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- Table of Contents
Plan paraffin AGBL2 IHC with the IHC-validated antibody at 1:100–1:300 (datasheet) and assess cytoplasmic staining (HPA tissue IHC). Interpret tissue staining cautiously because agreement with RNA expression is very low (HPA tissue IHC).
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
| Expected localisation | Cytoplasmic staining in tissue (HPA tissue IHC); centrosome and basal body localisation at the molecular level (UniProt) | |
| Staining pattern | General cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 44 tissues); use no-primary + isotype controls |
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Very low staining–RNA consistency; verify specificity (HPA tissue IHC) | |
| Regulation | No specific expression regulator annotated (UniProt) | |
| Isoform / epitope | Three isoforms; epitope differences unreported (UniProt) |
The catalog antibody’s IHC-P protocol is followed by published AGBL2 staining conditions for ovarian and renal tissues (PMC6781648; PMC7283229; PMC11417249).
| Sample | Paraffin-embedded human heart tissue; fixative not specified (datasheet A11275-1) |
| Fixation | Image fixative and duration unreported (datasheet A11275-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-AGBL2, 1:100-1:300 (datasheet A11275-1) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | AGBL2-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
AGBL2 is a cytosolic protein that also localizes to centrosomes, centrioles and ciliary basal bodies (UniProt Q5U5Z8 subcellular location; UniProt Q5U5Z8 topology). In paraffin sections, expect chiefly cytoplasmic staining in reported positive cells, including adrenal glandular cells and breast myoepithelial cells (HPA tissue IHC: High). Treat this as a provisional pattern: HPA rates tissue IHC reliability Uncertain because antibody staining and RNA expression have very low consistency (HPA tissue IHC: Uncertain).
| Cytoplasmic staining in adrenal glandular cells or breast myoepithelial cells. | This matches reported high staining in those cell types and the expected cytosolic compartment (HPA tissue IHC: High; UniProt Q5U5Z8 subcellular location). It is a plausible positive, but the HPA tissue pattern remains Uncertain; morphology alone does not establish antibody specificity (HPA tissue IHC: Uncertain). |
| Prominent staining confined to nuclei, cell membranes or extracellular material. | This differs from the expected IHC cytoplasmic pattern and from UniProt’s nonmembrane, intracellular annotation (HPA tissue IHC: General cytoplasmic expression; UniProt Q5U5Z8 topology). Investigate background or cross-reactivity before scoring it as AGBL2. Nucleolar localization has separate ICC-IF support and should not be assumed to validate a nuclear IHC pattern (HPA subcellular: Nucleoli approved). |
| Strong signal in an unexpected cell population within the section. | Compare the exact cell type with the HPA tissue record before assigning specificity (HPA tissue IHC: reported cell types; HPA tissue IHC: Uncertain). An unexpected chromogenic signal may reflect cross-reactivity or endogenous detection activity (standard IHC practice). HPA’s broad cytoplasmic profile does not define every unlisted cell type as negative (HPA tissue IHC: General cytoplasmic expression). |
| Diffuse colour across cells and surrounding tissue. | A poorly bounded deposit is difficult to score as cellular AGBL2 staining (standard IHC practice). Check background controls, blocking and detection chemistry; widespread colour may arise from nonspecific binding or endogenous activity (standard IHC practice). Compare with the reported cellular, chiefly cytoplasmic pattern (HPA tissue IHC: General cytoplasmic expression). |
| No detectable signal in an HPA high-staining cell type. | First confirm that the expected cells are present, then check section processing, retrieval, antibody dilution and detection controls (standard IHC practice). A negative result warrants investigation but cannot by itself disprove AGBL2 expression: the reported high pattern has Uncertain reliability and RNA discordance (HPA tissue IHC: High; HPA tissue IHC: Uncertain). |
| Tissue and cell choice | HPA reports high IHC staining in several cell types, including adrenal glandular, breast myoepithelial and bone marrow hematopoietic cells (HPA tissue IHC: High). Adipocytes are reported Low, not negative, so they are a limited contrast tissue (HPA tissue IHC: Low in adipocytes). |
| Evidence confidence | The tissue staining profile is Uncertain because antibody staining and RNA data have very low consistency; HPA also flags a splice or transcript discrepancy and pending external verification (HPA tissue IHC: reliability description). HPA007718 is rated Uncertain for IHC (HPA antibodies: HPA007718 IHC Uncertain). |
| Intracellular distribution | UniProt places AGBL2 in cytosol, centrioles and ciliary basal bodies, with no transmembrane segment (UniProt Q5U5Z8 subcellular location; UniProt Q5U5Z8 topology). Fine structures may be harder to resolve than broad cytoplasmic staining in routine chromogenic sections (standard IHC practice). |
| Isoforms and processing | UniProt lists three isoforms and a single chain spanning residues 1–902, with no signal peptide or propeptide (UniProt Q5U5Z8 isoforms; UniProt Q5U5Z8 processing). Without an antibody epitope map, isoform coverage cannot be inferred; no supplied evidence supports a shed extracellular staining pattern (UniProt Q5U5Z8 processing; UniProt Q5U5Z8 topology). |
| IF/ICC Q&A: What localization should I expect? | HPA ICC-IF places AGBL2 mainly in nucleoli, cytosol, basal bodies and flagellar centrioles; it also reports sperm mid, principal and end pieces (HPA subcellular). This informs localization interpretation only; HPA007718 has Uncertain ICC validation, and IF/ICC requires its own guide (HPA antibodies: HPA007718 ICC Uncertain). |
| Situation | Likely cause | Next action |
|---|---|---|
| The expected cytoplasmic cells are unstained. | The target cells may be absent from the section, or the staining run may have failed (standard IHC practice). The HPA positive call is itself Uncertain (HPA tissue IHC: Uncertain). | Confirm cell identity on the counterstained section and review the positive control, retrieval, primary dilution and detection run (standard IHC practice). Repeat with an independently supported antibody if available (standard IHC practice). |
| Staining is mainly nuclear in paraffin sections. | A nuclear-only IHC pattern conflicts with HPA’s general cytoplasmic tissue profile (HPA tissue IHC: General cytoplasmic expression). HPA’s approved nucleolar finding comes from ICC-IF (HPA subcellular: Nucleoli approved). | Check localization against a matched control and another antibody before calling the nuclear IHC signal specific (standard IHC practice; HPA antibodies: HPA007718 IHC Uncertain). |
| A low-staining comparison tissue shows colour. | Adipocytes are reported Low rather than negative; residual signal is possible (HPA tissue IHC: Low in adipocytes). Diffuse colour may also come from background detection activity (standard IHC practice). | Assess the exact stained cell type and compare with a no-primary control; do not treat adipose tissue as an established AGBL2-negative control (HPA tissue IHC: Low in adipocytes; standard IHC practice). |
| Colour appears without primary antibody. | The signal can arise from endogenous chromogenic detection activity or another reagent background source (standard IHC practice). | Review the no-primary control, endogenous activity blocking and detection reagents, then interpret the test section only after background is controlled (standard IHC practice). |
| Signal covers tissue boundaries and obscures cells. | Excess primary or detection signal, or insufficient blocking, can obscure cellular localization (standard IHC practice). | Recheck primary dilution and blocking, reduce detection development as appropriate, and retain a matched control for comparison (standard IHC practice). Score only distinguishable cellular staining (standard IHC practice). |
| High staining disagrees with the expected RNA pattern. | HPA reports very low consistency between antibody staining and RNA expression and flags a splice or transcript discrepancy (HPA tissue IHC: reliability description). | Record the exact tissue, cell type and compartment; interpret the IHC result as provisional and seek independent antibody or orthogonal support before making an expression claim (HPA tissue IHC: Uncertain; standard IHC practice). |
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Very low consistency between antibody staining and RNA expression data. Pending external verification. Caution, Splice and/or transcript discrepancy exists.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Myoepithelial cells | High | Protein (IHC) | HPA → |
| Caudate | Glial cells | High | Protein (IHC) | HPA → |
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: AGBL2 is detected in all 44 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Troubleshoot AGBL2 staining in paraffin sections by checking retrieval, cellular pattern, controls and scoring before interpreting signal as target specific.
Both anti-AGBL2 antibodies list IHC and IF for human and mouse (catalog: applications, reactivity). Each has a paraffin-section IHC image; neither has an IF image in the payload (catalog: image captions).
A11275-1 lists IHC and IF for human and mouse, with an IHC image of paraffin-embedded human heart and a peptide-blocked comparison (catalog: A11275-1 applications, reactivity, image caption). A30633 lists IHC, IF and ICC for human and mouse, with its own IHC image of paraffin-embedded human heart and a peptide-blocked comparison (catalog: A30633 applications, reactivity, image caption).
Which to pick: For tissue IHC, either SKU has a paraffin-embedded human heart example (A11275-1 and A30633 image captions); the fixative is unreported in both captions. For IF/ICC, choose A30633 because it lists both applications and is described as polyclonal; A11275-1 lists IF but not ICC (catalog: applications, A30633 dilution data). For human or mouse samples, both list reactivity, but their IHC images document human heart only (catalog: reactivity; A11275-1 and A30633 image captions).