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GLA / Alpha-galactosidase A · IHC design guide
Design Immunohistochemistry for GLA
Plan chromogenic GLA IHC around granular cytoplasmic staining, most abundant in glandular cells (HPA tissue IHC). The catalog antibody has human paraffin-section staining evidence (datasheet A01135-2); lung macrophages provide another high-staining cell population for interpretation (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 | Lysosomal location (UniProt); granular cytoplasm observed (HPA tissue IHC) | |
| 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 paraffin-section fixation consistent across samples (standard IHC practice; not target-specific) | |
| Caveat | Lung macrophages also show high staining (HPA tissue IHC) | |
| Regulation | Expression regulation not established (UniProt) | |
| Isoform / epitope | No isoforms annotated; mature chain starts at residue 32 (UniProt) |
Section 1
Recommended GLA IHC & IF Protocols
The catalog antibody’s IHC-P protocol is followed by published GLA staining protocols for human ESCC sections and NHP liver (datasheet A01135-2; PMC13414882; 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 EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A01135-2). The published protocols used sodium citrate or Tris-EDTA pH 9 (PMC13414882; PMC11646755).
Section 2
What Is the Expected GLA Staining Pattern?
GLA is a lysosomal enzyme with no transmembrane segment (UniProt P06280: location and topology). In paraffin-section IHC, expect granular cytoplasmic staining, especially in glandular cells; lung macrophages and kidney collecting ducts are also reported as high (HPA: tissue IHC). HPA rates the tissue pattern Enhanced, while reporting medium consistency between antibody staining and RNA expression (HPA: reliability).
| Discrete cytoplasmic granules in glandular cells, with stronger signal than adjacent unstained cells. | This matches the reported granular pattern and high glandular-cell staining in several tissues (HPA: tissue IHC). Interpret granules in cell context rather than requiring every cell in a section to stain equally (HPA: cell-specific levels). |
| Predominantly nuclear or sharply continuous plasma-membrane staining. | Treat this as a compartment mismatch: GLA is lysosomal and has no transmembrane segment (UniProt P06280: location and topology). Check a known-positive section and detection controls before assigning the unexpected pattern to GLA (general IHC practice). |
| Strong signal in a cell population reported as not detected, such as adipocytes. | HPA reports GLA as not detected in adipocytes, not throughout all adipose tissue (HPA: tissue IHC). Consider cross-reactivity or detection chemistry; compare cell identity, a positive control, and a no-primary control (general IHC practice). |
| Uniform haze across cells and extracellular spaces obscures cytoplasmic granules. | This cannot be scored as the reported granular cytoplasmic pattern (HPA: tissue IHC). Diffuse background can arise from nonspecific antibody binding or the detection system; use controls to locate its source (general IHC practice). |
| No signal in glandular cells of a supplied high-staining tissue. | A negative result conflicts with that cell-level reference pattern (HPA: tissue IHC). First confirm the expected cells are present, then review antibody and detection performance; one negative section does not establish absent GLA protein (general IHC practice). |
💡Expected GLA appearanceCall a section positive when the expected cells show distinct, often strong granular cytoplasmic signal (HPA: high cell-level staining and tissue profile); isolated nuclear, membrane-like, or diffuse extracellular color is a pattern mismatch (UniProt P06280: lysosome and topology; HPA: tissue profile).
| Choice of reference cells | HPA reports high staining in lung macrophages and kidney collecting ducts, among other listed cells (HPA: tissue IHC). Its not-detected calls apply to named cell populations, so inspect the cell type before scoring a tissue (HPA: tissue IHC). |
| Processing and glycosylation | GLA has a signal peptide at residues 1–31, a mature chain at 32–429, and three annotated glycosylation sites (UniProt P06280: processing and glycosylation). The supplied record gives no antibody epitope, so these annotations cannot predict staining strength or retrieval needs. |
| Antibody evidence | Two listed antibodies, HPA000237 and HPA000966, have Enhanced IHC status (HPA: antibody validation). This supports the reported tissue pattern, while HPA still notes medium staining–RNA consistency (HPA: reliability); assess each new run with its own controls (general IHC practice). |
| IF/ICC: what pattern should be expected? | A lysosomal, punctate cytoplasmic pattern is biologically plausible (UniProt P06280: lysosome), but HPA provides no main ICC-IF location or cell images and lists no ICC status for these antibodies (HPA: subcellular and antibody records). Treat IF localization as unverified here. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive cells are unstained. | The expected high cell-level signal is missing (HPA: tissue IHC); the supplied sources do not identify a GLA-specific fixation or retrieval failure. | Confirm cell identity and run controls; review the catalog antibody's IHC-P instructions for retrieval, dilution, and detection settings (general IHC practice). |
| Color appears mainly in nuclei or along cell borders. | The compartment conflicts with lysosomal GLA and its lack of a transmembrane segment (UniProt P06280: location and topology). | Compare with a known-positive section and no-primary control; repeat or reassess the stain if the mismatch persists (general IHC practice). |
| Unexpected cells stain strongly. | HPA calls some named populations not detected, including adipocytes and cardiomyocytes (HPA: tissue IHC); off-target binding or detection background is possible (general IHC practice). | Verify the stained cell population, compare an HPA high-staining cell population, and inspect no-primary controls (general IHC practice). |
| Brown precipitate or broad haze masks cellular detail. | The appearance differs from granular cytoplasmic GLA staining (HPA: tissue IHC). In chromogenic IHC, nonspecific binding or endogenous detection activity can add background (general IHC practice). | Inspect no-primary and detection-only controls; adjust blocking, washing, or detection according to the system used (general IHC practice). |
| Only faint signal appears in a proposed negative comparator. | HPA not-detected calls concern specified cells; other cells in the same tissue may differ (HPA: tissue IHC). Chromogen intensity alone cannot identify the source (general IHC practice). | Score the named cell population separately and compare its morphology and signal with the positive control and background controls (general IHC practice). |
| IF shows puncta but the IHC reference cannot be reproduced. | HPA provides tissue IHC observations but no ICC-IF images or main subcellular location (HPA: tissue IHC and subcellular record). | Judge the paraffin-section result against IHC cell patterns and controls; evaluate IF separately with its own validation controls (general IHC/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 score staining in its glandular cells, where GLA is High (HPA: breast glandular cells, High). Use adipose tissue adipocytes as the biological comparison, where GLA is Not detected (HPA: adipose tissue adipocytes, Not detected); on the breast slide, assess nonglandular 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 a no-primary, secondary-only control, a concentration-matched rabbit IgG control matched to the primary antibody’s clonality, and GLA-knockout tissue if available (selected-SKU caption: rabbit primary antibody). Quench endogenous peroxidase and check endogenous biotin background in breast sections when using the caption’s biotin-based DAB detection (selected-SKU caption: biotinylated secondary, streptavidin-biotin complex, DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state the fixative (selected-SKU caption: fixative not stated). The reported IHC method used heat-mediated EDTA retrieval at pH 8.0, but whether retrieval is required for GLA is unreported (selected-SKU caption: EDTA pH 8.0 retrieval). The supplied evidence does not establish whether frozen sections or IF are easier; biotin-related background is a practical concern with the reported detection system (selected-SKU caption: streptavidin-biotin complex).
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 staining in paraffin-section IHC by checking retrieval, cell type, lysosomal pattern, detection controls, and scoring before interpreting differences.
How should I optimize retrieval when GLA staining is weak in paraffin sections?
Use heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A01135-2). The selected tissue-IHC example used this retrieval before 10% goat-serum blocking and 2 µg/ml primary antibody overnight at 4°C (datasheet A01135-2). If signal is weak, vary heating duration on adjacent sections while keeping antibody concentration and DAB development consistent, then inspect both staining and tissue morphology (standard IHC practice). Only after that, compare another retrieval buffer as a fallback, alongside a known positive control and a no-primary section (standard IHC practice).
Can fixation differences explain inconsistent GLA staining between sections?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (datasheet A01135-2). Record each specimen’s fixative, fixation duration, processing history, and section age before comparing staining, because those variables can affect antigen preservation and accessibility in IHC (standard IHC practice). Compare sections processed together using the same EDTA pH 8.0 retrieval and primary-antibody conditions (datasheet A01135-2; standard IHC practice). If results differ, repeat staining with matched controls and assess morphology before attributing the difference to GLA expression; HPA tissue patterns do not establish a fixation effect (HPA tissue IHC; standard IHC practice).
What staining pattern should count as plausible GLA localisation?
Expect a granular cytoplasmic pattern in paraffin-section IHC, consistent with lysosomal GLA and the reported tissue-IHC profile (UniProt P06280; HPA tissue IHC). Prioritize the relevant cells within each specimen: HPA reports high staining in breast glandular cells, kidney collecting ducts, and lung macrophages (HPA tissue IHC). Diffuse nuclear or crisp plasma-membrane staining deserves scrutiny because the annotated protein is lysosomal and lacks a transmembrane segment (UniProt P06280 topology; standard IHC practice). Compare the pattern with a no-primary control and, where available, a lysosomal marker on an adjacent section before scoring ambiguous granules (standard IHC practice).
Could processing or glycosylation change which GLA epitope the antibody detects?
GLA has one annotated protein sequence, with a signal peptide at residues 1–31 and a mature chain spanning 32–429; no isoforms are annotated (UniProt P06280). Three glycosylation sites are annotated at residues 139, 192, and 215 (UniProt P06280). Because the supplied caption does not identify the antibody’s epitope, do not infer whether it recognizes the signal peptide, mature chain, or a glycosylation-sensitive region (datasheet A01135-2; UniProt P06280). For unexpected IHC staining, check available epitope documentation and compare retrieval conditions on serial sections with the same detection controls (standard IHC practice).
How can IF help assess an ambiguous chromogenic GLA pattern?
Use IF as a separate follow-up assay to ask whether GLA-like puncta occur in the expected cells and overlap a lysosomal marker; GLA is annotated in lysosomes (UniProt P06280). Multiplex with a marker identifying the cell population under study, such as glandular cells or macrophages where the tissue context supports them (HPA tissue IHC; standard IF practice). Choose fluorophores and exposure settings after checking the specimen’s autofluorescence, and include single-stain and no-primary controls (standard IF practice). Permeabilise sufficiently to reach the lysosomal lumenal epitope, while checking that the chosen conditions preserve punctate morphology; the antibody epitope is unspecified in the supplied caption (UniProt P06280 topology; datasheet A01135-2; standard IF practice).
How do I separate GLA signal from chromogenic background?
The selected IHC example uses a biotinylated secondary, streptavidin-biotin complex, and DAB, so assess background at each detection stage (datasheet A01135-2; standard IHC practice). Run a no-primary section and inspect whether staining persists after the peroxidase block; endogenous peroxidase can produce DAB signal (standard IHC practice). If background follows the biotin-based detection system, evaluate an endogenous-biotin block or a biotin-free detection system with matched controls (standard IHC practice). Compare those controls with the expected granular cytoplasmic pattern, especially in strongly stained cell populations, before adjusting the 2 µg/ml primary concentration (HPA tissue IHC; datasheet A01135-2; standard IHC practice).
How should I score GLA across specimens with different cell composition? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, because GLA staining varies by cell type in the HPA tissue-IHC profile (HPA tissue IHC). Within that population, record the percentage of positive cells and intensity on a 0–3 scale, then calculate an H-score from 0–300 if intensity-weighted comparison is useful (standard IHC practice). Alternatively, count positive cells per mm² when cell density is the outcome, and report the number of eligible cells or analyzed area alongside each result (standard IHC practice). Keep retrieval, DAB development, counterstain, and positivity threshold consistent across sections, and normalize comparisons to the same eligible cell type rather than whole-section area (standard IHC practice).
When is apparent GLA positivity more likely to be an artefact?
A credible positive shows granular cytoplasmic staining in an appropriate cell population, consistent with lysosomal GLA and the HPA tissue-IHC profile (UniProt P06280; HPA tissue IHC). Be cautious with isolated nuclear or surface staining, uniform staining at section edges, or signal concentrated in damaged or necrotic regions; inspect morphology and matched controls before scoring it (UniProt P06280 topology; standard IHC practice). Persistent DAB signal in a no-primary section suggests a detection artefact, including possible endogenous peroxidase activity (standard IHC practice). HPA rates its tissue evidence as Enhanced but reports only medium agreement between staining and RNA, so an unexpected cell-specific result needs independent validation (HPA tissue IHC).
Boster reagents
Best GLA / Alpha-galactosidase A IHC Antibodies
For human samples, A01135-2 has a paraffin-section IHC figure; M01135 is listed for IHC and IF/ICC, with no IF figure supplied (A01135-2 image caption; M01135 catalog applications).
Real IHC data
Anti-Galactosidase alpha/Gla Antibody ®
Cat # A01135-2
A01135-2 will render with IHC staining of a paraffin-embedded human mammary cancer section (A01135-2 image caption). M01135 will render as a human-reactive IHC and IF/ICC option, but its payload supplies no staining image (M01135 catalog applications and reactivity).
Which to pick: Choose A01135-2 for tissue IHC when a documented paraffin-section example matters; its caption reports EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody, but does not report the fixative (A01135-2 image caption). Choose M01135 for IF/ICC because that application is listed and it is a rabbit monoclonal, clone 23G29; no IF image is supplied (M01135 catalog applications and clone). Neither SKU has documented cross-species reactivity here: 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 Lysosomal location (UniProt); granular cytoplasm observed (HPA tissue IHC). 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 paraffin-section fixation consistent across samples (standard IHC practice; not target-specific); Caveat: Lung macrophages also show high staining (HPA tissue IHC); Isoform / epitope: No isoforms annotated; mature chain starts at residue 32 (UniProt); Regulation: Expression regulation not established (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.