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AGL / Glycogen debranching enzyme · IHC design guide
Design Immunohistochemistry for AGL
Plan chromogenic IHC on paraffin sections using cytoplasmic staining in hepatocytes and skeletal myocytes as reference patterns (HPA tissue IHC). Compare control tissues and interpret nuclear staining cautiously because the tissue IHC assessment considers it off target (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
AGL 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 several tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in hepatocytes and skeletal myocytes (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A02555) | |
| Positive control | Cervix+4 more · see all | |
| Negative control | Adrenal gland+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 | Disregard observed nuclear tissue staining as off target (HPA tissue IHC) | |
| Regulation | Glycogenolysis can redistribute AGL to nuclei (UniProt) | |
| Isoform / epitope | Three listed isoforms (1, 5, 6); epitope coverage unknown (UniProt) |
Section 1
Recommended AGL IHC & IF Protocols
The catalog antibody’s paraffin IHC protocol is paired with one published AGL staining protocol using frozen human muscle sections (PMC2753483).
| Sample | Paraffin-embedded human placenta tissue; fixative not specified (datasheet A02555) |
| Fixation | Image fixative and duration unreported (datasheet A02555); 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 A02555); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A02555) |
| Primary antibody | Rabbit anti-AGL, 2-5 μg/ml (datasheet A02555) |
| Primary incubation | Overnight at 4 °C (datasheet A02555) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A02555) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | AGL-positive staining in squamous epithelial cells of cervix (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in several tissues, including skeletal muscle and liver. Additional nuclear expression in squamous epithelia and brain. No signal in the no-primary control. |
💡Decision noteFor paraffin sections, start with heat-mediated EDTA pH 8.0 retrieval (datasheet A02555). The published frozen-section method uses acetone fixation (PMC2753483).
Section 2
What Is the Expected AGL Staining Pattern?
AGL is a soluble, non-membrane enzyme found mainly in the cytoplasm; UniProt also reports nuclear localisation under glycogenolytic conditions (UniProt P35573: localisation; topology). In paraffin-section IHC, expect cytoplasmic staining in hepatocytes and skeletal myocytes, each reported at medium intensity (HPA: liver and skeletal muscle). HPA rates its tissue IHC profile Approved but notes low agreement with RNA data and disregards the nuclear tissue staining as off-target (HPA: tissue IHC reliability).
| Cytoplasmic colour in hepatocytes or skeletal myocytes, with identifiable cell borders and little surrounding colour. | This fits the reported medium staining in both cell types (HPA: liver hepatocytes; skeletal muscle myocytes) and AGL’s principal location (UniProt P35573: cytoplasm). Compare cells within the same section before calling a weaker region negative (general IHC interpretation). |
| Strong nuclear colour dominates a tissue section, especially in squamous epithelium, with little cytoplasmic colour. | Treat it as suspect: HPA observed nuclear tissue staining but disregarded it as off-target (HPA: tissue IHC reliability). UniProt reports conditional nuclear localisation, which alone cannot authenticate this IHC pattern (UniProt P35573: localisation). |
| Colour appears in cells reported as unstained, such as bone-marrow hematopoietic cells, or follows blood, edges, or tissue debris. | HPA reports no detection in bone-marrow hematopoietic cells (HPA: bone marrow). A mismatched cellular pattern raises cross-reactivity or detection-background concerns; edge and debris staining also warrant a reagent-only control (general IHC practice). |
| Broad, hazy colour covers cells and extracellular areas without resolving cytoplasm. | This cannot establish AGL localisation (general IHC interpretation). Check whether the no-primary control also colours the section, then review blocking, wash stringency, antibody concentration, and chromogen development as general IHC variables (general IHC practice). |
| No cytoplasmic colour appears in adequately preserved liver hepatocytes or skeletal myocytes. | These are useful positive comparators because HPA reports medium staining in each (HPA: liver; skeletal muscle). A blank result warrants checking the antibody’s documented IHC-P conditions and detection controls; HPA supplies no AGL-specific fixation-sensitivity finding here (HPA: tissue IHC; general IHC practice). |
💡Expected AGL appearanceCall positive when hepatocytes or skeletal myocytes show discernible medium cytoplasmic colour (HPA: liver; skeletal muscle); dominant nuclear colour is a suspect tissue-IHC result (HPA: nuclear staining disregarded).
| Cell type and compartment | Use hepatocytes and skeletal myocytes as medium-staining references (HPA: tissue IHC); AGL has no transmembrane segment and is principally cytoplasmic (UniProt P35573: topology; localisation). |
| Tissue-IHC confidence | HPA labels its tissue IHC Approved while flagging low staining–RNA consistency and a splice/transcript discrepancy (HPA: reliability). Interpret borderline positives with that limitation; Approved does not resolve a disputed nuclear pattern (HPA: tissue IHC reliability). |
| Isoform context | UniProt reports differing isoform expression across liver and muscle (UniProt P35573: tissue specificity). The supplied record gives no antibody epitope or isoform coverage, so a difference in staining cannot be assigned to a particular isoform. |
| IF/ICC Q&A: should nuclear signal match tissue IHC? | No direct equivalence is established: ICC-IF lists supported nucleoplasm, nuclear bodies, and cytosol (HPA: subcellular ICC-IF), whereas HPA disregards nuclear tissue-IHC staining (HPA: tissue IHC reliability). Assess each application against its own validation. |
| Chromogenic detection | Endogenous enzyme activity can mimic signal when enzyme-based detection is used; a no-primary control and the matching detection block help identify it (general IHC practice). This is a workflow consideration, not an AGL-specific tissue claim. |
| Situation | Likely cause | Next action |
|---|---|---|
| Liver or skeletal muscle is blank. | A failed staining or detection step is possible because HPA reports medium signal in hepatocytes and myocytes (HPA: tissue IHC); a blank slide alone does not identify which step failed. | Confirm tissue morphology, the antibody’s documented IHC-P conditions, and a working detection control; review retrieval and dilution against the antibody instructions (general IHC practice). |
| Nuclei stain more strongly than cytoplasm in tissue IHC. | HPA observed nuclear tissue staining and explicitly disregarded it as off-target (HPA: tissue IHC reliability); UniProt’s conditional nuclear localisation does not settle this slide’s specificity (UniProt P35573: localisation). | Score cytoplasm separately, inspect the no-primary control, and compare with the expected positive cell types (HPA: liver; skeletal muscle; general IHC practice). |
| A reportedly negative cell population stains. | For example, hematopoietic cells in bone marrow were not detected by HPA (HPA: bone marrow); cross-reactivity or endogenous chromogenic activity may explain a discordant result (general IHC interpretation). | Check the no-primary and detection controls, then reassess whether colour is cellular and confined to the expected compartment (general IHC practice; UniProt P35573: cytoplasm). |
| The entire section has diffuse brown colour. | Residual detection background, concentrated antibody, or overdevelopment can obscure cell boundaries (general IHC practice); diffuse colour is not evidence of the reported cytoplasmic pattern (HPA: tissue IHC). | Compare a no-primary section; review blocking, wash steps, antibody dilution, and development time using the selected detection system’s instructions (general IHC practice). |
| Muscle and liver differ in intensity. | HPA reports medium staining in skeletal myocytes and hepatocytes but warns of low staining–RNA consistency (HPA: tissue IHC reliability). UniProt describes tissue-dependent isoform expression without establishing this antibody’s isoform recognition (UniProt P35573: tissue specificity). | Compare like cells under matched staining conditions and report the observed intensity; avoid assigning the difference to an isoform without epitope evidence (general IHC interpretation). |
| An ICC-IF image shows nuclei, while the paraffin IHC section is cytoplasmic. | HPA supports nucleoplasmic ICC-IF localisation but disregards nuclear staining in its tissue-IHC profile (HPA: subcellular ICC-IF; tissue IHC reliability). | Use the application-specific reference when scoring: assess this IHC section against the cytoplasmic tissue pattern, and evaluate ICC-IF separately under its own guide (HPA: tissue IHC; subcellular ICC-IF). |
Experiment evidence
IHC & IF images showing AGL staining
A02555 · IHCIHC analysis of AGL using anti-AGL antibody (A02555). AGL was detected in a paraffin-embedded section of human placenta tissue. Heat mediate
A02555 · IFIF analysis of AGL using anti-AGL antibody (A02555) and anti-Beta Tubulin antibody (M01857-3). AGL was detected in immunocytochemical sectio
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000162688-AGL/tissue/cerebral+cortex#imid_20841839
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000162688-AGL/tissue/colon#imid_20841749
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000162688-AGL/tissue/liver#imid_20841750
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000162688-AGL/tissue/kidney#imid_20841759
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA028498; green = AGL, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA028498; green = AGL, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for AGL IHC & IF
🧪Run placenta first and score trophoblastic cells, which show medium AGL staining (HPA: placenta, trophoblastic cells, Medium; datasheet: A02555 placenta IHC). Use adrenal glandular cells as the negative tissue (HPA: adrenal gland, glandular cells, Not detected); on the placenta slide, compare trophoblastic staining with adjacent non-trophoblastic cells, without assuming those cells are AGL-negative.
Positive control tissue: Cervix (Squamous epithelial cells, HPA Medium)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show AGL in A-431, U-251MG, U2OS, Hep-G2, NIH 3T3, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include no-primary (secondary-only), species- and immunoglobulin-class-matched rabbit isotype, and AGL-knockout material or peptide competition if a validated blocking peptide is available (datasheet: rabbit anti-AGL; standard IHC practice). Block endogenous peroxidase and inspect background in the placenta section when using the caption’s HRP/DAB detection (datasheet: placenta, HRP/DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A02555 placenta caption does not state the fixative (datasheet: A02555 placenta IHC). That paraffin-section example uses heat-mediated EDTA retrieval at pH 8.0, but it does not establish that retrieval is essential or that frozen-section IHC or IF is easier (datasheet: A02555 placenta IHC). Placental background from endogenous peroxidase is a practical concern with HRP/DAB detection (datasheet: placenta, HRP/DAB; standard IHC practice).
HPA tissue IHC evidence for AGL
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data. Caution, Splice and/or transcript discrepancy exists. Nuclear staining considered off target binding and disregarded.). 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 |
|---|---|---|---|---|
| Cervix | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Medium | Protein (IHC) | HPA → |
| Liver | Hepatocytes | Medium | Protein (IHC) | HPA → |
| Nasopharynx | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
| Placenta | Trophoblastic cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| 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 → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced AGL IHC Tips
Troubleshoot AGL staining in paraffin sections by checking retrieval, compartment, cell type, and controls before interpreting chromogenic signal.
How should I retrieve AGL in paraffin sections with weak staining?
Start with heat-mediated EDTA retrieval at pH 8.0 (datasheet A02555). The reported AGL image used a paraffin-embedded human placenta section, but its caption gives no heating time, so document your instrument’s temperature and duration rather than treating either as product-validated (datasheet A02555). If staining remains weak, compare a second retrieval condition on matched sections while holding antibody concentration and detection constant; judge improvement by signal in trophoblastic cells, where medium staining is reported (HPA tissue IHC). Excess retrieval can damage morphology or raise background, so inspect tissue integrity and a no-primary control before increasing primary incubation.
Could fixation explain inconsistent AGL staining between paraffin blocks?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (datasheet A02555). Record fixation type and duration for each block, then compare sections processed with the same EDTA pH 8.0 retrieval and the same staining run (datasheet A02555: retrieval condition). Overfixation can mask epitopes in routine IHC, while poor fixation can impair morphology; neither outcome has been demonstrated specifically for AGL here. Use a consistently processed reference section and assess both expected cellular staining and preservation of tissue architecture before attributing a between-block difference to AGL abundance. Do not infer fixation behavior from tissue-expression patterns or protein features.
Should nuclear AGL staining be scored as specific?
For paraffin IHC, prioritize cytoplasmic staining in the expected cells: AGL is annotated as cytoplasmic, with nuclear localization under glycogenolytic conditions (UniProt P35573), and tissue IHC reports cytoplasmic expression in several tissues (HPA tissue IHC). The tissue atlas explicitly disregards nuclear staining as off-target binding despite reporting nuclear signal in some tissues (HPA tissue IHC). An independent subcellular IF dataset supports nucleoplasm, nuclear bodies, and cytosol localization, which makes assay context relevant but does not validate nuclear signal from this IHC antibody (HPA subcellular). Score nuclear staining separately, compare it with no-primary controls, and require independent target-specific evidence before interpreting it biologically.
Can this antibody distinguish AGL isoforms or modified epitopes in tissue?
Do not assign an IHC signal to one AGL isoform without an epitope map and isoform-specific validation; neither is supplied for the catalog antibody. The record lists isoforms 1, 5, and 6, while its tissue-specificity annotation also describes muscle-specific isoform transcripts and isoforms 5 and 6 in liver and muscle (UniProt P35573). A phosphoserine at residue 64 is annotated, but no evidence establishes that this antibody recognizes or avoids that modification (UniProt P35573). Compare tissue staining with an independent method that resolves transcripts or protein variants, and report the chromogenic result as AGL immunoreactivity rather than isoform-specific expression.
How can I investigate AGL localization with multiplex immunofluorescence?
Treat multiplex IF as a separate optimization: the selected antibody’s supplied example is chromogenic paraffin IHC, while the subcellular atlas reports IF images without establishing validation for this catalog antibody (datasheet A02555; HPA subcellular). Pair AGL with a marker identifying the expected cell population, such as a validated trophoblastic marker in placenta, where trophoblastic staining is reported (HPA tissue IHC). Choose fluorophores in channels with low measured tissue autofluorescence and include single-stain and no-primary controls to assess bleed-through and background. AGL has no annotated transmembrane segment and is annotated in the cytoplasm (UniProt P35573); optimize permeabilization for intracellular access, since this antibody’s epitope position is unspecified.
What should I change when DAB appears throughout the section?
First inspect a no-primary section for endogenous peroxidase activity and apply a peroxidase block as part of the general chromogenic workflow. The selected example used a 10% goat-serum block, 2 μg/ml primary antibody overnight at 4°C, a peroxidase-conjugated secondary for 30 minutes at 37°C, and DAB development (datasheet A02555). If diffuse color persists after adequate washing, shorten chromogen development or titrate the primary on matched sections while retaining a reference tissue. Check whether signal follows section edges, damaged areas, or many unrelated cell types; those patterns warrant caution before assigning it to AGL. Compare with expected cytoplasmic staining (HPA tissue IHC).
How should I quantify AGL staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring: use cytoplasmic signal in the relevant cells, and keep nuclear signal in a separate category because the tissue atlas disregards its nuclear staining (HPA tissue IHC). Record the percentage of positive cells and intensity by category, then calculate an H-score from intensity-weighted percentages using one threshold set across the comparison. For spatial analyses, report positive-cell density per mm² of viable tissue and normalize counts to the sampled viable area. Compare matched tissue regions and staining runs, using a reference section to track assay drift. Report excluded necrotic areas and background thresholds alongside scores.
How can I distinguish true AGL staining from artefact?
Look for reproducible cytoplasmic signal in an expected cell type, such as hepatocytes or skeletal-muscle myocytes, which show medium tissue staining in the atlas (HPA tissue IHC). Treat isolated nuclear staining cautiously: nuclear localization under glycogenolytic conditions is annotated (UniProt P35573), but the tissue atlas considers its nuclear staining off target (HPA tissue IHC). Edge staining, necrotic areas, and widespread DAB deposition in a no-primary section favor processing or endogenous-enzyme artefact over target-specific signal. Compare serial sections, morphology, and appropriate controls before calling a specimen positive; the selected placenta image establishes a staining example, not specificity in every tissue (datasheet A02555).
Boster reagents
Best AGL / Glycogen debranching enzyme IHC Antibodies
A02555 has IHC data from a human placenta paraffin section and IF data from U2OS cells (A02555 image captions). Both antibodies list human, mouse and rat reactivity (catalog: A02555, M02555).
Real IHC data
Anti-AGL Antibody ®
Cat # A02555
A02555 lists IHC and IF and has images from a human placenta paraffin section and U2OS cells, respectively (catalog: A02555; A02555 image captions). M02555 lists IHC and ICC/IF for human, mouse and rat, but has no IHC or IF image caption in this payload (catalog: M02555).
Which to pick: For tissue IHC, choose A02555: its own caption documents a human placenta paraffin section, EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody; the fixative is unreported (A02555 IHC caption). For IF/ICC, A02555 has a U2OS IF image at 5 μg/ml (A02555 IF caption); M02555 is a rabbit monoclonal that lists ICC/IF but has no image caption here (catalog: M02555). Both list human, mouse and rat reactivity for cross-species planning, while the supplied IHC and IF images document only the A02555 samples named above (catalog: A02555, M02555; A02555 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P35573 (GDE_HUMAN, Glycogen debranching enzyme).
- Human Protein Atlas. AGL tissue IHC expression (reliability: Approved).
- Human Protein Atlas. AGL subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the nuclear bodies and cytosol..
- Human Protein Atlas. AGL antibody validation summary (2 antibodies).
- Fast-twitch sarcomeric and glycolytic enzyme protein loss in inclusion body myositis. Muscle & nerve 2009 — PMC2753483.
- A strain-programmed lignin-based Janus patch for rapid healing of postoperative rectal wounds. Theranostics 2025 — PMC12315808.
- PubMed PMID:1374391 — UniProt-cited evidence.
- PubMed PMID:8954797 — UniProt-cited evidence.
- PubMed PMID:9332391 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for AGL (Glycogen debranching enzyme). Expected localisation Cytoplasmic staining in several tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A02555). Validated IHC antibody: A02555 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Cervix (Squamous epithelial cells); negative Adrenal gland. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human placenta tissue; fixative not specified (datasheet A02555); 2. Fixation: Image fixative and duration unreported (datasheet A02555); 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 A02555); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A02555); 8. Primary antibody: Rabbit anti-AGL, 2-5 μg/ml (datasheet A02555); 9. Primary incubation: Overnight at 4 °C (datasheet A02555). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Disregard observed nuclear tissue staining as off target (HPA tissue IHC); Isoform / epitope: Three listed isoforms (1, 5, 6); epitope coverage unknown (UniProt); Regulation: Glycogenolysis can redistribute AGL to nuclei (UniProt). Evidence: UniProt P35573, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet A02555, PMC2753483. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/AGL/immunohistochemistry-ihc-guide.