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ATG5 / Autophagy protein 5 · IHC design guide
Design Immunohistochemistry for ATG5
Plan ATG5 IHC-P using the reported cytoplasmic staining in intestinal glandular cells (HPA tissue IHC). Compare colon glandular cells reported as high with cardiomyocytes reported as undetected, and account for stronger protein expression in apoptotic cells (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ATG5 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 intestinal glandular cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Bone marrow+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A00240) | |
| Caveat | Apoptotic cells may stain much more strongly (UniProt) | |
| Regulation | Protein rises sharply in apoptotic cells (UniProt) | |
| Isoform / epitope | 2 isoforms, Long and Short; epitope coverage unknown (UniProt) |
Section 1
Recommended ATG5 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with 3 published ATG5 IHC protocols (PMC7609390; PMC5489357; PMC5458246).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A00240); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-ATG5, 2.5 μg/mL (datasheet A00240) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ATG5-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues, mainly in intestines and thyroid gland. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min, as specified for this page’s cytoplasmic antigen (page retrieval rule; UniProt Q9H1Y0).
Section 2
What Is the Expected ATG5 Staining Pattern?
ATG5 is cytoplasmic and associates with preautophagosomal membranes; it has no transmembrane segment (UniProt Q9H1Y0). In paraffin-section IHC, expect cytoplasmic staining in several tissues, including glandular cells of the colon, duodenum and rectum and respiratory epithelial cells of the bronchus (HPA: tissue IHC). HPA rates the tissue staining Approved, with medium consistency between staining and RNA expression; interpret intensity alongside cell identity and controls (HPA: reliability).
| Clear cytoplasmic staining in colon or duodenal glandular cells, with identifiable cell boundaries. | This fits the reported high signal in those cells and ATG5's cytoplasmic location (HPA: High in colon and duodenum glandular cells; UniProt Q9H1Y0). Compare cells within the same section before judging overall intensity. |
| A predominantly nuclear pattern replaces the expected cytoplasmic signal. | Treat dominant nuclear staining as an unexpected pattern requiring a specificity check, rather than as evidence of ATG5 redistribution (UniProt Q9H1Y0: cytoplasm; HPA: cytoplasmic tissue profile). Review the negative control and staining distribution. |
| Strong staining appears mainly in a cell population reported as not detected. | For example, signal in heart muscle cardiomyocytes conflicts with the HPA observation and warrants checks for cross-reactivity or endogenous detection activity (HPA: cardiomyocytes Not detected). A discordant cell alone does not prove either cause. |
| Diffuse colour covers stroma, lumens or the slide without a clear cellular pattern. | This is difficult to score as ATG5: the reference pattern is cellular and cytoplasmic (HPA: tissue IHC profile; UniProt Q9H1Y0). Uneven background can arise from detection or wash conditions (general IHC practice). |
| No cytoplasmic signal is visible in an otherwise intact positive tissue section. | First check the assay with a reported high-signal cell population, such as colon glandular cells (HPA: High in colon glandular cells). A blank slide is inconclusive until section integrity, detection and antibody performance are checked (general IHC practice). |
💡Expected ATG5 appearanceCall a result positive when identifiable glandular or respiratory epithelial cells show cytoplasmic signal, strongest in HPA-reported high groups; dominant nuclear staining or broad acellular colour is suspect (HPA: tissue IHC profile; UniProt Q9H1Y0).
| Cell-type reference and reliability | HPA reports high signal in appendix, colon, duodenum and rectum glandular cells and bronchus respiratory epithelium; its Approved tissue profile has medium staining–RNA consistency (HPA: tissue IHC). Use these as pattern references, not guaranteed results for every specimen. |
| Low or undetected reference groups | HPA reports low signal in kidney tubule cells and adipocytes, and no detected signal in cardiomyocytes or skeletal myocytes (HPA: tissue IHC). These observations help assess contrast, but HPA also reports low tissue RNA specificity; absence in one cell group does not establish a universal negative. |
| Subcellular context | ATG5 is cytoplasmic and associates with preautophagosomal membranes; its conjugate can leave those membranes near autophagosome completion (UniProt Q9H1Y0). Routine chromogenic IHC should be judged by cellular compartment and morphology rather than requiring resolvable puncta (general IHC practice). |
| Molecular forms | UniProt lists Long and Short isoforms and ATG12 conjugation (UniProt Q9H1Y0). The supplied evidence gives no antibody epitope or isoform coverage, so an intensity difference cannot be assigned to one form from IHC appearance alone. |
| IF/ICC Q&A | Q: Should IF/ICC show the same broad pattern as tissue IHC? A: HPA reports approved localization at the centrosome, primary cilium transition zone and basal body, with additional vesicles (HPA: subcellular ICC-IF). These higher-resolution observations inform interpretation; they do not set a required chromogenic IHC puncta pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known high-signal glandular cells are blank. | The result conflicts with the HPA reference, but the supplied sources do not identify a target-specific fixation or retrieval failure (HPA: High in colon glandular cells). | Check tissue preservation, primary-antibody application, detection reagents and a concurrent positive control; review the assay's validated retrieval conditions if available (general IHC practice). Do not infer ATG5 fixation sensitivity from this result. |
| Nuclei stain more strongly than cytoplasm. | The compartment conflicts with the reported cytoplasmic tissue pattern (HPA: tissue IHC profile; UniProt Q9H1Y0). Background or cross-reactivity remains possible; the pattern alone cannot identify which. | Compare a no-primary control and a reported high-signal tissue, then score signal by compartment and cell type (general IHC practice; HPA: High in colon glandular cells). |
| Broad brown haze obscures cell boundaries. | Acellular or diffuse colour does not match the HPA cellular cytoplasmic profile (HPA: tissue IHC); detection background or insufficient washing can obscure localization (general IHC practice). | Inspect the no-primary control and tissue edges, review blocking and wash steps, and only interpret cells whose boundaries remain clear (general IHC practice). |
| A reported undetected cell population stains strongly. | Signal in cardiomyocytes or skeletal myocytes disagrees with those HPA observations (HPA: Not detected in cardiomyocytes and skeletal myocytes). Cross-reactivity or endogenous detection activity is possible, not proven. | Check a no-primary control, compare a reported high-signal cell group in the same run, and investigate whether the unexpected signal follows tissue rather than cellular boundaries (general IHC practice; HPA: tissue IHC). |
| Only a few fine puncta are visible, without convincing cytoplasmic cell staining. | UniProt describes preautophagosomal association, while HPA's centrosome and cilium observations come from ICC-IF (UniProt Q9H1Y0; HPA: subcellular ICC-IF). Puncta alone are insufficient to establish the expected tissue IHC result. | Reassess morphology and the broader cytoplasmic pattern in a reported high-signal cell type; compare controls before scoring puncta as specific (HPA: tissue IHC; general IHC practice). |
| Different specimens show unequal staining intensity. | HPA records high, low and undetected cell groups, and rates tissue staining–RNA consistency as medium (HPA: tissue IHC; HPA: reliability). Intensity variation alone does not demonstrate a change in autophagic activity. | Compare the same cell type under matched staining and scoring conditions, document the compartment, and use appropriate controls before assigning biological meaning (general IHC practice; UniProt Q9H1Y0: cytoplasm). |
Experiment evidence
IHC & IF images showing ATG5 staining
A00240 · IHCImmunohistochemistry of ATG5 in human spleen tissue with ATG5 antibody at 2.5 μg/mL.
A00240 · IFImmunofluorescence of ATG5 in Human Spleen cells with ATG5 antibody at 20 μg/mL.
M00240 · IHCImmunohistochemical analysis of paraffin-embedded human ovarian cancer, using ATG5 Antibody.
M00240 · IFImmunofluorescent analysis of Raji cells, using ATG5 Antibody.
M00240-2 · IHCHuman colon was stained with anti-ATG5 rabbit antibody
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000057663-ATG5/tissue/cerebral+cortex#imid_7669704
HPA · Thyroid glandHuman Protein Atlas IHC, Thyroid gland. Source: https://www.proteinatlas.org/ENSG00000057663-ATG5/tissue/thyroid+gland#imid_7669664
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000057663-ATG5/tissue/colon#imid_7669790
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000057663-ATG5/tissue/liver#imid_7669783
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA042973; green = ATG5, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA042973; green = ATG5, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ATG5 IHC & IF
🧪Run colon first; glandular cells should stain strongly (HPA: High in colon glandular cells). Use heart muscle cardiomyocytes as a negative comparator (HPA: Not detected in cardiomyocytes); on the colon slide, cells outside the stained glands should show only background signal if used as internal negative comparators, since their ATG5 status is not specified by the supplied HPA row.
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ATG5 in A-431, U-251MG, U2OS, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), Sperm, with annotated localisation: Primary cilium transition zone (approved), Centrosome (approved), Basal body (approved) (HPA subcellular).
Technical controls: Include a no-primary (secondary-only) slide, a control immunoglobulin matched to the primary antibody’s host species and class or clonality as applicable, and ATG5-knockout material or a validated peptide-block control (standard IHC practice). Block endogenous peroxidase for chromogenic detection and check colon mucin and inflammatory cells for background staining (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and retrieval dependency are unreported in the supplied evidence; optimize antigen retrieval empirically for paraffin sections. IF may help resolve punctate or ciliary signal (HPA subcellular: vesicles and primary cilium transition zone), but the evidence does not establish that frozen sections are easier; colon mucin can contribute background staining (standard IHC practice). The fixative is unreported in the selected A00240 spleen IHC caption (caption: fixative not stated).
HPA tissue IHC evidence for ATG5
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — 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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | 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 → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ATG5 IHC Tips
Troubleshoot ATG5 staining in paraffin sections by checking retrieval, cellular pattern, controls and scoring before interpreting changes in signal.
Which retrieval conditions should I try first for weak ATG5 staining?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval rule: cytoplasmic/membrane antigen). Let sections cool in the retrieval solution before washing, and keep section thickness, antibody concentration and detection conditions constant while comparing runs (standard IHC practice). ATG5 lacks a transmembrane segment, so a weak result alone does not establish that a membrane epitope needs harsher retrieval (UniProt Q9H1Y0 topology). Include a previously stained positive section and a primary-antibody omission control to separate retrieval failure from detection background (standard IHC practice). If staining remains weak, test an alternative retrieval buffer as a documented comparison and check tissue preservation before selecting it (standard IHC practice).
Could fixation explain inconsistent ATG5 staining between paraffin blocks?
Target-specific ATG5 sensitivity to fixation is unknown from the supplied evidence; the selected spleen caption does not state its fixative (A00240 tissue-IHC caption). Record each block’s fixative and fixation duration, then compare sections using the same pH 6.0, 95–98 °C, 20 min retrieval conditions (page retrieval rule: cytoplasmic/membrane antigen; standard IHC practice). Examine morphology alongside staining: poorly preserved cells, detached sections or uneven staining limit comparisons between blocks (standard IHC practice). Run a reference block through each staining batch to identify changes in processing or detection (standard IHC practice). Do not assign a particular ATG5 signal change to fixation until matched material supports that conclusion (standard IHC practice).
Where should convincing ATG5 staining appear in tissue sections?
Expect predominantly cytoplasmic tissue staining, while recognising that ATG5 also associates with preautophagosomal structures (HPA tissue IHC: cytoplasmic expression; UniProt Q9H1Y0 subcellular localisation). Evaluate the pattern within intact cells rather than counting diffuse chromogen over stroma, folds or torn section edges (standard IHC practice). HPA reports high staining in colon glandular cells and bronchial respiratory epithelial cells, providing specified cell populations for pattern comparison (HPA tissue IHC: High). ATG5 can localise to discrete ciliary puncta, but routine chromogenic resolution may not establish their identity without an appropriate companion marker (UniProt Q9H1Y0 subcellular localisation; standard IHC practice). Strong, isolated nuclear staining warrants review with omission controls and another validated readout (standard IHC practice).
Can this stain distinguish ATG5 isoforms or conjugated ATG5?
The record lists 2 isoforms, Long and Short, but supplies no antibody epitope map or isoform-specific staining evidence (UniProt Q9H1Y0 isoforms; supplied antibody evidence). ATG5 also forms an ATG12–ATG5 conjugate, so chromogenic signal cannot by itself identify which molecular form contributed to staining (UniProt Q9H1Y0 subunit; standard IHC interpretation). Check the catalog antibody’s epitope documentation before making isoform claims, and keep retrieval and detection settings matched across specimens (standard IHC practice). Compare a positive tissue area with a primary-antibody omission control to assess whether the cellular pattern is credible (standard IHC practice). Describe results as ATG5 immunoreactivity unless form-specific validation is available (standard IHC interpretation).
How should I investigate ATG5 localisation by multiplex IF?
On the separate IF/ICC workflow, pair ATG5 with a marker identifying the expected cell population and use a nuclear counterstain to assign signal to individual cells (standard IF practice). Choose fluorophores after checking the specimen’s autofluorescence, reserving a spectrally distinct, brighter channel for weak ATG5 signal (standard IF practice). ATG5 has no transmembrane segment; permeabilisation should therefore be selected for the antibody’s documented epitope accessibility, with a mild condition tested before increasing detergent exposure (UniProt Q9H1Y0 topology; standard IF practice). HPA reports approved centrosome, basal-body and primary-cilium transition-zone localisation, with vesicles as an additional location (HPA subcellular). Include single-colour and secondary-only controls when evaluating apparent puncta or overlap (standard IF practice).
What should I check when ATG5 chromogen obscures cellular staining?
First inspect a primary-antibody omission section to identify signal from detection reagents or endogenous tissue activity (standard IHC practice). For peroxidase-based chromogenic detection, check the peroxidase block and shorten chromogen development if the control accumulates colour (standard IHC practice). If background appears only with the primary antibody, titrate its concentration and review blocking and wash conditions while keeping retrieval fixed at pH 6.0 (page retrieval rule: cytoplasmic/membrane antigen; standard IHC practice). Judge improvement by clearer cytoplasmic boundaries in an expected positive cell population, such as colon glandular cells (HPA tissue IHC: High). Avoid scoring pigment, section folds or edge deposits as ATG5-positive cells (standard IHC practice).
How can I compare ATG5 staining across paraffin specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and analysis region before scoring; HPA reports high ATG5 staining in colon glandular cells, making that population an evidence-based example (HPA tissue IHC: High). For chromogenic sections, report the percentage of positive cells and, when intensity is reproducible, an H-score across the same cell type (standard IHC practice). For discrete positive profiles, density per mm² can supplement cell-based scoring if the denominator is clearly defined (standard IHC practice). Normalise counts to eligible, viable cells or measured tissue area, and exclude necrosis, folds and section edges consistently (standard IHC practice). Keep staining batches, imaging settings and scoring thresholds aligned, with reference sections to monitor drift (standard IHC practice).
When is an ATG5-positive IHC pattern convincing rather than artefactual?
A convincing result shows cellular cytoplasmic staining in a plausible population, such as glandular cells of the colon or appendix (HPA tissue IHC: cytoplasmic expression; HPA tissue IHC: High). Compare intact tissue with section edges and necrotic areas; signal restricted to damaged regions is less persuasive (standard IHC practice). Review a primary-antibody omission control for endogenous enzyme or detection signal, especially when colour appears outside cells (standard IHC practice). Predominantly nuclear staining needs independent validation because the supplied localisation evidence describes cytoplasm and preautophagosomal structures, with ciliary sites also reported (UniProt Q9H1Y0 subcellular localisation). Interpret intensity cautiously: HPA rates tissue staining as Approved with medium consistency against RNA expression (HPA tissue IHC reliability).
Boster reagents
Best ATG5 / Autophagy protein 5 IHC Antibodies
Anti-ATG5 antibodies have IHC images from human spleen, ovarian cancer paraffin sections, and colon (catalog IHC captions); IF images show human spleen cells, HeLa cells, and Raji cells (catalog IF captions).
Real IHC data
Anti-Autophagy protein 5 ATG5 Antibody
Cat # A00240
Real IHC data
Anti-ATG5/Apg5 Rabbit Monoclonal Antibody
Cat # M00240
Real IHC data
Anti-ATG5 Rabbit Monoclonal Antibody
Cat # M00240-2
A00240 has human spleen IHC and human spleen cell and HeLa IF images; M00240 has a paraffin-embedded human ovarian cancer IHC image and a Raji cell IF image (catalog image captions). M00240-2 has a human colon IHC image and lists IF among its applications, without an IF image in the payload (catalog image captions; catalog applications).
Which to pick: For paraffin tissue IHC, choose M00240: its own image caption identifies a paraffin-embedded human ovarian cancer section; the fixative is unreported (M00240 IHC image caption). For IF/ICC, choose M00240 because both applications are listed and its IF image shows Raji cells (M00240 catalog applications; M00240 IF image caption). For work across species, A00240, M00240, and M00240-2 each list Human, Mouse, and Rat reactivity, while their supplied IHC images show human samples (catalog reactivity; catalog IHC image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9H1Y0 (ATG5_HUMAN, Autophagy protein 5).
- Human Protein Atlas. ATG5 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ATG5 subcellular location (ICC-IF): Mainly localized to the centrosome, primary cilium transition zone and basal body. In addition localized to vesicles..
- Human Protein Atlas. ATG5 antibody validation summary (3 antibodies).
- Pathophysiological role of Atg5 in human ulcerative colitis. Intestinal research 2020 — PMC7609390.
- Regulated in Development and DNA Damage Response 1 Deficiency Impairs Autophagy and Mitochondrial Biogenesis in Articular Cartilage and Increases the Severity of Experimental Osteoarthritis. Arthritis & rheumatology (Hoboken, N.J.) 2017 — PMC5489357.
- Ligustrazine protects against chronic hypertensive glaucoma in rats by inhibiting autophagy via the PI3K-Akt/mTOR pathway. Molecular vision 2021 — PMC8711580.
- Negative LC3b immunoreactivity in cancer cells is an independent prognostic predictor of prostate cancer specific death. Oncotarget 2017 — PMC5458246.
- PubMed PMID:9563500 — UniProt-cited evidence.
- PubMed PMID:17974005 — UniProt-cited evidence.
- PubMed PMID:14574404 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ATG5 (Autophagy protein 5). Expected localisation Cytoplasmic staining in several tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A00240 at 2.5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Bone marrow. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A00240); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-ATG5, 2.5 μg/mL (datasheet A00240); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A00240); Caveat: Apoptotic cells may stain much more strongly (UniProt); Isoform / epitope: 2 isoforms, Long and Short; epitope coverage unknown (UniProt); Regulation: Protein rises sharply in apoptotic cells (UniProt). Evidence: UniProt Q9H1Y0, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the centrosome, primary cilium transition zone…, Datasheet A00240, PMC7609390, PMC5489357. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ATG5/immunohistochemistry-ihc-guide.