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AGL / Glycogen debranching enzyme · IHC design guide
Design Immunohistochemistry for AGL
Plan paraffin section IHC for AGL using cytoplasmic staining in hepatocytes and skeletal myocytes as reference patterns (HPA tissue IHC). Compare tissues with consistent fixation, and interpret nuclear staining cautiously because HPA 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 | Nuclear staining may be off target; disregard it (HPA tissue IHC) | |
| Regulation | Isoform expression varies by tissue (UniProt) | |
| Isoform / epitope | 3 annotated isoforms; verify antibody epitope coverage (UniProt) |
Section 1
Recommended AGL IHC & IF Protocols
Compare the catalog antibody’s paraffin IHC protocol (datasheet: A02555) with a published AGL protocol on human muscle cryosections (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 cryosection protocol uses cold acetone fixation (PMC2753483).
Section 2
What Is the Expected AGL Staining Pattern?
AGL is primarily cytoplasmic, with no transmembrane segment (UniProt P35573: subcellular location and topology). In paraffin IHC, expect cytoplasmic staining in hepatocytes and skeletal muscle myocytes, both scored Medium by HPA (HPA: tissue IHC). Treat tissue nuclear staining cautiously: HPA disregards it as off-target and reports low agreement between staining and RNA expression (HPA: Approved reliability note).
| Cytoplasmic chromogen in hepatocytes or skeletal muscle myocytes, around HPA's Medium level (HPA: tissue IHC). | This matches the expected compartment and the two useful reference cell types (UniProt P35573: cytoplasm; HPA: liver and skeletal muscle). Compare staining within identified cells, since other cell populations in a section need not share the reported pattern (HPA: cell-specific tissue IHC). |
| Predominantly nuclear staining in a paraffin section, especially with little cytoplasmic signal (HPA: tissue IHC reliability note). | Do not score nuclear chromogen alone as confirmed AGL. HPA observes nuclear staining in squamous epithelia and brain but disregards tissue nuclear staining as off-target (HPA: tissue IHC). UniProt reports nuclear localization under glycogenolytic conditions; that exception does not validate a particular nuclear IHC result (UniProt P35573: subcellular location). |
| Strong staining in cells expected to be undetected, such as bone marrow hematopoietic cells or appendix glandular cells (HPA: tissue IHC). | Consider cross-reactivity or detection chemistry before assigning the signal to AGL. HPA reports Not detected for those specific cell types, while its overall antibody staining has low agreement with RNA data (HPA: tissue IHC reliability note). Endogenous enzyme activity is a possible chromogenic IHC artifact (general IHC practice). |
| Diffuse color across cells, stroma, and section edges without clear cytoplasmic boundaries. | This is background rather than a readable cell-specific AGL pattern (general IHC practice). Check a no-primary control to assess detection-system and endogenous-activity contributions; compare its distribution with the test slide under the same imaging conditions (general IHC practice). |
| No signal in hepatocytes or skeletal muscle myocytes on an otherwise intact section (HPA: tissue IHC). | A negative result in these HPA Medium reference cells is inconclusive until staining performance is checked (HPA: liver and skeletal muscle; general IHC practice). Review the antibody's IHC-P validation, detection reagents, and a run control; HPA's reliability note also limits confidence in any single tissue comparison (HPA: Approved reliability note). |
💡Expected AGL appearanceCall a paraffin IHC result consistent with AGL when hepatocytes or skeletal muscle myocytes show discernible cytoplasmic staining near HPA's Medium reference level; isolated nuclear or diffuse section-wide color is suspect (UniProt P35573: cytoplasm; HPA: tissue IHC and reliability note; general IHC practice).
| Tissue and cell selection | HPA records Medium staining in hepatocytes and skeletal muscle myocytes, but Not detected in selected cell types such as bone marrow hematopoietic cells (HPA: tissue IHC). Use named cell populations when comparing slides; a whole-organ label alone can hide differences among cells (general IHC practice). |
| Antibody evidence | HPA054340 is IHC Approved; HPA028498 has no stated IHC status in the supplied record (HPA: antibody validation). HPA describes low agreement between tissue staining and RNA expression, with a splice or transcript discrepancy; an Approved designation does not resolve that caveat (HPA: tissue IHC reliability note). |
| Subcellular interpretation and IF/ICC Q&A | Q: Does supported nucleoplasmic IF/ICC signal make nuclear IHC positive? A: No. HPA IF/ICC supports nucleoplasm, nuclear bodies, and cytosol, whereas HPA explicitly disregards nuclear staining in tissue IHC (HPA: subcellular ICC-IF; HPA: tissue IHC reliability note). Interpret the assays in their own validation contexts. |
| Isoforms and epitope coverage | AGL has reported isoforms and tissue-dependent transcript expression, including muscle-specific isoform mRNAs (UniProt P35573: isoforms and tissue specificity). If results differ by tissue, check the antibody's documented epitope and isoform coverage before assuming every variant is detected equally; the supplied record does not establish that coverage (UniProt P35573: isoforms). |
| Situation | Likely cause | Next action |
|---|---|---|
| Nuclear-only IHC staining | Tissue nuclear staining may reflect off-target binding; HPA explicitly disregards it (HPA: tissue IHC reliability note). | Score cytoplasm separately from nuclei, compare a matched no-primary control, and seek cytoplasmic staining in HPA reference cells before interpreting the section (HPA: liver and skeletal muscle; general IHC practice). |
| Color in an HPA Not detected cell type | Cross-reactivity or endogenous chromogenic activity is possible (HPA: tissue IHC; general IHC practice). | Confirm the cell identity and compare no-primary and detection controls. Review blocking of endogenous activity appropriate to the chromogen system before scoring cell-specific signal (general IHC practice). |
| Diffuse background obscures cell borders | Nonspecific reagent binding, insufficient washing, or detection-system background can obscure localization (general IHC practice). | Compare the no-primary control; optimize blocking, antibody concentration, washes, and detection exposure within the assay's validated workflow (general IHC practice). Interpret AGL only when a cell-specific pattern is readable. |
| No cytoplasmic signal in HPA reference cells | An assay or reagent failure is possible; HPA reports Medium staining in hepatocytes and skeletal muscle myocytes (HPA: tissue IHC; general IHC practice). | Check the run control, antibody IHC-P validation, retrieval conditions, and detection reagents, then repeat alongside a positive reference section (general IHC practice). No AGL-specific retrieval condition or fixation sensitivity is supplied. |
| Uneven signal across the section | Section handling or uneven reagent coverage may produce regional staining differences (general IHC practice). | Inspect tissue integrity and reagent coverage, repeat with uniform processing, and compare the same identified cell type across regions (general IHC practice). Avoid treating patchiness alone as a tissue-specific AGL distribution. |
| IF/ICC shows nuclear signal while IHC is cytoplasmic | The supplied HPA assays have different localization summaries: supported nucleoplasmic IF/ICC and disregarded nuclear tissue IHC (HPA: subcellular ICC-IF; HPA: tissue IHC reliability note). | Interpret each result against its own assay-specific HPA evidence. For this paraffin IHC guide, use the cytoplasmic tissue pattern for the staining decision (HPA: tissue IHC; UniProt P35573: cytoplasm). |
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: trophoblastic cells should stain (HPA: Medium in placental trophoblastic cells; selected A02555 tissue-IHC caption: placental staining), then use adrenal gland glandular cells as a negative tissue (HPA: Not detected). On the placenta slide, cells outside the listed trophoblastic compartment should show only background staining, but should not be assumed AGL-negative without independent validation (HPA: placental trophoblastic cells listed).
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 a no-primary, secondary-only control and a matched rabbit IgG isotype control (selected A02555 tissue-IHC caption: rabbit primary antibody), plus AGL-knockout material or a cognate-peptide competition control if available. Quench endogenous peroxidase and check for background DAB signal in placenta (selected A02555 tissue-IHC caption: placental section with peroxidase/DAB detection).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A02555 tissue-IHC caption does not state the fixative. Placental paraffin-section staining was reported after heat retrieval in EDTA at pH 8.0; whether retrieval is required for AGL is unreported (selected A02555 tissue-IHC caption). There is no supplied comparison showing that frozen sections or IF are easier; for the reported placental IHC workflow, assess endogenous peroxidase background before scoring DAB signal (selected A02555 tissue-IHC caption: placental peroxidase/DAB detection).
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
Use the documented paraffin-section conditions as the IHC starting point, and interpret staining against AGL’s reported tissue and compartment patterns.
What retrieval should I start with for AGL in paraffin sections?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A02555). The documented placenta IHC used this retrieval before overnight incubation at 4°C with 2 μg/ml primary antibody (datasheet A02555). If staining is weak, first check deparaffinisation, heating consistency and tissue preservation, then compare a cautiously adjusted retrieval run against the documented condition (general IHC practice). Evaluate changes using cytoplasmic signal in expected cells and background in adjacent structures; increased nuclear colour alone is unconvincing because HPA disregards nuclear tissue staining as off-target (HPA tissue IHC).
How should I troubleshoot fixation-related loss of AGL staining?
The selected paraffin-section caption does not report its fixative, so AGL-specific fixation sensitivity is unknown (datasheet A02555). Record the actual fixative and fixation duration for each specimen, and compare sections processed together before attributing a weak result to fixation (general IHC practice). Use the documented EDTA pH 8.0 retrieval and 2 μg/ml primary incubation as reference conditions when making that comparison (datasheet A02555). Check morphology and cytoplasmic staining together: poor preservation or uneven staining can indicate a processing problem, but neither the tissue expression pattern nor AGL’s cytoplasmic localisation establishes a fixation effect (HPA tissue IHC; UniProt P35573).
Should AGL staining be cytoplasmic or nuclear in tissue IHC?
Prioritise cytoplasmic staining when assessing paraffin-section IHC: AGL is annotated as cytoplasmic, and HPA reports cytoplasmic expression across several tissues (UniProt P35573; HPA tissue IHC). UniProt also notes nuclear localisation under glycogenolytic conditions, so a biological nuclear signal is possible in a defined context (UniProt P35573). Nevertheless, HPA disregards nuclear staining in its tissue IHC as off-target, despite reporting nuclear expression in some tissues (HPA tissue IHC). Score nuclear colour separately from cytoplasmic colour, inspect its distribution and controls, and seek independent validation before treating it as AGL in this chromogenic assay (HPA tissue IHC; general IHC practice).
Can one AGL antibody distinguish tissue-specific isoforms?
Do not assign an isoform from this stain without an epitope map and evidence that the antibody distinguishes splice products (UniProt P35573; general IHC practice). The record lists isoforms 1, 5 and 6, while its tissue-specificity note also describes muscle-specific isoform transcripts; liver and kidney predominantly express isoform 1 (UniProt P35573). AGL has no annotated transmembrane segment, and the record does not establish which sequence the catalog antibody recognises across isoforms (UniProt P35573; datasheet A02555). If staining differs between liver and muscle, compare matched processing and cellular localisation before proposing an isoform explanation, then confirm that explanation with an isoform-resolving method (general IHC practice).
How should I assess AGL by IF alongside the chromogenic IHC result?
Treat IF/ICC as a separate application and verify antibody performance there before comparing it with the documented paraffin-section IHC (datasheet A02555; general IF practice). Multiplex AGL with a marker for the expected cell type, such as a hepatocyte marker in liver, and choose a far-red fluorophore when tissue autofluorescence obscures shorter wavelengths (HPA tissue IHC; general IF practice). Because AGL lacks a transmembrane segment and has intracellular localisations, permeabilise to assess its cytosolic or nuclear signal, while confirming that the marker remains interpretable (UniProt P35573; HPA subcellular; general IF practice). HPA supports nucleoplasmic IF localisation, but its tissue IHC assessment disregards nuclear staining, so interpret the two assays separately (HPA subcellular; HPA tissue IHC).
What should I check when AGL DAB staining is diffuse or widespread?
Compare the test section with a no-primary control and inspect whether colour follows tissue edges, damaged areas or expected cells (general IHC practice). The documented assay uses a peroxidase-conjugated secondary and DAB, so include a peroxidase-blocking step and check secondary-only signal as general chromogenic controls (datasheet A02555; general IHC practice). The caption reports 10% goat serum blocking and 2 μg/ml primary antibody; use these as reference conditions when adjusting blocking or primary concentration (datasheet A02555). Interpret widespread nuclear colour cautiously because HPA considers nuclear staining off-target in tissue IHC, even where cytoplasmic expression is reported (HPA tissue IHC).
How can I quantify AGL IHC across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and cytoplasmic compartment before scoring, using the same thresholds, imaging conditions and region-selection rules across specimens (general IHC practice; HPA tissue IHC). For intensity-based analysis, record the percentage of cells at each staining grade and calculate a cytoplasmic H-score on a 0–300 scale; report percentage-positive cells separately when useful (general IHC practice). Normalise counts to the number of eligible cells, or stained area to measured tissue area, and exclude folds, necrosis and edges consistently (general IHC practice). Keep nuclear staining in a separate field rather than combining it with cytoplasmic signal, because HPA disregards nuclear tissue staining as off-target (HPA tissue IHC).
How do I distinguish credible AGL staining from an artefact?
A credible IHC result shows reproducible cytoplasmic signal in an appropriate cell population, with liver hepatocytes, skeletal-muscle myocytes and placental trophoblastic cells among HPA’s reported positive examples (HPA tissue IHC). Compare that pattern with no-primary and secondary controls, and inspect edges, necrotic regions and tissue damage before assigning cellular staining (general IHC practice). Endogenous peroxidase can mimic DAB signal, so evaluate the peroxidase block and control section when colour persists outside plausible cells (general IHC practice). Treat isolated nuclear signal cautiously: UniProt allows condition-dependent nuclear localisation, but HPA specifically disregards nuclear staining in tissue IHC as off-target (UniProt P35573; HPA tissue IHC).
Boster reagents
Best AGL / Glycogen debranching enzyme IHC Antibodies
A02555 has real IHC data from a human placenta paraffin section and IF data from U2OS cells (catalog image captions); its listed reactivity is human, mouse, and rat (catalog).
Real IHC data
Anti-AGL Antibody ®
Cat # A02555
A02555 will render with its own human placenta paraffin-section IHC figure; its IF caption documents U2OS cells (catalog image captions). M02555 will render as a rabbit monoclonal listed for IHC and ICC/IF in human, mouse, and rat, but has no IHC or IF figure in the payload (catalog).
Which to pick: Choose A02555 for tissue IHC because its own caption documents staining in a human placenta paraffin section; the fixative is unreported (A02555 IHC caption). For IF/ICC, A02555 has a U2OS cell IF example, while M02555 offers a rabbit monoclonal option listed for ICC/IF without a supplied figure (catalog). Both list human, mouse, and rat reactivity, but the supplied IHC image documents human tissue only (catalog; A02555 IHC caption).
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: Nuclear staining may be off target; disregard it (HPA tissue IHC); Isoform / epitope: 3 annotated isoforms; verify antibody epitope coverage (UniProt); Regulation: Isoform expression varies by tissue (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.