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GLA / Alpha-galactosidase A · IHC design guide
Design Immunohistochemistry for GLA
Plan chromogenic IHC for GLA in paraffin sections with an IHC-validated antibody (datasheet A01135-2). Assess granular cytoplasmic staining in glandular cells and use reported high and undetected cell populations to interpret the result (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
GLA 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 | Granular cytoplasm in tissue sections (HPA tissue IHC); lysosomal protein (UniProt) | |
| Staining pattern | Granular cytoplasm, most abundant in glandular cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A01135-2) | |
| Positive control | Adrenal gland+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 fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | No expression regulation annotated (UniProt) | |
| Isoform / epitope | No isoforms annotated; mature chain spans aa 32–429 (UniProt) |
Section 1
Recommended GLA IHC & IF Protocols
The catalog antibody protocol uses EDTA pH 8.0 retrieval (datasheet: A01135-2). The published IHC protocols below cover ESCC tissue (PMC13414882) and non-human primate liver (PMC11646755).
| Sample | Paraffin-embedded human mammary cancer tissue; fixative not specified (datasheet A01135-2) |
| Fixation | Image fixative and duration unreported (datasheet A01135-2); 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 A01135-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01135-2) |
| Primary antibody | Rabbit anti-GLA, 2μg/ml (datasheet A01135-2) |
| Primary incubation | Overnight at 4 °C (datasheet A01135-2) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A01135-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | GLA-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Granular cytoplasmic expression, most abundant in glandular cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet: A01135-2); the cited studies used different retrieval buffers with their own antibodies (PMC13414882; PMC11646755).
Section 2
What Is the Expected GLA Staining Pattern?
GLA is a soluble lysosomal enzyme with no transmembrane segment (UniProt P06280 topology). In paraffin-section IHC, expect granular cytoplasmic staining, most abundant in glandular cells, including those in breast and endometrium (HPA: tissue IHC). Collecting ducts in kidney and macrophages in lung also stain strongly (HPA: High). HPA rates the tissue IHC evidence Enhanced, with medium consistency between staining and RNA expression (HPA: reliability).
| Granular cytoplasmic signal in glandular cells, kidney collecting ducts, or lung macrophages (HPA: tissue IHC). | This fits the reported cell distribution and lysosomal location (HPA: High; UniProt P06280: lysosome). Judge granularity within identified cells, alongside a negative control (general IHC practice). |
| Predominantly nuclear, membranous, or extracellular staining (UniProt P06280: lysosome; no transmembrane segment). | This differs from the expected granular cytoplasm (HPA: tissue IHC). Check antibody specificity and detection background before treating it as GLA localization (general IHC practice). |
| Strong signal in adipocytes, cardiomyocytes, or oral squamous cells (HPA: Not detected in these cells). | The cell-specific mismatch raises concern for cross-reactivity or endogenous detection activity (HPA: tissue IHC; general IHC practice). Confirm morphology and examine a control without primary antibody (general IHC practice). |
| Diffuse color across cells and surrounding tissue, without distinct cytoplasmic granules (HPA: granular cytoplasm). | Broad haze does not match the reported pattern (HPA: tissue IHC). Compare a control without primary antibody; assess blocking, washes, and chromogen development (general IHC practice). |
| No signal in kidney collecting ducts or lung macrophages (HPA: High in these cells). | A known-positive cell population failing to stain makes the run hard to interpret (HPA: tissue IHC). Review controls, retrieval, antibody dilution, and detection steps (general IHC practice). |
💡Expected GLA appearanceCall a positive result when defined cells show granular cytoplasmic staining, strong in reported High populations; predominant nuclear signal or diffuse tissue-wide color warrants review (HPA: tissue IHC; UniProt P06280: lysosome).
| Compartment and processing (UniProt P06280). | GLA has a signal peptide at residues 1–31 and a mature chain at 32–429; its reported location is lysosomal (UniProt P06280). |
| Cell-type contrast (HPA: tissue IHC). | High staining in kidney collecting ducts contrasts with undetected cardiomyocytes; assess the named cell populations, not whole-section darkness (HPA: tissue IHC). |
| Antibody evidence (HPA: antibody validation). | HPA000237 and HPA000966 each have Enhanced IHC status (HPA: antibodies). That supports the reported pattern without establishing specificity for every sample. |
| IF/ICC applicability (HPA: subcellular record). | Can this pattern be carried over to IF/ICC? HPA lists no main IF location or ICC-IF images; confirm localization independently in that application (HPA: subcellular). |
| Target-specific fixation sensitivity (UniProt P06280; HPA: supplied records). | No target-specific fixation effect is reported here. Retrieval decisions therefore require run controls and method validation (general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells show no stain (HPA: High in kidney collecting ducts or lung macrophages). | The run may have failed, or the cells may be missing from the section (general IHC practice). | Verify cell identity and a stained positive control, then review retrieval, antibody dilution, detection reagents, and timing (general IHC practice). |
| Only uniform weak color appears where granules are expected (HPA: granular cytoplasm). | Low specific signal or excess background can obscure the reported pattern (HPA: tissue IHC; general IHC practice). | Compare positive and no-primary controls; adjust antibody dilution, washes, or chromogen time against both controls (general IHC practice). |
| Strong staining appears in cardiomyocytes or adipocytes (HPA: Not detected in those cells). | Cross-reactivity or endogenous detection activity is possible; HPA's negative call is cell-specific (HPA: tissue IHC; general IHC practice). | Check a no-primary control and, if using peroxidase detection, its endogenous-activity block; compare an independently validated antibody (general IHC practice). |
| Signal is mainly nuclear or outlines the plasma membrane (UniProt P06280: lysosome; no transmembrane segment). | That compartment conflicts with the reported granular cytoplasm (HPA: tissue IHC). Nonspecific binding or detection background may contribute (general IHC practice). | Review morphology and no-primary controls, then check antibody specificity with a positive cell population (general IHC practice; HPA: High populations). |
| Glandular cells vary in intensity across tissues (HPA: High in breast; Low in colon). | HPA reports different staining levels despite its broad glandular profile (HPA: tissue IHC). | Score comparable cell types against the tissue-specific HPA level; do not require every glandular tissue to match breast intensity (HPA: tissue IHC). |
| IF/ICC does not show the expected puncta (UniProt P06280: lysosome). | HPA provides no ICC-IF images or main IF location for GLA (HPA: subcellular). | Treat the IHC pattern as a hypothesis for IF/ICC and validate that application with its own controls (HPA: tissue IHC and subcellular; general IF practice). |
Experiment evidence
IHC & IF images showing GLA staining
A01135-2 · IHCFigure 2. IHC analysis of Galactosidase alpha/Gla using anti-Galactosidase alpha/Gla antibody (A01135-2). Galactosidase alpha/Gla was detect
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000102393-GLA/tissue/cerebral+cortex#imid_337030
HPA · Adrenal glandHuman Protein Atlas IHC, Adrenal gland. Source: https://www.proteinatlas.org/ENSG00000102393-GLA/tissue/adrenal+gland#imid_124754
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000102393-GLA/tissue/colon#imid_124696
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000102393-GLA/tissue/liver#imid_124705
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for GLA IHC & IF
🧪Run breast first and look for staining in its glandular cells (HPA: High in breast glandular cells). Use adipose tissue adipocytes as a negative comparator (HPA: Not detected in adipocytes); on the breast slide, assess adjacent non-glandular cells for background, without assuming they are GLA-negative.
Positive control tissue: Adrenal gland (Glandular 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 GLA; derive a cell-line control from the positive tissue's cell type (Glandular cells) and confirm it by RNA or western blot first.
Technical controls: Include no-primary (secondary-only) and host-species, isotype, and clonality-matched antibody controls, plus GLA-knockout material as a biological negative. Quench endogenous peroxidase and check for endogenous biotin background because the selected tissue-IHC caption uses biotin-based detection with DAB (selected-SKU caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported; the selected-SKU tissue-IHC caption does not state the fixative (selected-SKU caption: fixative unreported). Retrieval dependency is unestablished, although that caption uses heat-mediated EDTA retrieval at pH 8.0 (selected-SKU caption). The supplied evidence does not establish that frozen sections or IF are easier, and the biotin-based detection workflow warrants a background check in the chosen tissue (selected-SKU caption: SABC and DAB).
HPA tissue IHC evidence for GLA
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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
| Fallopian tube | Ciliated cells (cell body) | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced GLA IHC Tips
Troubleshoot GLA chromogenic IHC by checking retrieval, cell type, and granular cytoplasmic staining against matched controls (datasheet A01135-2; HPA: tissue profile).
How should I optimize retrieval when GLA staining is weak?
Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A01135-2). Apply the same heating and cooling schedule to adjacent sections, then assess granular cytoplasmic staining in glandular cells alongside tissue morphology (HPA: granular cytoplasmic expression; standard IHC practice). Use 10% goat serum blocking and 2 μg/mL primary antibody overnight at 4°C as documented comparison conditions, with a no-primary section alongside (datasheet A01135-2; standard IHC practice). If signal remains weak, adjust heating duration consistently across controls; test another buffer only as a fallback while monitoring background and tissue damage (standard IHC practice).
Which fixation variables could explain inconsistent GLA staining between paraffin blocks?
The selected paraffin-section caption does not report a fixative, so GLA-specific fixation sensitivity and an optimal fixation duration are unknown (datasheet A01135-2). Record fixative, delay before fixation, duration, and block age, then compare sections under identical EDTA pH 8.0 retrieval and detection settings (datasheet A01135-2; standard IHC practice). Assess preserved morphology and granular cytoplasmic signal in comparable glandular regions, or in kidney collecting ducts when available (HPA: granular cytoplasmic expression; HPA: high in collecting ducts). Hold section thickness, antibody concentration, incubation, and DAB development constant before assigning a staining difference to fixation (standard IHC practice).
What GLA staining pattern should I expect in paraffin sections?
GLA is annotated as lysosomal and has no transmembrane segment, supporting an intracellular granular cytoplasmic pattern rather than a plasma membrane rim (UniProt P06280: lysosome, topology; HPA: tissue profile). Glandular cells show the most abundant granular expression in the tissue profile; kidney collecting ducts and lung macrophages are also reported as high (HPA: profile; HPA: collecting ducts and macrophages). Score staining within intact cells, keeping epithelial, immune, and stromal compartments separate so their different signals remain visible (standard IHC practice). Predominantly nuclear, surface, or extracellular staining warrants review of morphology and detection controls before assigning it to GLA (UniProt P06280: lysosome; standard IHC practice).
Could processing or glycosylation change how this antibody stains GLA?
GLA has a 429-aa precursor with a 1–31 signal peptide and a 32–429 mature chain; no isoforms are annotated (UniProt P06280: processing, isoforms). Three glycosylation sites are annotated at residues 139, 192, and 215, but the supplied antibody caption does not map its epitope (UniProt P06280: glycosylation; datasheet A01135-2). Therefore, a staining change cannot be assigned specifically to signal-peptide removal or glycosylation without epitope information and supporting experiments (UniProt P06280: processing, glycosylation; standard IHC practice). Compare matched retrieval conditions and cellular patterns before attributing discordant sections to a molecular form of GLA (standard IHC practice).
How can a companion IF experiment help assess the IHC pattern?
Use companion IF to compare GLA with a marker for an expected cell population, such as an epithelial marker in glandular regions or a macrophage marker in lung (HPA: glandular cells and lung macrophages; standard IF practice). Choose separated fluorophores and inspect unstained tissue in each channel, favoring a longer-wavelength channel when tissue autofluorescence interferes (standard IF practice). Because GLA is lysosomal with no transmembrane segment, permeabilise fixed cells for access to an expected intracellular epitope, while titrating detergent because the antibody epitope is unmapped (UniProt P06280: lysosome, topology; datasheet A01135-2; standard IF practice). Interpret apparent overlap with a lysosomal marker against single-label controls; the supplied HPA record has no ICC/IF images for direct validation (HPA: subcellular record; standard IF practice).
How do I separate GLA signal from chromogenic background?
The selected image used a biotinylated secondary antibody, streptavidin–biotin complex, and DAB, so inspect no-primary and detection-reagent controls for staining unrelated to primary-antibody binding (datasheet A01135-2; standard IHC practice). Include a peroxidase block in the DAB workflow, and consider endogenous biotin if background persists with the biotin-based detection system (standard IHC practice; datasheet A01135-2). Compare background in tissue edges, folds, and damaged areas with staining inside intact cells (standard IHC practice). Reduce excessive primary-antibody concentration or DAB development only after confirming that expected granular cytoplasmic staining remains visible (HPA: tissue profile; standard IHC practice).
How should I quantify GLA staining across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and region before imaging, because GLA staining varies by cell type and glandular cells are prominent in the tissue profile (HPA: tissue profile; standard IHC practice). Report the percentage of positive cells with intensity categories, or calculate an H-score by summing each intensity category’s percentage multiplied by its 0–3 score (standard IHC practice). For dispersed positive cells, report positive-cell density per mm² of evaluable tissue and state which cell type was counted (standard IHC practice). Normalize comparisons to the same eligible cell population, viable area, imaging settings, and DAB development conditions; retain matched negative controls (standard IHC practice).
When is a GLA-positive stain convincing rather than artefactual?
A convincing signal sits in intact cells as granular cytoplasmic staining, consistent with lysosomal GLA and the tissue staining profile (UniProt P06280: lysosome; HPA: tissue profile). Check cell identity: high staining is reported in glandular cells, kidney collecting ducts, and lung macrophages, whereas HPA reports cardiomyocytes as not detected (HPA: positive and negative tissue profiles). Treat nuclear rims, extracellular deposits, section-edge gradients, folds, and necrotic areas cautiously, especially when a no-primary control shares the pattern (UniProt P06280: lysosome; standard IHC practice). With DAB detection, exclude endogenous peroxidase staining using the peroxidase block and detection controls before assigning brown signal to GLA (datasheet A01135-2; standard IHC practice).
Boster reagents
Best GLA / Alpha-galactosidase A IHC Antibodies
A01135-2 has pictured IHC in paraffin-embedded human mammary cancer tissue (catalog image caption); M01135 lists human IHC and ICC/IF applications without a figure (catalog).
Real IHC data
Anti-Galactosidase alpha/Gla Antibody ®
Cat # A01135-2
A01135-2 will render with its IHC image from a paraffin-embedded human mammary cancer section (catalog image caption). M01135 will render with human IHC and ICC/IF applications listed, but no IHC or IF image is supplied (catalog).
Which to pick: For tissue IHC, pick polyclonal A01135-2: its own caption documents staining in a paraffin-embedded human mammary cancer section, with the fixative unreported (catalog image caption; catalog clonality). For IF/ICC, consider monoclonal M01135 because those applications are listed, although no IF figure is supplied (catalog applications; catalog clonality). Neither SKU has stated cross-species reactivity; both list human only (catalog reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P06280 (AGAL_HUMAN, Alpha-galactosidase A).
- Human Protein Atlas. GLA tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. GLA subcellular location (ICC-IF): Highest expression in A-549: 142.0 nTPM.
- Human Protein Atlas. GLA antibody validation summary (2 antibodies).
- GLA is associated with ESCC progression and chemotherapy response via DNA damage repair-related pathways. Frontiers in oncology 2026 — PMC13414882.
- Dietary intervention rescues a bone porosity phenotype in a murine model of Neurofibromatosis Type 1 (NF1). PloS one 2024 — PMC11195983.
- Preclinical efficacy and safety of adeno-associated virus 5 alpha-galactosidase: A gene therapy for Fabry disease. Molecular therapy. Methods & clinical development 2024 — PMC11646755.
- Thrombospondin 2, matrix Gla protein and digital analysis identified distinct fibroblast populations in fibrostenosing Crohn's disease. Scientific reports 2024 — PMC11178913.
- PubMed PMID:3036505 — UniProt-cited evidence.
- PubMed PMID:2542896 — UniProt-cited evidence.
- PubMed PMID:7626884 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for GLA (Alpha-galactosidase A). Expected localisation Granular cytoplasm in tissue sections (HPA tissue IHC); lysosomal protein (UniProt). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A01135-2). Validated IHC antibody: A01135-2 at 2μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human mammary cancer tissue; fixative not specified (datasheet A01135-2); 2. Fixation: Image fixative and duration unreported (datasheet A01135-2); 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 A01135-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01135-2); 8. Primary antibody: Rabbit anti-GLA, 2μg/ml (datasheet A01135-2); 9. Primary incubation: Overnight at 4 °C (datasheet A01135-2). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: No isoforms annotated; mature chain spans aa 32–429 (UniProt); Regulation: No expression regulation annotated (UniProt). Evidence: UniProt P06280, HPA tissue IHC · Enhanced, HPA cell-line RNA · Highest expression in A-549: 142.0 nTPM, Datasheet A01135-2, PMC13414882, PMC11646755. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/GLA/immunohistochemistry-ihc-guide.