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CALCA / Calcitonin · IHC design guide
Design Immunohistochemistry for CALCA
Plan chromogenic CALCA IHC in paraffin sections using thyroid and bronchial epithelial subsets as positive reference tissue (HPA tissue IHC). This guide covers fixation, controls, and interpretation of a secreted target (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CALCA 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 | Secreted (UniProt); tissue compartment unreported (HPA tissue IHC) | |
| Staining pattern | Thyroid and bronchial epithelial subsets; compartment unclear (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB9936) | |
| Positive control | Bronchus+1 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 paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Secreted protein: RNA and protein locations may differ (HPA tissue IHC) | |
| Regulation | No expression regulator specified (UniProt) | |
| Isoform / epitope | 3 isoforms; check epitopes across processing regions (UniProt) |
Section 1
Recommended CALCA IHC & IF Protocols
The catalog antibody protocol uses EDTA pH 8.0 retrieval (datasheet PB9936). The published CALCA IHC protocol below uses citrate retrieval (PMC5716875).
| Sample | Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet PB9936) |
| Fixation | Image fixative and duration unreported (datasheet PB9936); 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 PB9936); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9936) |
| Primary antibody | Rabbit anti-CALCA, 0.5-1μg/ml recommended; image 2 μg/ml (datasheet PB9936) |
| Primary incubation | Overnight at 4 °C (datasheet PB9936) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet PB9936) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CALCA-positive staining in respiratory epithelial cells of bronchus (HPA tissue IHC: Medium). HPA tissue profile: Expressed in a subset of thyroid epithelial cells and bronchial epithelial neuroendocrine cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet PB9936); consider microwave citrate retrieval when adapting the published protocol (PMC5716875).
Section 2
What Is the Expected CALCA Staining Pattern?
CALCA is a secreted peptide precursor with no transmembrane segment (UniProt P01258: secreted; topology). In paraffin-section IHC, expect selective staining in a subset of thyroid epithelial cells and bronchial epithelial neuroendocrine cells; HPA reports medium staining in thyroid glandular and bronchial respiratory epithelial cells (HPA tissue IHC: Enhanced). HPA does not assign CALCA a precise intracellular compartment (HPA subcellular: main location unavailable).
| A subset of thyroid glandular cells stains at moderate intensity while neighbouring cells remain unstained. | This fits the reported thyroid pattern (HPA tissue IHC: thyroid glandular cells, Medium; subset of thyroid epithelial cells). Score the fraction of stained cells as well as intensity: uniform staining of every glandular cell would be less consistent with the reported subset pattern. HPA does not define a required nuclear, cytoplasmic or granular distribution (HPA subcellular: main location unavailable). |
| Scattered bronchial epithelial cells stain at moderate intensity within a mostly unstained lining. | This fits the reported subset of bronchial epithelial neuroendocrine cells (HPA tissue IHC: bronchus respiratory epithelial cells, Medium; profile). Use cell identity and the selective distribution together when reading the slide. A positive bronchial section need not show continuous staining along the epithelium (HPA tissue IHC: subset pattern). |
| Staining appears mainly as a sharp membrane rim or isolated nuclear signal. | Review it as a compartment mismatch: CALCA is secreted and has no transmembrane segment (UniProt P01258: secreted; topology). Neither source specifies a precise intracellular staining pattern (HPA subcellular: main location unavailable). A membrane rim or nuclear-only result therefore needs further validation rather than a confident CALCA localisation call. |
| Broad staining appears in cells outside the reported positive subsets. | This is less consistent with the selective tissue pattern (HPA tissue IHC: thyroid and bronchus profiles). In chromogenic IHC, cross-reactivity or endogenous detection activity can produce apparent positives (general IHC practice). Check the cell type, negative controls and detection controls before attributing widespread colour to CALCA. |
| The slide shows diffuse colour, or a known-positive thyroid or bronchus section has no signal. | Diffuse colour that does not resolve into the reported cell subsets is background rather than a readable positive pattern (HPA tissue IHC: subset pattern; general IHC practice). An unstained section is inconclusive until a suitable positive control works. HPA reports medium staining, not universal staining of every cell in these tissues (HPA tissue IHC: positive entries). |
💡Expected CALCA appearanceCall the result positive when cell-associated chromogenic signal marks a subset of thyroid glandular or bronchial epithelial cells at approximately medium intensity (HPA tissue IHC: positive entries and profile); diffuse field colour, widespread unrelated-cell staining or nuclear-only signal warrants review (general IHC practice; UniProt P01258: secreted; HPA subcellular: main location unavailable).
| Secreted protein and tissue interpretation (UniProt P01258: secreted). | RNA and protein locations may differ, so do not use tissue RNA enrichment alone to accept or reject a stained section (HPA tissue IHC: reliability description; RNA group enriched in parathyroid and thyroid gland). HPA provides no internal RNA correlation for this staining assessment (HPA tissue IHC: reliability description). |
| Antibody validation (HPA antibodies: HPA064453 IHC Enhanced; HPA059886 IHC Supported). | The validation labels differ by antibody. Record which antibody produced the image when comparing results; the overall tissue reliability is Enhanced, while the two listed antibodies do not carry identical IHC validation labels (HPA tissue IHC: reliability; HPA antibodies). |
| Precursor processing and alternative isoforms (UniProt P01258: signal 1–25, propeptide 26–82; isoforms 1, 2 and 3). | The record does not locate the antibody epitope or show which processed form or isoform it detects (UniProt P01258: processing and isoforms; HPA antibodies: validation summary). Interpret staining as antibody reactivity to CALCA-related antigen without assigning a particular molecular form from the slide alone. |
| Subcellular resolution (HPA subcellular: Secreted; main location unavailable; no ICC-IF images). | A specific intracellular organelle or punctate pattern is not established by the supplied sources. Use the documented tissue and cell distribution as the primary IHC expectation (HPA tissue IHC: thyroid and bronchus profile), and avoid treating a proposed compartment as an independent pass criterion. |
| Situation | Likely cause | Next action |
|---|---|---|
| A thyroid section is entirely unstained, including the expected positive subset. | A failed IHC run or weak detection is possible (general IHC practice); the reported positive population is only a subset and has medium staining (HPA tissue IHC: thyroid). | Check that a separate known-positive control develops and that the counterstain does not obscure weak chromogen. Review antibody incubation, antigen retrieval and detection settings as general IHC variables; the sources provide no CALCA-specific retrieval condition or fixation-sensitivity claim. |
| Bronchial staining is continuous across most epithelial cells. | That distribution exceeds the reported neuroendocrine subset (HPA tissue IHC: bronchus profile). Cross-reactivity or excessive detection may broaden apparent staining (general IHC practice). | Compare the cell distribution with the positive control, reduce nonspecific detection through routine IHC optimisation, and inspect a negative control. Do not score a continuous epithelial sheet as matching the HPA subset solely because the tissue is bronchus. |
| Colour appears in expected-negative cells or across the entire section. | HPA reports several cell populations as not detected, including adipocytes in adipose tissue and glandular cells in adrenal gland (HPA tissue IHC: negative entries). Endogenous enzyme activity or nonspecific binding can add chromogenic colour (general IHC practice). | Inspect a no-primary detection control and use an appropriate endogenous-activity block for the detection chemistry (general IHC practice). Compare tissue and cell identity with the HPA negative entries before calling cross-reactivity; a negative entry applies to the listed cell population. |
| Only nuclear staining or a crisp cell-edge rim is visible. | Neither distribution is established as a CALCA pattern; the protein is secreted without a transmembrane segment (UniProt P01258: localisation and topology; HPA subcellular: main location unavailable). | Check morphology, background and detection controls, then repeat with an independently validated antibody if available (general IHC practice). Report the compartment mismatch rather than reclassifying the pattern as confirmed CALCA. |
| Diffuse haze prevents scoring the expected positive subset. | Background may arise from nonspecific antibody binding, incomplete blocking or detection chemistry (general IHC practice). The HPA pattern requires a discernible subset of cells (HPA tissue IHC: profile). | Compare no-primary and positive-control sections, then optimise blocking, washes and antibody concentration as general IHC steps. Reassess whether distinct thyroid or bronchial cells remain stained above the local background (HPA tissue IHC: positive entries). |
| What localisation should an IF/ICC image show? | A precise IF/ICC compartment cannot be inferred: HPA lists CALCA as secreted but provides no main location or cell-line ICC-IF images (HPA subcellular: summary and image list). | Use the separate IF/ICC guide for that application. Here, interpret the paraffin-section chromogenic IHC result by the documented thyroid and bronchial cell subsets (HPA tissue IHC: profile), without assigning an IF/ICC-specific pattern. |
Experiment evidence
IHC & IF images showing CALCA staining
PB9936 · IHCIHC analysis of CGRP using anti-CGRP antibody (PB9936). CGRP was detected in a paraffin-embedded section of human lung cancer tissue. Heat m
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000110680-CALCA/tissue/cerebral+cortex#imid_20608491
HPA · Thyroid glandHuman Protein Atlas IHC, Thyroid gland. Source: https://www.proteinatlas.org/ENSG00000110680-CALCA/tissue/thyroid+gland#imid_1591
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000110680-CALCA/tissue/colon#imid_20608409
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000110680-CALCA/tissue/liver#imid_20608410
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CALCA IHC & IF
🧪Run thyroid gland first: its glandular cells should stain (HPA: Medium in thyroid glandular cells). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); neighboring nonglandular cells on the thyroid slide should remain at background as an internal check.
Positive control tissue: Bronchus (Respiratory epithelial cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for CALCA; derive a cell-line control from the positive tissue's cell type (Respiratory epithelial cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control, an isotype-matched rabbit IgG control (selected-SKU caption: rabbit primary antibody), and a validated CALCA-knockout specimen as a biological negative. Quench endogenous peroxidase in thyroid sections before HRP/DAB detection (standard IHC practice; selected-SKU caption: HRP/DAB detection).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported in the supplied evidence, and the exact selected-SKU paraffin-section caption does not report a fixative (selected-SKU caption: fixative unreported). Heat retrieval with EDTA at pH 8.0 was used, but retrieval dependence was not established (selected-SKU caption: EDTA retrieval); thyroid endogenous peroxidase may produce DAB background (standard IHC practice). Frozen sections or IF cannot be judged easier from these data; HPA lists no ICC-IF cell-line images (HPA: no ICC-IF cell-line images).
HPA tissue IHC evidence for CALCA
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Secreted protein, tissue location of RNA and protein is expected to differ. 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 |
|---|---|---|---|---|
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Thyroid gland | Glandular 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 CALCA IHC Tips
Troubleshooting CALCA staining in paraffin-section IHC, with one entry on multiplex immunofluorescence.
Which retrieval should I use when CALCA staining is weak in paraffin sections?
Start with heat-mediated EDTA retrieval at pH 8.0 for paraffin sections (datasheet PB9936). The illustrated section used 2 μg/ml primary antibody overnight at 4°C after retrieval, so assess that tested combination before changing conditions (datasheet PB9936). If staining remains weak, compare citrate buffer at pH 6.0 on adjacent sections as an empirical fallback, while keeping detection and development time constant (general IHC practice). Include a known positive thyroid or bronchial section and a primary-omission control; focal staining is plausible because CALCA protein occurs in subsets of epithelial cells (HPA tissue IHC).
How should I handle fixation when CALCA signal varies between paraffin blocks?
The selected paraffin-section caption does not state a fixative, and target-specific fixation sensitivity is unknown (datasheet PB9936). Record fixative, fixation duration, tissue thickness, and time to fixation for each block, then compare sections processed under matched conditions (general IHC practice). If material permits, evaluate a fixation-duration series with identical pH 8.0 EDTA retrieval and 2 μg/ml primary antibody; score signal and morphology together (datasheet PB9936; general IHC practice). Avoid assigning weak staining to fixation alone: low abundance or sampling can also matter when expression is limited to cell subsets (HPA tissue IHC).
Where should convincing CALCA staining appear in a tissue section?
Expect signal in a subset of thyroid epithelial cells or bronchial epithelial neuroendocrine cells, with a cellular secretory pattern rather than membrane outlining (HPA tissue IHC; UniProt P01258 secreted, no transmembrane segment). Compare stained cells with morphology and nearby unstained cells on the same section before calling diffuse extracellular color positive (general IHC practice). The HPA reports medium staining in thyroid glandular cells and bronchial respiratory epithelial cells, while its profile specifies narrower cell subsets (HPA tissue IHC). Because secreted protein can leave the producing cell, absent overlap with local RNA would not by itself invalidate a cellular IHC signal (HPA tissue IHC; UniProt P01258 secreted).
Can this stain distinguish calcitonin from other CALCA products or isoforms?
Do not interpret this image as selective mature calcitonin staining: its caption calls the antibody anti-CGRP, while CALCA has 3 annotated isoforms (datasheet PB9936; UniProt P01258 isoforms). The precursor has a signal peptide at residues 1–25 and a propeptide at 26–82, and is secreted after processing (UniProt P01258 processing, subcellular). Ask for the antibody immunogen or mapped epitope and verify which processed product it recognizes before assigning a peptide identity (general IHC practice). Use independently validated peptide-specific reagents or orthogonal expression data when a distinction between CALCA-derived products is essential (general IHC practice).
How can I adapt CALCA detection to multiplex immunofluorescence?
Treat IF as a separate assay requiring titration and controls; the caption documents paraffin-section chromogenic IHC, not IF performance (datasheet PB9936). Multiplex with a validated marker of thyroid epithelial or bronchial neuroendocrine cells, and inspect whether CALCA signal lies in the expected cellular subset (HPA tissue IHC; general IF practice). Choose a fluorophore and imaging channel after measuring tissue autofluorescence with an unstained section, favoring a spectral region with cleaner background (general IF practice). For an intracellular precursor or stored secretory peptide, test mild permeabilisation; for extracellular deposited antigen, compare without permeabilisation, since CALCA is secreted and has no transmembrane segment (UniProt P01258 topology, subcellular; general IF practice).
What should I change when DAB signal spreads beyond expected cells?
First inspect a primary-omission section and an unstained section for endogenous peroxidase, pigment, and nonspecific DAB deposition (general IHC practice). Apply a peroxidase block and matched serum or protein blocking, then titrate the primary below the demonstrated 2 μg/ml if diffuse staining persists (datasheet PB9936; general IHC practice). The illustrated protocol used 10% goat serum, an overnight primary incubation at 4°C, and a peroxidase-linked secondary before DAB development (datasheet PB9936). Because CALCA is secreted, extracellular staining requires morphology and control-based assessment rather than automatic rejection, especially near expected positive epithelial subsets (UniProt P01258 secreted; HPA tissue IHC).
How should I quantify heterogeneous CALCA staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define a region of interest and score morphologically identified thyroid epithelial or bronchial neuroendocrine cells rather than averaging the entire section (HPA tissue IHC; general IHC practice). Report the percentage of positive target cells and an H-score using intensity categories 0–3; alternatively count positive cells per mm² when cell density is the biological question (general IHC practice). Normalize counts to the number of eligible cells or sampled tissue area, and keep threshold, illumination, retrieval, and DAB development consistent across cases (general IHC practice). Record glandular or epithelial compartment and exclude folds, necrosis, and section edges before scoring (general IHC practice).
How do I distinguish true CALCA positivity from artefact in tissue IHC?
Favor staining in morphologically appropriate thyroid epithelial or bronchial neuroendocrine subsets, consistent with the reported tissue profile (HPA tissue IHC). Be cautious with isolated nuclear color or sharp membrane outlining, because CALCA is secreted and lacks a transmembrane segment (UniProt P01258 subcellular, topology). Exclude edge staining, necrotic zones, folds, pigment, and endogenous peroxidase by reviewing morphology, a primary-omission control, and the peroxidase-block result (general IHC practice). Compare a positive thyroid or bronchial section with a negative adipose or adrenal section, but interpret discordance in light of the antibody's CGRP label and unestablished peptide-level epitope specificity (HPA tissue IHC; datasheet PB9936).
Boster reagents
Best CALCA / Calcitonin IHC Antibodies
PB9936 has a human paraffin-section IHC image (PB9936 IHC image caption); M02352-6 lists IHC and IF/ICC for human, mouse and rat (catalog applications/reactivity).
Real IHC data
Anti-Calcitonin/CALCA Antibody
Cat # PB9936
PB9936 is the rendered card, with IHC imagery from a human lung cancer paraffin section (PB9936 IHC image caption). M02352-6 lists human, mouse and rat IHC/IF/ICC applications, but has no IHC or IF image in the payload (catalog applications/reactivity/image alts).
Which to pick: Choose PB9936 for human paraffin-section chromogenic IHC, supported by its DAB-stained image; the caption does not report the fixative (PB9936 IHC image caption). For IF/ICC or mouse and rat samples, consider rabbit monoclonal M02352-6 based on its listed applications and reactivity (catalog host/clone/applications/reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P01258 (CALC_HUMAN, Calcitonin).
- Human Protein Atlas. CALCA tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CALCA subcellular location (ICC-IF): Secreted.
- Human Protein Atlas. CALCA antibody validation summary (2 antibodies).
- TRP53 Mutants Drive Neuroendocrine Lung Cancer Through Loss-of-Function Mechanisms with Gain-of-Function Effects on Chemotherapy Response. Molecular cancer therapeutics 2017 — PMC5716875.
- Synergistic Anti-Tumor Effects of Sulfatinib and Kaempferol on Pancreatic Neuroendocrine Tumors via CALCA-mediated PI3K/AKT/mTOR Pathway. International journal of biological sciences 2025 — PMC12631220.
- Methylation of CALCA and CALCB in Pancreatic Ductal Adenocarcinoma. Oxidative medicine and cellular longevity 2021 — PMC8357496.
- The role of the transient receptor potential ankyrin type-1 (TRPA1) channel in migraine pain: evaluation in an animal model. The journal of headache and pain 2017 — PMC5589714.
- PubMed PMID:6546550 — UniProt-cited evidence.
- PubMed PMID:3872459 — UniProt-cited evidence.
- PubMed PMID:3034287 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CALCA (Calcitonin). Expected localisation Secreted (UniProt); tissue compartment unreported (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB9936). Validated IHC antibody: PB9936 at 0.5-1μg/ml recommended; image 2 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bronchus (Respiratory epithelial cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissue; fixative not specified (datasheet PB9936); 2. Fixation: Image fixative and duration unreported (datasheet PB9936); 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 PB9936); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9936); 8. Primary antibody: Rabbit anti-CALCA, 0.5-1μg/ml recommended; image 2 μg/ml (datasheet PB9936); 9. Primary incubation: Overnight at 4 °C (datasheet PB9936). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Secreted protein: RNA and protein locations may differ (HPA tissue IHC); Isoform / epitope: 3 isoforms; check epitopes across processing regions (UniProt); Regulation: No expression regulator specified (UniProt). Evidence: UniProt P01258, HPA tissue IHC · Enhanced, HPA ICC-IF · Secreted, Datasheet PB9936, PMC5716875. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CALCA/immunohistochemistry-ihc-guide.