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CA2 / Carbonic anhydrase 2 · IHC design guide
Design Immunohistochemistry for CA2
Plan chromogenic CA2 IHC in paraffin sections with catalog antibody M00143 at 0.5–1 μg/mL (datasheet M00143). Use the observed cytoplasmic and nuclear tissue pattern to guide staining assessment (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CA2 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 | Tissue IHC: cytoplasm/nucleus; UniProt: cytoplasm/membrane (HPA tissue IHC; UniProt) | |
| Staining pattern | GI tract, kidney tubules, gallbladder, glia: cytoplasm/nucleus (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M00143) | |
| Positive control | Appendix+4 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 across samples. (standard IHC practice; not target-specific) | |
| Caveat | Antibody staining has medium concordance with RNA expression (HPA tissue IHC) | |
| Regulation | PKC/PMA displaces membrane-associated CA2 (UniProt) | |
| Isoform / epitope | No isoforms; chain 2–260; no extracellular domain (UniProt) |
Section 1
Recommended CA2 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: M00143) is accompanied by published CA2 staining protocols for chicken uterine tissue (PMC9513254) and ovarian granulosa cell areas (PMC13592854).
| Sample | Paraffin-embedded human gastric cancer tissue; fixative not specified (datasheet M00143) |
| Fixation | Image fixative and duration unreported (datasheet M00143); 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 M00143); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M00143) |
| Primary antibody | Mouse monoclonal (clone 10E11) anti-CA2, 0.5-1μg/ml (datasheet M00143) |
| Primary incubation | Overnight at 4 °C (datasheet M00143) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet M00143) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CA2-positive staining in endocrine cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nuclear expression in gastrointestinal tract, kidney tubules, gallbladder and glial cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet: M00143). Neither published excerpt specifies retrieval (PMC9513254; PMC13592854).
Section 2
What Is the Expected CA2 Staining Pattern?
In paraffin-section IHC, expect CA2 staining chiefly in cytoplasm, with nuclear staining reported in gastrointestinal cells, kidney tubules, gallbladder cells and glial cells (HPA tissue IHC). CA2 is cytoplasmic and can associate with the cell membrane; it has no transmembrane segment (UniProt P00918). HPA rates the tissue pattern Enhanced, while describing only medium consistency between antibody staining and RNA expression (HPA tissue IHC).
| Distinct cytoplasmic staining in gallbladder glandular cells, intestinal endocrine cells or glial cells. | This fits reported positive populations; HPA lists gallbladder glandular cells, appendix, colon and duodenum endocrine cells, and several glial populations as High (HPA tissue IHC). Judge the named cells within their tissue, rather than treating every cell on the section as an expected positive. |
| Nuclear staining accompanies cytoplasmic staining, or cerebellar granular-cell nuclei stain strongly. | Do not reject nuclear signal solely because UniProt lists cytoplasm and cell membrane: HPA reports nuclear expression in its tissue profile and High staining in cerebellar granular-cell nuclei (HPA tissue IHC; UniProt P00918). Check whether the signal follows those reported cells and retains clear boundaries. |
| Staining is confined to extracellular material or appears only as a continuous outline around every cell. | That distribution does not match the reported cytoplasmic and nuclear tissue pattern or establish CA2 membrane association (HPA tissue IHC; UniProt P00918). Review the negative control and detection steps before assigning the signal to CA2 (standard IHC practice). |
| Strong signal appears in adipocytes or bronchial respiratory epithelial cells while expected positive cells are also stained. | HPA reports CA2 as Not detected in those specific cell populations (HPA tissue IHC). Consider antibody cross-reactivity or endogenous detection activity, and compare a reagent-omission control; a positive control elsewhere does not validate every stained cell (standard IHC practice). |
| Known positive cells lack staining, or the whole section has a diffuse brown haze. | Absent signal in a reported High population makes a negative interpretation inconclusive; uniform haze makes cell-level scoring unreliable (HPA tissue IHC; standard IHC practice). Inspect section integrity, detection controls and background before interpreting either result. |
💡Expected CA2 appearanceCall a result positive when identifiable reported cell populations show distinct cytoplasmic staining, with compatible nuclear signal and clear surrounding morphology; HPA lists several such populations as High, while staining of listed Not detected cells or uniform haze warrants investigation (HPA tissue IHC; standard IHC practice).
| Compartment and tissue context | UniProt places CA2 in cytoplasm and at the cell membrane, whereas HPA tissue IHC also reports nuclear staining; interpret nuclear signal in the reported cells in that context, especially cerebellar granular-cell nuclei (UniProt P00918; HPA tissue IHC). |
| Choice of positive and comparison tissue | Gallbladder glandular cells, intestinal endocrine cells and glial cells provide reported High populations; adipocytes and bronchial respiratory epithelial cells are listed as Not detected (HPA tissue IHC). These are cell-specific comparisons, not claims that an entire tissue is uniformly positive or negative. |
| IHC evidence versus IF/ICC evidence: can the pattern be transferred? | HPA lists two antibodies with Enhanced IHC validation, HPA001550 and CAB010102, but gives neither an ICC validation status nor ICC/IF images or a main subcellular location (HPA antibodies; HPA subcellular). Use the tissue pattern as IHC evidence; an IF/ICC result needs its own validation. |
| Processing and isoforms | UniProt lists one CA2 chain spanning residues 2–260, no signal peptide or propeptide, and no annotated isoforms (UniProt P00918). The supplied record therefore gives no basis for expecting a distinct processed or isoform-specific tissue staining pattern. |
| Chromogenic detection background | Endogenous peroxidase activity or nonspecific reagent binding can generate brown signal independently of the primary antibody; blocking and reagent-omission controls help identify it (standard IHC practice). This is a general detection concern, not a reported CA2-specific effect. |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain in gallbladder glandular cells or another reported High population. | The run may have failed, or the chosen preparation and detection settings may need optimization; HPA's High rating describes observed staining, not guaranteed performance in every run (HPA tissue IHC; standard IHC practice). | Check tissue preservation and a run control, then review the IHC-validated antibody's supplied IHC-P conditions, retrieval step and detection reagents before scoring the sample negative (standard IHC practice). |
| Brown precipitate is widespread, including areas without identifiable positive cells. | Excess chromogen development, incomplete washing or endogenous detection activity can obscure a cell-specific pattern (standard IHC practice). | Compare a primary-antibody omission control; inspect wash and detection steps, and reduce nonspecific background before interpreting CA2 localisation (standard IHC practice). |
| Adipocytes or bronchial respiratory epithelial cells stain strongly. | Those cells are listed as Not detected, so cross-reactivity or detection background is plausible; HPA status alone cannot identify the cause in this run (HPA tissue IHC; standard IHC practice). | Compare the omission control and a reported High cell population on the same run; investigate antibody specificity if the unexpected cellular signal persists (HPA tissue IHC; standard IHC practice). |
| Only nuclei stain, across nearly all cell types. | HPA supports nuclear staining in some populations, notably cerebellar granular cells, but a uniform, tissue-wide nuclear pattern is not established by its CA2 profile (HPA tissue IHC). | Identify the stained cell types, compare their cytoplasm and the omission control, and avoid calling the pattern CA2-specific until the cellular distribution is supported (HPA tissue IHC; standard IHC practice). |
| An IF/ICC image seems to disagree with the paraffin-section IHC result. | HPA supplies Enhanced IHC antibody status but no ICC validation status, ICC/IF images or main ICC/IF location for CA2 (HPA antibodies; HPA subcellular). | Treat the IF/ICC observation as a separate result requiring validation; do not use this IHC tissue profile as proof of an IF/ICC compartment pattern (HPA tissue IHC; HPA subcellular). |
Experiment evidence
IHC & IF images showing CA2 staining
M00143 · IHCIHC analysis of CA2 using anti-CA2 antibody (M00143). CA2 was detected in paraffin-embedded section of human gastric cancer tissue. Heat med
M00143 · IHCIHC analysis of CA2 using anti-CA2 antibody (M00143). CA2 was detected in paraffin-embedded section of human colon cancer tissue. Heat media
M00143 · IFIF analysis of CA2 using anti-CA2 antibody (M00143). CA2 was detected in immunocytochemical section of HEPG2 cells. Enzyme antigen retrieval
PB9989 · IHCIHC analysis of CA2 using anti-CA2 antibody (PB9989). CA2 was detected in paraffin-embedded section of human gastric cancer tissues. Heat me
PB9989 · IHCIHC analysis of CA2 using anti-CA2 antibody (PB9989). CA2 was detected in paraffin-embedded section of human liver cancer tissues. Heat medi
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000104267-CA2/tissue/cerebral+cortex#imid_2844876
HPA · StomachHuman Protein Atlas IHC, Stomach. Source: https://www.proteinatlas.org/ENSG00000104267-CA2/tissue/stomach+1#imid_2844784
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000104267-CA2/tissue/colon#imid_2844818
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000104267-CA2/tissue/liver#imid_2844803
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CA2 IHC & IF
🧪Run caudate first: glial cells should stain (HPA: High in caudate glial cells). Use adipose tissue as the negative tissue (HPA: adipocytes Not detected); on the caudate slide, cells without specific CA2 staining should show counterstain without brown DAB, although HPA does not identify a verified internal negative cell type (HPA: caudate glial-cell row).
Positive control tissue: Appendix (Endocrine cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for CA2; derive a cell-line control from the positive tissue's cell type (Endocrine cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) control, a matched mouse IgG isotype control, and CA2 knockout tissue as a biological negative (M00143 IHC caption: mouse primary; standard IHC practice). For DAB detection, control endogenous peroxidase; if using the caption’s avidin–biotin detection, check endogenous biotin background, and distinguish neuronal pigment from DAB in caudate (M00143 IHC caption: SABC/DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the M00143 paraffin-section caption does not state a fixative (M00143 IHC caption). Retrieval dependence is unestablished; that caption uses heat-mediated EDTA retrieval at pH 8.0 (M00143 IHC caption). The supplied evidence does not establish whether frozen sections or IF/ICC would be easier; caudate pigment may complicate chromogenic scoring (M00143 IHC caption: paraffin IHC only; standard IHC practice).
HPA tissue IHC evidence for CA2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium 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 |
|---|---|---|---|---|
| Appendix | Endocrine cells | High | Protein (IHC) | HPA → |
| Caudate | Glial cells | High | Protein (IHC) | HPA → |
| Cerebellum | Granular cells - nucleus | High | Protein (IHC) | HPA → |
| Cerebral cortex | Glial cells | High | Protein (IHC) | HPA → |
| Colon | Endocrine cells | High | 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 → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CA2 IHC Tips
Troubleshoot CA2 staining in paraffin sections by checking retrieval, cell type, compartment, detection controls, and scoring (datasheet M00143; HPA tissue IHC; UniProt P00918).
How should I retrieve CA2 in paraffin sections when staining is weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet M00143). The selected paraffin-section image used that retrieval before overnight incubation at 4°C with 1 μg/mL catalog antibody, so keep those conditions together for the first comparison (datasheet M00143). If staining remains weak, vary heating time on adjacent sections while keeping buffer, antibody concentration, and detection constant, and inspect tissue integrity after each change (standard IHC practice). Include a positive reference section, such as gallbladder glandular cells, and compare signal with a no-primary control (HPA tissue IHC: High in gallbladder glandular cells; standard IHC practice).
Could fixation explain weak or uneven CA2 staining?
The selected CA2 paraffin-section caption does not report its fixative, and target-specific fixation sensitivity is unknown from the supplied evidence (datasheet M00143). Record the fixative, fixation duration, section thickness, and storage history for each specimen before comparing staining across blocks (standard IHC practice). Assess matched sections with the same EDTA pH 8.0 retrieval and 1 μg/mL primary incubation used in the selected image, changing one preparation variable at a time (datasheet M00143; standard IHC practice). If staining differs between blocks, check morphology and a positive reference on each run before attributing the difference to CA2 expression (standard IHC practice).
Where should CA2 signal appear, and when is membrane staining plausible?
Expect predominantly cytoplasmic staining, while allowing signal at the cell membrane because both locations are annotated for CA2 (UniProt P00918: Cytoplasm; Cell membrane). CA2 has no transmembrane segment and can colocalize with SLC26A6 at the cell surface, so a peripheral rim alone does not establish membrane insertion (UniProt P00918). HPA also reports cytoplasmic and nuclear staining in gastrointestinal tract, kidney tubules, gallbladder, and glial cells (HPA tissue IHC). Compare each compartment with its surrounding cell morphology, a no-primary control, and a positive reference; regard a dominant extracellular or necrotic-edge deposit as suspect (HPA tissue IHC; standard IHC practice).
How should I assess an unexpected CA2 staining pattern without a mapped epitope?
The supplied record lists 0 isoforms, one alpha-carbonic-anhydrase domain spanning residues 3–259, and no glycosylation sites (UniProt P00918). It lists acetylation or phosphorylation at residue 2 and phosphorylation at residues 165 and 172, but supplies no catalog-antibody epitope (UniProt P00918; datasheet M00143). Therefore, do not assign weak staining to a specific modification or epitope without independent mapping evidence (standard IHC practice). First compare retrieval intensity and antibody concentration on adjacent sections, then assess whether the changed signal remains in plausible cells and compartments (datasheet M00143; HPA tissue IHC; standard IHC practice).
How can I adapt CA2 assessment to multiplex immunofluorescence?
Treat IF as a separate assay because the supplied selected-antibody evidence describes chromogenic staining of a paraffin section, with no IF validation stated (datasheet M00143). Pair CA2 with a validated marker for the expected cell type, such as a glial-cell marker when assessing glia, and verify each single-stain channel before multiplexing (HPA tissue IHC: High in glial cells; standard IF practice). Choose fluorophores after inspecting unstained tissue for autofluorescence, and use a spectrally separated channel for the weaker signal (standard IF practice). Because CA2 is cytoplasmic and can occur at the cytosolic membrane surface, titrate permeabilisation to expose the intracellular epitope while preserving cell boundaries (UniProt P00918; standard IF practice).
What should I check when CA2 DAB staining spreads beyond expected cells?
Check a no-primary section and the tissue edges before increasing antibody concentration; diffuse color in both suggests detection background or section artefact (standard IHC practice). The selected image used biotinylated secondary antibody, a streptavidin-biotin complex, and DAB, so evaluate secondary-only and detection-reagent controls when that workflow is used (datasheet M00143; standard IHC practice). Include an endogenous peroxidase block before DAB development and compare blocked with unblocked controls if enzyme activity is suspected (standard IHC practice). Reassess blocking and washes while retaining the reported 10% goat-serum block and 1 μg/mL primary concentration as reference conditions (datasheet M00143; standard IHC practice).
How should I score CA2 staining across heterogeneous tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because HPA reports CA2 in several epithelial and neural cell populations and both cytoplasmic and nuclear staining (HPA tissue IHC). For each annotated region, record the percentage of positive cells and an intensity-based H-score using identical thresholds across slides (standard IHC practice). Normalize positive counts to total eligible cells in the same region, or report positive-cell density per mm² of viable tissue when cell segmentation is unreliable (standard IHC practice). Exclude folds, necrosis, and edges by a prespecified rule, and report how many regions and sections contributed to each specimen's summary (standard IHC practice).
How can I distinguish true CA2 positivity from misleading staining?
A credible result follows cell identity and compartment: cytoplasmic signal is consistent with UniProt, and HPA reports high staining in gallbladder glandular cells and glial cells (UniProt P00918; HPA tissue IHC). Treat staining restricted to a torn edge, necrotic debris, or acellular material as suspect, and compare it with a no-primary section (standard IHC practice). If DAB appears broadly in enzyme-rich areas, check the endogenous peroxidase block before interpreting the color as CA2 (standard IHC practice). HPA labels its tissue evidence “Enhanced” but describes only medium agreement between staining and RNA, so resolve discordant cell patterns with an independent control (HPA tissue IHC; standard IHC practice).
Boster reagents
Best CA2 / Carbonic anhydrase 2 IHC Antibodies
Anti-CA2 IHC images cover paraffin sections of human gastric, colon and liver cancers and mouse/rat intestine; IF imagery covers HEPG2 cells (catalog image captions).
Real IHC data
Anti-CA2 Antibody ® (monoclonal, 10E11)
Cat # M00143
Real IHC data
Anti-Carbonic Anhydrase II/CA2 Antibody ®
Cat # PB9989
M00143 has IHC images from human gastric and colon cancers and mouse/rat intestine, plus IF imagery from HEPG2 cells (M00143 image captions). PB9989 has IHC images from human gastric and liver cancers and lists IF/ICC among its applications (PB9989 image captions; catalog applications).
Which to pick: For tissue IHC, M00143’s paraffin-section caption uses EDTA retrieval at pH 8, while PB9989’s uses citrate at pH 6; neither caption reports the fixative (M00143 and PB9989 IHC captions). For IF/ICC, choose M00143 for its HEPG2 IF image; PB9989 lists IF/ICC but has no IF image in the payload (M00143 IF caption; PB9989 applications and image payload). For cross-species IHC, M00143 is a mouse monoclonal with human, mouse and rat tissue images; PB9989 is rabbit-hosted and lists those species as reactive, but its IHC images show human tissue only (catalog host, clone and reactivity; M00143 and PB9989 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P00918 (CAH2_HUMAN, Carbonic anhydrase 2).
- Human Protein Atlas. CA2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CA2 subcellular location (ICC-IF): Variation in transcript expression is correlated to the cell cycle..
- Human Protein Atlas. CA2 antibody validation summary (2 antibodies).
- Hippocampal Beta-Amyloid Accumulation In Aluminium Chloride and D-Galactose-Induced Rats: Establishing a Nontransgenic Alzheimer's Model. Journal of pharmacy & bioallied sciences 2025 — PMC12782526.
- Morphological and molecular markers of mouse area CA2 along the proximodistal and dorsoventral hippocampal axes. Hippocampus 2023 — PMC10443601.
- Circadian miR-218-5p targets gene CA2 to regulate uterine carbonic anhydrase activity during egg shell calcification. Poultry science 2022 — PMC9513254.
- Identification of CA2 as a potential candidate gene involved in the therapeutic effect of Huatan Huoxue decoction on polycystic ovary syndrome: An integrated study of network pharmacology, Mendelian randomization, and experimental validation. Journal of Food and Drug Analysis 2026 — PMC13592854.
- PubMed PMID:3108857 — UniProt-cited evidence.
- PubMed PMID:3121496 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CA2 (Carbonic anhydrase 2). Expected localisation Tissue IHC: cytoplasm/nucleus; UniProt: cytoplasm/membrane (HPA tissue IHC; UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M00143). Validated IHC antibody: M00143 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Endocrine cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human gastric cancer tissue; fixative not specified (datasheet M00143); 2. Fixation: Image fixative and duration unreported (datasheet M00143); 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 M00143); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M00143); 8. Primary antibody: Mouse monoclonal (clone 10E11) anti-CA2, 0.5-1μg/ml (datasheet M00143); 9. Primary incubation: Overnight at 4 °C (datasheet M00143). Watch for: Fixation: Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific); Caveat: Antibody staining has medium concordance with RNA expression (HPA tissue IHC); Isoform / epitope: No isoforms; chain 2–260 ; no extracellular domain (UniProt); Regulation: PKC/PMA displaces membrane-associated CA2 (UniProt). Evidence: UniProt P00918, HPA tissue IHC · Enhanced, HPA cell-line RNA · Variation in transcript expression is correlated to the cell cycle., Datasheet M00143, PMC9513254, PMC13592854. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CA2/immunohistochemistry-ihc-guide.