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CALB2 / Calretinin · IHC design guide
Design Immunohistochemistry for CALB2
Plan chromogenic CALB2 IHC in paraffin sections using the IHC-validated antibody (datasheet A04255). The guide uses cytoplasmic tissue staining and documented positive cell populations to help select controls and interpret results (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CALB2 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 staining in tissue IHC (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in brain, adipose tissue and Leydig cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A04255) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Appendix+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 | Brain staining varies by region and cell type (HPA tissue IHC) | |
| Regulation | RNA is tissue enhanced in adipose tissue and brain (HPA tissue RNA) | |
| Isoform / epitope | No annotated isoforms; one 1–271 chain (UniProt) |
Section 1
Recommended CALB2 IHC & IF Protocols
The catalog antibody protocol uses EDTA pH 8.0 heat retrieval (datasheet A04255). One published IHC protocol stains Japanese flounder testis and ovary (PMC7552167).
| Sample | Paraffin-embedded human rectal cancer tissue; fixative not specified (datasheet A04255) |
| Fixation | Image fixative and duration unreported (datasheet A04255); 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 A04255); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A04255) |
| Primary antibody | Rabbit anti-CALB2, 0.5-1μg/ml (datasheet A04255) |
| Primary incubation | Overnight at 4 °C (datasheet A04255) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A04255) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CALB2-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues and cells, including brain, adipose tissue, fallopian tube, retina, endocrine pancreas and Leydig cells. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval (datasheet A04255); the published room-temperature antigen repair lasts 10 min but does not specify a reagent (PMC7552167).
Section 2
What Is the Expected CALB2 Staining Pattern?
CALB2 should appear chiefly as cytoplasmic staining in selected cells, including cortical and caudate neurons, cerebellar GLUC cells, and testicular Leydig cells (HPA: tissue IHC, Enhanced). Staining may extend into neuronal projections (HPA: hypothalamus, High; UniProt P22676: dendrites and synapse). CALB2 has no transmembrane segment (UniProt P22676: topology). Assess the stained cell type as well as its compartment.
| Distinct cytoplasmic staining in selected cortical or caudate neurons, with surrounding cells less stained. | This fits the reported high neuronal staining in those regions (HPA: cerebral cortex and caudate, High). Score the identified cells and their cytoplasm; a uniformly stained field would need a separate explanation. |
| Staining follows neuronal projections as well as cell bodies. | Projection staining can fit the expected pattern (HPA: hypothalamic neuronal projections, High; UniProt P22676: dendrites and synapse). Check that the signal tracks recognizable structures rather than coating the whole section. |
| Prominent nuclear staining with little cytoplasmic signal. | Question the compartment assignment: HPA describes tissue expression as cytoplasmic, and UniProt places CALB2 at dendrites and synapses (HPA: tissue IHC profile; UniProt P22676: subcellular location). Review counterstain and controls before scoring nuclei as positive. |
| Strong staining in appendix glandular cells or heart cardiomyocytes. | These cells are reported as not detected (HPA: appendix glandular cells and heart cardiomyocytes). Unexpected staining warrants checks for antibody cross-reactivity, endogenous detection activity, and whether the apparent signal belongs to another cell type. |
| Diffuse colour across cells and tissue spaces, or no signal in a known-positive region. | Diffuse colour lacks the cell-restricted pattern reported for CALB2 (HPA: tissue IHC profile). A blank positive control conflicts with reported high staining (HPA: cerebral cortex neurons, High); check the staining run before interpreting the study section. |
💡Expected CALB2 appearanceCall a result positive when selected cells show distinct cytoplasmic staining, potentially extending into neuronal projections, with strong signal in an HPA high-staining cell type; uniform background or dominant nuclear colour is suspect (HPA: tissue IHC profile and High neuronal projections; UniProt P22676: dendrites and synapse).
| Compartment and tissue context | Expect cytoplasmic signal in selected cells (HPA: tissue IHC profile). Projection staining has specific support in hypothalamus (HPA: neuronal projections, High); do not require every positive tissue to show the same structure. |
| Choice of comparison cells | Cortical neurons and Leydig cells are reported High, while appendix glandular cells are not detected (HPA: tissue IHC). Compare the specified cells within each section; a tissue label alone does not define every cell as positive. |
| Antibody evidence | HPA grades tissue staining Enhanced and lists HPA007305, HPA007306, and CAB000106 as IHC Enhanced (HPA: tissue IHC reliability; HPA: antibody validation). This supports pattern comparison but does not validate an unlisted antibody. |
| Retrieval and epitope information | No CALB2-specific retrieval condition, epitope location, or fixation sensitivity is supplied (UniProt P22676 and HPA: supplied records). Treat retrieval changes as general IHC optimization; judge them against positive and negative cell controls. |
| IF/ICC interpretation | For IF/ICC, is cytosolic signal plausible? Yes: HPA reports supported cytosolic localization (HPA: subcellular ICC-IF). That observation can inform compartment interpretation here, but it supplies no IHC retrieval or detection settings. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive cortical neurons are blank. | The result conflicts with reported High staining (HPA: cerebral cortex neuronal cells); the supplied sources do not identify a CALB2-specific fixation cause. | Confirm that the expected cells are present, then check the IHC run's positive control, primary antibody step, retrieval record, and detection reagents as general workflow checks. |
| Colour appears mainly over nuclei. | The compartment differs from the reported cytoplasmic tissue pattern and supported ICC-IF cytosol (HPA: tissue IHC profile; HPA: subcellular ICC-IF). | Compare the stain with the counterstain and a reagent control; score only signal whose compartment and cell identity can be resolved. |
| Appendix glands stain strongly. | That cell type is reported as not detected (HPA: appendix glandular cells); cross-reactivity or endogenous detection activity are possibilities, not established causes. | Inspect a control omitting primary antibody to assess detection background, then compare the cell-specific pattern with a known-positive section. |
| The entire section has a diffuse brown haze. | The haze obscures the selective cytoplasmic pattern (HPA: tissue IHC profile); nonspecific reagent binding or detection background may contribute. | Use a reagent control, review blocking and washing, and adjust antibody concentration only against a positive control; these are general IHC checks. |
| A testis section appears negative despite a working neuronal control. | The reported high testis signal belongs specifically to Leydig cells (HPA: testis Leydig cells, High), so the chosen field or cell identification may matter. | Locate and assess Leydig cells before calling the tissue negative; compare their signal with nearby cells and the control section. |
| An IF/ICC image looks more diffuse than the IHC section. | HPA supports cytosolic ICC-IF localization, while tissue IHC describes cytoplasmic expression in selected cells (HPA: subcellular ICC-IF; HPA: tissue IHC profile). | Compare cell identity and cytosolic localization; use the separate IF/ICC guide for its workflow rather than transferring IF settings to IHC. |
Experiment evidence
IHC & IF images showing CALB2 staining
A04255 · IHCIHC analysis of Calretinin/CALB2 using anti-Calretinin/CALB2 antibody (A04255). Calretinin/CALB2 was detected in paraffin-embedded section o
A04255 · IHCIHC analysis of Calretinin/CALB2 using anti-Calretinin/CALB2 antibody (A04255). Calretinin/CALB2 was detected in paraffin-embedded section o
A04255 · IFIF analysis of Calretinin/CALB2 using anti-Calretinin/CALB2 antibody (A00346). Calretinin/CALB2 was detected in immunocytochemical section o
PA1015 · IHCIHC analysis of Calretinin/CALB2 using anti-Calretinin/CALB2 antibody (PA1015). Calretinin/CALB2 was detected in a paraffin-embedded section
PA1015 · IHCIHC analysis of Calretinin/CALB2 using anti-Calretinin/CALB2 antibody (PA1015). Calretinin/CALB2 was detected in a paraffin-embedded section
PA1015 · IFIF analysis of Calretinin/CALB2 using anti-Calretinin/CALB2 antibody (PA1015). Calretinin/CALB2 was detected in a paraffin-embedded section
PB9248 · IHCAnti-Calretinin vendor antibody, PB9248, IHC(P) IHC(P): Mouse Brain Tissue
PB9248 · IHCAnti-Calretinin vendor antibody, PB9248, IHC(P) IHC(P): Rat Brain Tissue
HPA · HypothalamusHuman Protein Atlas IHC, Hypothalamus. Source: https://www.proteinatlas.org/ENSG00000172137-CALB2/tissue/hypothalamus#imid_4374
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000172137-CALB2/tissue/cerebral+cortex#imid_2065368
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000172137-CALB2/tissue/colon#imid_2065294
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000172137-CALB2/tissue/liver#imid_2065295
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA007305; green = CALB2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA007305; green = CALB2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CALB2 IHC & IF
🧪Run caudate first and score neuronal cells, which HPA rates High for CALB2 (HPA: caudate neuronal cells, High). Use appendix glandular cells as the negative tissue (HPA: appendix glandular cells, Not detected); on the caudate slide, compare neuronal staining with adjacent cells lacking neuronal morphology as an internal background check.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Appendix (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CALB2 in A-431, AF22, U2OS, U-251MG, NIH 3T3, with annotated localisation: Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a host- and class-matched nonimmune rabbit IgG isotype control (caption: rabbit primary antibody). Use CALB2 knockout material as a biological negative where available; for chromogenic detection, control endogenous peroxidase and biotin because the selected caption uses a streptavidin–biotin complex with DAB (A04255 tissue-IHC caption).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the fixative for the selected paraffin-section caption is unreported (A04255 tissue-IHC caption). That caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required (A04255 tissue-IHC caption). No matched evidence establishes that frozen sections or IF are easier; assess neuronal lipofuscin autofluorescence if using IF on caudate (HPA: caudate neuronal cells, High; standard IF practice).
HPA tissue IHC evidence for CALB2
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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | GLUC cells - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | Basal cells | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CALB2 IHC Tips
Troubleshoot CALB2 staining in paraffin sections by checking retrieval, cell type, intracellular pattern, and controls before comparing chromogenic signal across samples.
What retrieval should I try first when CALB2 staining is weak?
Use heat-mediated retrieval in EDTA at pH 8.0 first (datasheet A04255). The selected paraffin-section example used this retrieval before staining with 1 μg/ml antibody overnight at 4°C, so keep those conditions consistent while assessing retrieval (datasheet A04255). Include a section with expected neuronal staining and compare it with a no-primary control on the same run (HPA: High in caudate neuronal cells; standard IHC practice). If signal remains weak, test another retrieval buffer or heating duration on adjacent sections, recording tissue damage and background alongside signal (standard IHC practice).
Could fixation explain absent or uneven CALB2 staining?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative, so its staining cannot establish an optimal fixation condition (datasheet A04255). Record fixative, fixation duration, section thickness, and storage history for each block before comparing staining outcomes (standard IHC practice). Examine adjacent sections for poorly preserved morphology or uneven staining, then repeat staining with matched processing and the documented EDTA pH 8.0 retrieval (standard IHC practice; datasheet A04255). Include a tissue area expected to stain and a no-primary control to distinguish a processing problem from widespread detection background (HPA: High in caudate neuronal cells; standard IHC practice).
Which staining pattern is credible for CALB2 in tissue sections?
Prioritise cellular cytoplasmic staining and assess processes where morphology permits: CALB2 is supported in the cytosol, with synaptic and dendritic localisation also annotated (HPA subcellular: Cytosol supported; UniProt P22676 localisation). Its lack of a transmembrane segment makes a sharp, continuous plasma-membrane-only pattern questionable (UniProt P22676 topology). Check a neuronal area and, when available, peripheral nerve or neuronal projections as spatial references rather than scoring diffuse tissue pigment as positive cells (HPA: High in caudate neuronal cells, soft-tissue peripheral nerve, and hypothalamic neuronal projections; standard IHC practice). Compare the candidate pattern with a no-primary section and preserved cell morphology before accepting unusual nuclear or extracellular signal (standard IHC practice).
How should I troubleshoot an unexpected CALB2 epitope pattern?
The supplied record lists one CALB2 chain spanning residues 1–271 and no annotated isoforms; it does not identify this antibody’s epitope (UniProt P22676 processing and isoforms; datasheet A04255). CALB2 contains six EF-hand regions and an annotated phosphotyrosine at residue 214, but neither establishes how this antibody reacts after retrieval (UniProt P22676 domains and modified residues). Keep antibody concentration, section processing, and EDTA pH 8.0 retrieval constant while comparing staining across adjacent sections (datasheet A04255; standard IHC practice). If the pattern remains discordant, use an independently characterised CALB2 antibody or orthogonal expression evidence and verify that the candidate signal occurs in expected cells (standard IHC practice; HPA: Enhanced tissue-IHC reliability).
How can I check CALB2 by IF alongside chromogenic IHC?
Treat IF as a separate assay and establish its controls and imaging settings independently of the paraffin-section chromogenic example (datasheet A04255; standard IF practice). Multiplex CALB2 with a marker identifying the expected cell type, such as a neuronal marker when examining neuronal tissue, and compare cellular overlap rather than fluorescence intensity alone (HPA: High in caudate neuronal cells; standard IF practice). Choose fluorophores and filter sets after checking unstained tissue for autofluorescence, favouring a spectrally separated channel when background is strong (standard IF practice). Because CALB2 is cytosolic and has no transmembrane segment, optimise permeabilisation for intracellular access while monitoring tissue morphology and no-primary background (HPA subcellular: Cytosol supported; UniProt P22676 topology; standard IF practice).
What should I change if DAB background obscures CALB2-positive cells?
Run a no-primary section to identify detection-system staining, and inspect unstained tissue for native pigment before adjusting the primary antibody (standard IHC practice). The selected example used 10% goat serum, a biotinylated secondary, streptavidin–biotin detection, and DAB, so evaluate blocking and reagent-specific background within that workflow (datasheet A04255). Include an endogenous-peroxidase block and, if background persists with biotin-based detection, assess an appropriate biotin-blocking or alternative detection workflow (standard IHC practice). Shorten chromogen development or adjust antibody concentration only after controls identify the background source, then confirm that expected cytoplasmic cells remain visible (standard IHC practice; HPA subcellular: Cytosol supported).
How should I quantify CALB2 staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue compartment and cell population before scoring, because CALB2 staining is reported in several distinct cell types and tissues (HPA tissue IHC: expression profile). For cellular staining, report the percentage of positive cells and an intensity-based H-score using a fixed positivity threshold; for sparse neuronal profiles, consider positive-cell density per mm² (standard IHC practice). Normalise counts to the assessed area or the number of eligible cells, and apply the same section selection, counterstain, and imaging conditions throughout (standard IHC practice). Record cytoplasmic staining separately from process-associated signal so differences in tissue architecture do not masquerade as changes in cell positivity (HPA subcellular: Cytosol supported; UniProt P22676 localisation; standard IHC practice).
How do I distinguish true CALB2 staining from tissue artefact?
Accept staining when it follows intact cell morphology and a plausible cytoplasmic or neuronal-process pattern, supported by an appropriate positive area (HPA subcellular: Cytosol supported; UniProt P22676 localisation; HPA: High in caudate neuronal cells). Scrutinise isolated nuclear, extracellular, or membrane-only signal, since those patterns do not match the supplied cytosolic localisation or lack of a transmembrane segment (HPA subcellular: Cytosol supported; UniProt P22676 topology). Check section edges and necrotic regions for nonspecific accumulation, and use no-primary and peroxidase controls to assess detection artefact (standard IHC practice). Interpret a negative cell population against its tissue context; expression differs by cell type, and the selected rectal-cancer image alone does not define a universal positivity threshold (HPA tissue IHC: expression profile; datasheet A04255).
Boster reagents
Best CALB2 / Calretinin IHC Antibodies
CALB2 IHC images show paraffin sections of human rectal cancer and brain, plus mouse and rat brain (A04255/PA1015 image captions); IF is shown in mouse brain (PA1015 IF caption).
Real IHC data
Anti-Calretinin/CALB2 Antibody ®
Cat # A04255
Real IHC data
Anti-Calretinin/CALB2 Antibody ®
Cat # PA1015
Real IHC data
Anti-Calretinin/CALB2 Antibody ®
Cat # PB9248
A04255 and PA1015 each have IHC images from human rectal cancer and mouse and rat brain paraffin sections; A04255 also has a human brain IHC image, while PA1015 has a mouse brain IF image (A04255/PA1015 image captions). PB9248 has IHC images labeled mouse brain, rat brain, and human intestinal cancer (PB9248 image captions).
Which to pick: For tissue IHC, start with PA1015: its paraffin section caption documents EDTA retrieval at pH 8.0 and 1 μg/ml primary antibody (PA1015 IHC caption); the fixative is unreported (PA1015 IHC caption). For IF, PA1015 has a mouse brain image; for ICC, A04255 lists that application, but its supplied IF caption identifies a different SKU, so that image does not verify A04255 (PA1015 IF caption; A04255 applications and IF caption). For cross-species tissue IHC, A04255 has paraffin section images from human, mouse, and rat samples, with fixatives unreported (A04255 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P22676 (CALB2_HUMAN, Calretinin).
- Human Protein Atlas. CALB2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CALB2 subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. CALB2 antibody validation summary (3 antibodies).
- Characterization and Localization of Calb2 in Both the Testis and Ovary of the Japanese Flounder (Paralichthys olivaceus). Animals : an open access journal from MDPI 2020 — PMC7552167.
- Hypertrophy of the Anterior External Arcuate Fasciculus: A Rare Variant With Implications for the Development of the Arcuate Nucleus. Frontiers in neuroanatomy 2020 — PMC7729007.
- Unraveling Spermatogenesis in Molly Fish (Poecilia sphenops): An Integrative Study of Testicular Ultrastructure and Immunohistochemistry. Veterinary sciences 2025 — PMC12567713.
- RNA-programmable cell type monitoring and manipulation in the human cortex with CellREADR. bioRxiv : the preprint server for biology 2025 — PMC11642864.
- PubMed PMID:2618861 — UniProt-cited evidence.
- PubMed PMID:2001709 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CALB2 (Calretinin). Expected localisation Cytoplasmic staining in tissue IHC (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A04255). Validated IHC antibody: A04255 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Appendix. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human rectal cancer tissue; fixative not specified (datasheet A04255); 2. Fixation: Image fixative and duration unreported (datasheet A04255); 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 A04255); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A04255); 8. Primary antibody: Rabbit anti-CALB2, 0.5-1μg/ml (datasheet A04255); 9. Primary incubation: Overnight at 4 °C (datasheet A04255). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Brain staining varies by region and cell type (HPA tissue IHC); Isoform / epitope: No annotated isoforms; one 1–271 chain (UniProt); Regulation: RNA is tissue enhanced in adipose tissue and brain (HPA tissue RNA). Evidence: UniProt P22676, HPA tissue IHC · Enhanced, HPA ICC-IF · cytosol., Datasheet A04255, PMC7552167. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CALB2/immunohistochemistry-ihc-guide.