAB/immunohistochemistry-ihc-guide.