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CNN2 / Calponin-2 · IHC design guide
Design Immunohistochemistry for CNN2
Plan CNN2 staining in paraffin sections using the catalog antibody’s documented tonsil IHC conditions (datasheet A08680-2). Compare the result with cytoplasmic immune-cell staining and medium staining in tonsil germinal center cells (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CNN2 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 immune cells (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic immune cells, including germinal center cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A08680-2) | |
| Positive control | Lung+2 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep paraffin-section fixation consistent. (standard IHC practice; not target-specific) | |
| Caveat | Immune-cell staining may complicate interpretation (HPA tissue IHC) | |
| Regulation | No expression regulator specified (UniProt) | |
| Isoform / epitope | 2 isoforms; antibody epitope coverage is unspecified (UniProt) |
Section 1
Recommended CNN2 IHC & IF Protocols
The catalog antibody uses EDTA retrieval (datasheet A08680-2); one published CNN2 IHC protocol uses citrate retrieval (PMC10298237).
| Sample | Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A08680-2) |
| Fixation | Image fixative and duration unreported (datasheet A08680-2); 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 A08680-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A08680-2) |
| Primary antibody | Rabbit anti-CNN2, 2-5 μg/ml (datasheet A08680-2) |
| Primary incubation | Overnight at 4 °C (datasheet A08680-2) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A08680-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CNN2-positive staining in alveolar cells of lung (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in immune cells. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A08680-2). Citrate retrieval is a published alternative (PMC10298237).
Section 2
What Is the Expected CNN2 Staining Pattern?
CNN2 is reported as cytoplasmic in immune cells by tissue IHC, with medium staining in lung alveolar cells and germinal center cells of lymph node and tonsil (HPA tissue IHC). ICC-IF places it mainly on actin filaments (HPA subcellular). It has no annotated subcellular location or transmembrane segment in UniProt (UniProt Q99439). The tissue IHC profile is Approved but pending external verification (HPA tissue IHC).
| Cytoplasmic staining in germinal center cells, or in the reported lung alveolar cells, at about medium intensity (HPA tissue IHC). | This matches the reported cell types and intensity (HPA tissue IHC). Score the named cells separately from surrounding tissue; a positive cell population does not make every cell in that section positive. |
| Predominantly nuclear, surface-restricted, or extracellular signal in place of cytoplasmic signal (HPA tissue IHC: cytoplasmic profile). | Treat this as a localization mismatch requiring review, rather than confirmed CNN2. Check morphology, the negative control, and whether detection signal follows tissue edges or deposits (general IHC practice). |
| Signal in adipocytes or bronchial respiratory epithelial cells, both reported as not detected (HPA tissue IHC). | Consider cross-reactivity or endogenous detection activity, especially if a control also stains (general IHC practice). These HPA calls concern the specified cell types; they do not establish that the whole tissue is negative. |
| Weak color spread across many structures, with little separation between reported positive and negative cells (HPA tissue IHC). | This is difficult to score as cell-specific CNN2. Review blocking, washing, detection reagent, and counterstain balance using matched controls (general IHC practice); HPA does not identify a CNN2-specific cause. |
| No signal in the reported medium germinal center cells of lymph node or tonsil (HPA tissue IHC). | The run lacks its expected reference pattern. Check the section and staining controls, antibody dilution, retrieval and detection steps (general IHC practice) before interpreting other cells as negative. |
💡Expected CNN2 appearanceA convincing positive is cytoplasmic staining of the specified lung alveolar or germinal center cells at roughly medium intensity (HPA tissue IHC); isolated nuclear, extracellular, or broadly cell-independent color is suspect (HPA tissue IHC; general IHC practice).
| Cell and compartment reference | Use the HPA cell-level calls: medium in the named lung and germinal center cells, not detected in listed adipocytes and bronchial respiratory epithelial cells (HPA tissue IHC). Do not transfer a cell-level call to an entire organ. |
| Localization and topology | The cytoplasmic tissue profile agrees with the actin-filament ICC-IF location (HPA tissue IHC; HPA subcellular). CNN2 has no transmembrane segment or signal peptide (UniProt Q99439); tissue IHC need not resolve individual filaments. |
| Strength of antibody evidence | The tissue profile is Approved and pending external verification; HPA049095 is Approved for IHC, with no Enhanced IHC status supplied (HPA tissue IHC; HPA antibodies). Compare unexpected staining with the controls before treating it as a new pattern. |
| Isoforms and epitope uncertainty | UniProt lists 2 CNN2 isoforms (UniProt Q99439). No antibody epitope or isoform-specific IHC result is supplied, so a staining difference cannot be assigned to an isoform from this evidence. |
| IF/ICC Q: where should CNN2 appear? | A: Mainly on actin filaments (HPA subcellular, ICC-IF). That observation supports an intracellular interpretation but does not establish that filament detail will be visible in chromogenic paraffin-section IHC (HPA subcellular; general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| The reported positive germinal center cells are unstained (HPA tissue IHC). | An unsuccessful staining run or an unsuitable section is possible; HPA supplies no CNN2-specific retrieval or fixation sensitivity. | Confirm tissue morphology and run controls, then review antibody dilution, retrieval and detection records (general IHC practice). Avoid calling test sections negative until a reference stains. |
| Signal is chiefly nuclear, despite the cytoplasmic tissue profile (HPA tissue IHC). | Localization mismatch may reflect nonspecific detection or misleading counterstain (general IHC practice). | Compare the negative control and counterstain, then assess whether cytoplasmic signal is actually present in reported positive cells (HPA tissue IHC; general IHC practice). |
| Adipocytes or bronchial respiratory epithelial cells stain strongly (HPA tissue IHC: not detected). | Cross-reactivity or endogenous detection activity is possible; the HPA call is specific to these cells (HPA tissue IHC; general IHC practice). | Inspect a matched negative control and check endogenous-activity blocking where relevant to the detection system (general IHC practice). Recheck cell identity before scoring. |
| Color covers the section diffusely and obscures cell boundaries. | Background from blocking, washing, or detection conditions can limit interpretation (general IHC practice); no CNN2-specific background mechanism is supplied. | Review matched controls and the blocking, wash, detection and counterstain steps (general IHC practice). Score only cells with distinguishable signal and morphology. |
| Extracellular deposits or edge staining dominate the slide. | This distribution conflicts with the cytoplasmic tissue profile (HPA tissue IHC) and may reflect a staining artifact (general IHC practice). | Check the negative control and section quality, and separate deposited color from intracellular signal in the named positive cells (HPA tissue IHC; general IHC practice). |
| Low-level thyroid glandular staining is called a strong positive (HPA tissue IHC: Low). | The intensity call may have been overinterpreted; HPA reports low staining for these cells (HPA tissue IHC). | Rescore intensity against matched controls and the reported medium reference cells, recording cell type and compartment as well as color strength (HPA tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing CNN2 staining
A08680-2 · IHCIHC analysis of Calponin 2/CNN2 using anti-Calponin 2/CNN2 antibody (A08680-2). Calponin 2/CNN2 was detected in a paraffin-embedded section
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000064666-CNN2/tissue/cerebral+cortex#imid_13667283
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000064666-CNN2/tissue/colon#imid_13759802
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000064666-CNN2/tissue/liver#imid_13759750
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000064666-CNN2/tissue/kidney#imid_13759792
HPA · ICC-IF RT-4Human Protein Atlas immunofluorescence, RT-4, antibody HPA075297; green = CNN2, blue = nucleus, red = microtubules.
HPA · ICC-IF RT-4Human Protein Atlas immunofluorescence, RT-4, antibody HPA075297; green = CNN2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CNN2 IHC & IF
🧪Run tonsil first and assess staining in germinal center cells (HPA: Medium in tonsil germinal center cells; selected-SKU caption: paraffin-section tonsil staining). Use adipose tissue as a negative comparator, focusing on adipocytes, while cells within the tonsil section that lack specific signal should show counterstain without DAB deposits (HPA: Not detected in adipocytes; standard IHC interpretation).
Positive control tissue: Lung (Alveolar cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CNN2 in RT-4, SiHa, U2OS, with annotated localisation: Actin filaments (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a concentration-matched rabbit IgG isotype control matched to the primary antibody’s clonality if known; and CNN2 knockout material or an immunizing-peptide block if available (selected-SKU caption: rabbit primary; standard IHC controls). For tonsil, quench endogenous peroxidase and assess endogenous biotin background when using the caption’s biotin–streptavidin/DAB detection (selected-SKU caption: SABC with DAB; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative unreported). The demonstrated IHC conditions use heat retrieval in EDTA at pH 8.0, followed by 2 μg/ml primary antibody overnight at 4°C; retrieval dependence beyond those conditions is unreported (selected-SKU caption). The supplied evidence does not establish whether frozen sections or IF are easier; in tonsil, background from endogenous peroxidase or biotin warrants attention with SABC/DAB detection (selected-SKU caption: tonsil and SABC/DAB; standard IHC practice).
HPA tissue IHC evidence for CNN2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Pending external verification. Antibody staining in cells/structures not annotated, view images.). 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 |
|---|---|---|---|---|
| Lung | Alveolar cells | Medium | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Medium | Protein (IHC) | HPA → |
| Tonsil | Germinal center 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 → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CNN2 IHC Tips
Troubleshoot CNN2 staining in paraffin sections by checking retrieval, cell identity, cytoplasmic pattern, and controls before comparing chromogenic signal across samples.
What retrieval should I start with when CNN2 staining is weak?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A08680-2). The selected tonsil example used this retrieval before overnight incubation at 4°C with 2 μg/ml catalog antibody (caption A08680-2). If staining remains weak, check that sections were fully deparaffinised and compare a controlled range of heating times while keeping the buffer and pH 8.0 constant (standard IHC practice). Run the same tonsil control alongside test sections, and judge improvement by staining of germinal center cells and background together (HPA: medium in tonsil germinal center cells; standard IHC practice).
How should I investigate inconsistent CNN2 staining after fixation?
The selected paraffin-section caption does not report a fixative, so CNN2-specific sensitivity to fixation is unknown for this antibody (caption A08680-2). Record each sample’s fixative, fixation duration, processing history, and section age before comparing signal (standard IHC practice). Test matched sections with the stated EDTA pH 8.0 retrieval and the same antibody concentration of 2 μg/ml, using tonsil as a concurrent control (datasheet A08680-2; caption A08680-2; HPA: medium in tonsil germinal center cells). If the control stains consistently while samples vary, investigate specimen handling and cell composition before attributing the difference to a CNN2-specific fixation effect (standard IHC practice).
Where should convincing CNN2 staining appear in a paraffin section?
Assess CNN2 as a cellular cytoplasmic signal, with attention to its association with actin filaments (HPA: cytoplasmic expression in immune cells; HPA: approved actin-filament localisation). The protein has no annotated transmembrane segment, and its calponin-homology domain spans residues 28–132 (UniProt Q99439 topology and domains). In tonsil, compare signal with the morphology of germinal center cells, where medium staining is reported, rather than scoring every DAB-positive area as target expression (HPA: tonsil germinal center cells, medium; standard IHC practice). A predominantly nuclear or extracellular deposit requires review of counterstain, section quality, and detection controls before it is called CNN2 (HPA: cytoplasmic expression; standard IHC practice).
Could isoforms or epitope accessibility explain a changed CNN2 staining pattern?
CNN2 has 2 listed isoforms, but the supplied antibody caption does not identify its epitope or establish isoform selectivity (UniProt Q99439 isoforms; caption A08680-2). Its calponin-homology domain occupies residues 28–132, and reported modifications include acetylation and phosphoserine at residue 138 (UniProt Q99439 domains and modified residues). Those features alone do not establish which form this antibody detects in a tissue section (UniProt Q99439 record; caption A08680-2). If staining changes, compare matched sections under identical EDTA pH 8.0 retrieval and antibody conditions, then seek epitope or isoform documentation before assigning the difference to splicing or modification (datasheet A08680-2; standard IHC practice).
How can IF help check the cell identity and distribution of CNN2 staining?
Use IF as a separate confirmation experiment: pair CNN2 with a validated germinal center cell marker when examining tonsil, where those cells have medium IHC staining (HPA: tonsil germinal center cells, medium; standard IF practice). Choose a fluorophore and imaging channel after measuring tissue autofluorescence with an unstained control, and include single-label controls for bleed-through in multiplex images (standard IF practice). CNN2 has no annotated transmembrane segment and is mainly associated with actin filaments, so evaluate cytoplasmic access with a controlled permeabilisation step appropriate to the IF specimen and antibody epitope (UniProt Q99439 topology; HPA: approved actin-filament localisation; standard IF practice). Do not transfer the paraffin-section retrieval or incubation conditions directly into IF (caption A08680-2; standard IF practice).
How do I reduce diffuse or misleading DAB background?
Start by comparing the stained section with a no-primary control and checking whether DAB signal follows cells or spreads across empty tissue and section edges (standard IHC practice). The selected tonsil example used 10% goat serum, biotinylated goat anti-rabbit secondary, a streptavidin–biotin complex, and DAB (caption A08680-2). With that detection format, assess endogenous peroxidase and endogenous biotin as possible background sources, and confirm that the peroxidase block and wash steps work in your workflow (caption A08680-2; standard IHC practice). If background persists, titrate primary antibody around the reported 2 μg/ml condition while retaining a concurrent positive control (caption A08680-2; standard IHC practice).
What should I measure when comparing CNN2 IHC across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue region and eligible cell population before scoring, then record the percentage of positive cells and staining intensity as an H-score, or report positive-cell density per mm² (standard IHC practice). For tonsil, score germinal center cells separately from other structures because medium staining is reported specifically for that population (HPA: tonsil germinal center cells, medium). Normalise positive-cell counts to the number of eligible cells, or density to the measured tissue area, and keep threshold and imaging settings consistent across sections (standard IHC practice). Report the no-primary background and concurrent control result so differences in DAB development are not mistaken for biological changes (standard IHC practice).
How can I distinguish true CNN2 staining from artefact?
Look for reproducible cytoplasmic staining in identifiable cells, consistent with reported immune-cell expression and actin-filament localisation (HPA: cytoplasmic expression in immune cells; HPA: approved actin-filament localisation). In tonsil, germinal center cell staining has tissue-level support, whereas staining in an unexpected cell population needs independent verification (HPA: tonsil germinal center cells, medium; HPA: reliability approved, pending external verification). Treat predominantly nuclear deposits, section-edge enhancement, and necrotic areas cautiously, and compare them with morphology and a no-primary control (HPA: cytoplasmic expression; standard IHC practice). Check whether signal survives adequate endogenous peroxidase control before calling diffuse DAB product CNN2 (standard IHC practice).
Boster reagents
Best CNN2 / Calponin-2 IHC Antibodies
A08680-2 has real IHC data from a paraffin-embedded human tonsil section; no IF figure is supplied (catalog: A08680-2 image captions).
Real IHC data
Anti-Calponin 2/CNN2 Antibody ®
Cat # A08680-2
A08680-2 is listed for human IHC (catalog: A08680-2 applications and reactivity). Its IHC figure shows a paraffin-embedded human tonsil section stained with 2 μg/ml primary antibody after EDTA pH 8.0 retrieval (A08680-2 image caption).
Which to pick: Choose A08680-2 for human paraffin-section IHC: it is a rabbit antibody listed for human IHC at 2–5 μg/ml, and its own figure documents staining in human tonsil (catalog: A08680-2; A08680-2 image caption). No SKU in the payload is listed for IF/ICC or non-human reactivity, and clonality is not reliably specified (catalog: A08680-2 applications, reactivity, and dilution_raw). The IHC caption does not report the fixative, so fixation cannot be inferred (A08680-2 image caption). The selected A08680-2 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image A08680-2).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q99439 (CNN2_HUMAN, Calponin-2).
- Human Protein Atlas. CNN2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CNN2 subcellular location (ICC-IF): Mainly localized to the actin filaments..
- Human Protein Atlas. CNN2 antibody validation summary (2 antibodies).
- The Role of H2-Calponin Antigen in Cancer Metastasis: Presence of Autoantibodies in Liver Cancer Patients. International journal of molecular sciences 2023 — PMC10298237.
- The potential role of DNA methylation in abdominal aortic aneurysms. International journal of molecular sciences 2015 — PMC4463699.
- Loss of Calponin 2 causes premature ovarian insufficiency in mice. Journal of ovarian research 2024 — PMC10854048.
- CNN2 silencing inhibits colorectal cancer development through promoting ubiquitination of EGR1. Life science alliance 2023 — PMC10185810.
- PubMed PMID:8889829 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15057824 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CNN2 (Calponin-2). Expected localisation Cytoplasmic staining in immune cells (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A08680-2). Validated IHC antibody: A08680-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Lung (Alveolar cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A08680-2); 2. Fixation: Image fixative and duration unreported (datasheet A08680-2); 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 A08680-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A08680-2); 8. Primary antibody: Rabbit anti-CNN2, 2-5 μg/ml (datasheet A08680-2); 9. Primary incubation: Overnight at 4 °C (datasheet A08680-2). Watch for: Fixation: Keep paraffin-section fixation consistent. (standard IHC practice; not target-specific); Caveat: Immune-cell staining may complicate interpretation (HPA tissue IHC); Isoform / epitope: 2 isoforms; antibody epitope coverage is unspecified (UniProt); Regulation: No expression regulator specified (UniProt). Evidence: UniProt Q99439, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the actin filaments., Datasheet A08680-2, PMC10298237. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CNN2/immunohistochemistry-ihc-guide.