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ATG14 / Beclin 1-associated autophagy-related key regulator · IHC design guide
Design Immunohistochemistry for ATG14
ATG14 shows cytoplasmic and nuclear staining across tissues (HPA tissue IHC). Plan paraffin-section staining with the IHC-validated antibody PB9481 at 0.5–1 μg/mL, then score cellular distribution and intensity (datasheet PB9481).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ATG14 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 and nuclear staining in tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic and nuclear signal across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet PB9481) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation conditions consistent between samples. (standard IHC practice; not target-specific) | |
| Caveat | Autophagy stimuli may shift cytosolic signal to foci (UniProt) | |
| Regulation | Broad tissue expression (HPA tissue IHC) | |
| Isoform / epitope | 2 isoforms; antibody epitope coverage is unknown (UniProt) |
Section 1
Recommended ATG14 IHC & IF Protocols
The catalog antibody protocol uses citrate pH 6 retrieval (datasheet: PB9481). The published kidney section protocol below uses microwave Tri-EDTA retrieval (PMC7241494).
| Sample | Paraffin-embedded Rat Spleen tissue; fixative not specified (datasheet PB9481) |
| Fixation | Image fixative and duration unreported (datasheet PB9481); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet PB9481) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB9481) |
| Primary antibody | Rabbit anti-ATG14, 0.5-1μg/ml (datasheet PB9481) |
| Primary incubation | Overnight at 4 °C (datasheet PB9481) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9481) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | ATG14-positive staining in adipocytes of adipose tissue (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic and nuclear expression in all tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 retrieval for the catalog antibody (datasheet: PB9481); consider microwave Tri-EDTA retrieval when adapting the published kidney section protocol (PMC7241494).
Section 2
What Is the Expected ATG14 Staining Pattern?
ATG14 is associated with the cytoplasm, endoplasmic reticulum and autophagic membranes, and has no transmembrane segment (UniProt Q6ZNE5 topology and subcellular location). Expect staining across many cell types: HPA reports cytoplasmic and nuclear staining in all tissues, with medium staining in several listed populations (HPA tissue IHC). HPA rates its tissue IHC evidence Approved, with medium consistency against RNA expression (HPA tissue IHC).
| Cytoplasmic staining in adipocytes, respiratory epithelium or glandular cells; nuclear staining may accompany it. | This fits HPA's reported cytoplasmic and nuclear tissue pattern; those listed populations show Medium staining (HPA tissue IHC). Score each compartment separately. UniProt places ATG14 mainly in cytoplasmic and membrane-associated locations, so nuclear IHC alone is less persuasive (UniProt Q6ZNE5 subcellular location). |
| Distinct cytoplasmic foci appear, especially in a sample with documented autophagy induction. | Foci are biologically plausible: ATG14 shifts from cytosolic distribution toward isolation membranes and autophagosomes after starvation or rapamycin stimulation (UniProt Q6ZNE5 subcellular location). A routine tissue section has no stated induction status, so absence of obvious puncta does not invalidate otherwise appropriate staining. |
| Signal is confined to an unexpected compartment, such as sharply outlined cell borders, without a cytoplasmic pattern. | Treat this as a possible artefact and check controls. UniProt lists cytoplasmic and autophagic membrane locations (UniProt Q6ZNE5 subcellular location); HPA tissue IHC reports cytoplasmic and nuclear expression (HPA tissue IHC). HPA's plasma-membrane ICC-IF assignment is uncertain and does not establish a border-only IHC pattern (HPA subcellular). |
| Strong staining appears only in an unexpected cell population, or color spreads diffusely over cells and empty spaces. | An isolated cell-type mismatch raises cross-reactivity or endogenous detection activity; diffuse staining raises nonspecific background. Compare intact cell morphology and a no-primary control before assigning positivity (general IHC practice). HPA reports broad tissue expression, so an unfamiliar positive cell type alone cannot prove cross-reactivity (HPA tissue IHC). |
| No signal is visible in a section containing a listed Medium-staining population. | First check the assay and tissue quality rather than calling ATG14 absent. HPA reports Medium staining in, for example, adipocytes and bronchial respiratory epithelial cells (HPA tissue IHC). HPA's Approved rating has medium RNA agreement, so these are useful comparison populations, not guaranteed positive controls for every specimen (HPA tissue IHC). |
💡Expected ATG14 appearanceA convincing positive shows cellular cytoplasmic staining, sometimes with nuclear staining, in an HPA-listed population at an interpretable level around its reported Medium category; diffuse noncellular color or a border-only pattern is suspect (HPA tissue IHC; UniProt Q6ZNE5 subcellular location; general IHC practice).
| Compartment and stimulation | UniProt describes cytosolic ATG14 under nutrient-rich conditions and cytoplasmic foci after autophagy stimulation (UniProt Q6ZNE5 subcellular location). Record the specimen's treatment before interpreting a change from diffuse staining to puncta; tissue IHC alone does not establish autophagic flux. |
| Tissue comparator | HPA lists Medium staining in selected adipocytes, glandular cells, respiratory epithelial cells, glia and other populations, but Low staining in spleen red-pulp and marrow hematopoietic cells (HPA tissue IHC). Use these as relative expectations; neither Low group is a verified ATG14-negative control. |
| Antibody application | CAB037242 is Approved for IHC, whereas CAB080659 has Supported ICC evidence and no listed IHC status (HPA antibodies). Use IHC evidence to judge paraffin-section staining; ICC support does not validate an IHC result. The IHC rating is Approved, with medium RNA-expression consistency (HPA tissue IHC). |
| Isoforms and epitope information | UniProt lists two ATG14 isoforms but supplies no antibody epitope or isoform coverage here (UniProt Q6ZNE5 isoforms; HPA antibodies). An unexplained difference between antibodies cannot be assigned to isoform recognition from these records. No target-specific fixation or retrieval sensitivity is reported in the supplied sources. |
| IF/ICC Q: Should vesicle staining match IHC? | A: HPA supports vesicle localisation in U2OS ICC-IF images and labels plasma-membrane localisation uncertain (HPA subcellular). These images can inform compartment interpretation, but they do not set an IHC staining requirement or provide a paraffin-section protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| A listed Medium-staining population is blank. | The tissue or detection run may have failed; a blank section does not establish biological absence (HPA tissue IHC; general IHC practice). | Check section integrity, positive-run control and detection reagents; review the catalog antibody's IHC-P instructions for retrieval and working dilution (general IHC practice). |
| Every compartment looks uniformly brown. | Diffuse signal can reflect excess primary antibody, incomplete blocking or endogenous enzyme activity (general chromogenic IHC practice). | Run a no-primary control; optimize dilution and blocking, and quench endogenous peroxidase when using an HRP-based detector (general IHC practice). |
| Only nuclei stain intensely. | HPA reports nuclear expression, but nuclear-only staining lacks the accompanying cytoplasmic pattern in its tissue summary (HPA tissue IHC). | Check morphology and controls, then compare with cytoplasmic staining in a listed Medium population before accepting a nuclear-only result (HPA tissue IHC; general IHC practice). |
| Only crisp cell borders stain. | A border-only result is weakly supported: HPA calls plasma-membrane ICC-IF localisation uncertain (HPA subcellular). | Inspect a no-primary control and background at tissue edges; seek cellular cytoplasmic signal before scoring ATG14 positive (HPA tissue IHC; general IHC practice). |
| Spleen red pulp or marrow hematopoietic cells stain faintly. | Low staining is reported for these populations and need not mean assay failure (HPA tissue IHC). | Compare the run with a listed Medium population and keep intensity categories relative; do not use these Low populations as absolute negatives (HPA tissue IHC). |
| Puncta are absent despite a readable cytoplasmic signal. | Prominent foci are associated with autophagy stimulation; the supplied tissue IHC data do not specify that condition (UniProt Q6ZNE5 subcellular location; HPA tissue IHC). | Record treatment and score the observed cytoplasmic pattern. Avoid using puncta alone as a pass/fail criterion or as proof of autophagic flux (UniProt Q6ZNE5 subcellular location; general IHC practice). |
Experiment evidence
IHC & IF images showing ATG14 staining
PB9481 · IHCIHC analysis of ATG14L using anti-ATG14L antibody (PB9481). ATG14L was detected in paraffin-embedded section of Rat Spleen Tissue. Heat medi
PB9481 · IHCIHC analysis of ATG14L using anti-ATG14L antibody (PB9481). ATG14L was detected in paraffin-embedded section of Human Lung Cancer Tissue. He
PB9481 · IFIF analysis of ATG14L using anti-ATG14L antibody (PB9481). ATG14L was detected in an immunocytochemical section of U2OS cells. Enzyme antige
A03546 · IFImmunofluorescence of ATG14 in human small intestine tissue with ATG14 antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000126775-ATG14/tissue/cerebral+cortex#imid_9439312
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000126775-ATG14/tissue/colon#imid_9439222
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000126775-ATG14/tissue/liver#imid_9439223
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000126775-ATG14/tissue/kidney#imid_9439232
HPA · ICC-IF U2OSHuman Protein Atlas immunofluorescence, U2OS, antibody CAB080659; green = ATG14, blue = nucleus, red = microtubules.
HPA · ICC-IF U2OSHuman Protein Atlas immunofluorescence, U2OS, antibody CAB080659; green = ATG14, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ATG14 IHC & IF
🧪Run bronchus first and score respiratory epithelial cells, which show Medium ATG14 staining (HPA: bronchus, respiratory epithelial cells, Medium). HPA detects ATG14 in all 45 scored tissues, so use no-primary and isotype controls for negative staining; cells without specific signal on the bronchus slide should show counterstain alone, but no bronchial cell type is established as ATG14-negative (HPA: no negative tissue rows; standard IHC practice).
Positive control tissue: Adipose tissue (Adipocytes, HPA Medium)
Negative control tissue: None in HPA: ATG14 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ATG14 in U2OS, with annotated localisation: Vesicles (supported), Plasma membrane (uncertain) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide, a rabbit IgG control matched to the primary antibody’s clonality, and an ATG14 knockout specimen or validated peptide-block control (PB9481 caption: rabbit primary; standard IHC practice). Quench endogenous peroxidase and check endogenous biotin background when using the caption’s biotin–streptavidin DAB detection on bronchus (PB9481 caption: biotinylated secondary, SABC, DAB; standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected PB9481 paraffin-section caption does not state a fixative (PB9481 caption: fixative unreported). Heat retrieval in citrate buffer at pH 6 for 20 minutes is a documented starting condition, but the caption does not establish that retrieval is required (PB9481 caption: retrieval conditions). The supplied evidence does not establish whether frozen sections or IF are easier; in bronchus, distinguish intracellular epithelial staining from luminal deposits, and assess autofluorescence if using IF (standard IHC/IF practice).
HPA tissue IHC evidence for ATG14
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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Adipocytes | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: ATG14 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced ATG14 IHC Tips
Troubleshoot ATG14 staining in paraffin sections by controlling retrieval and detection, then interpreting signal against its expected localisation and tissue context.
How should I adjust retrieval when ATG14 staining is weak?
Start with heat-mediated citrate retrieval at pH 6 for 20 minutes in paraffin sections (datasheet PB9481; caption PB9481). The selected rat spleen image used that retrieval before 10% goat-serum blocking and 1 μg/ml primary antibody overnight at 4°C (caption PB9481). If staining is weak, first check section adherence, heating consistency, and whether the detection controls worked (standard IHC practice). Then compare retrieval durations on adjacent sections while holding antibody concentration and detection constant (standard IHC practice). Judge any gain by specific cellular staining and background together, since stronger DAB alone does not establish improved specificity (standard IHC practice).
Could fixation explain weak or variable ATG14 staining?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not report a fixative or fixation duration (caption PB9481). Record the fixative and processing history for each block before attributing a staining difference to ATG14 abundance (standard IHC practice). For a controlled comparison, use two differently fixed samples of the same specimen type when available, and process their sections together (standard IHC practice). Keep citrate retrieval at pH 6 for 20 minutes and the primary at 1 μg/ml during that comparison (caption PB9481). Compare cellular signal, morphology, and background; fixation effects cannot be established from tissue staining patterns alone (standard IHC practice).
Should ATG14 appear diffuse, punctate, or nuclear in tissue sections?
Expect cytoplasmic staining to be plausible because ATG14 is reported in cytoplasm and on endoplasmic-reticulum, preautophagosomal, and autophagosome membranes (UniProt Q6ZNE5 subcellular location). Nutrient-rich conditions favor cytosolic localisation, whereas autophagy stimuli can produce cytoplasmic foci (UniProt Q6ZNE5; PubMed:19050071). Human tissue IHC also reports cytoplasmic and nuclear expression across tissues, with medium consistency between staining and RNA data (HPA tissue IHC). Thus, nuclear DAB should be evaluated with cellular morphology and controls, rather than rejected solely by the UniProt localisation summary (HPA tissue IHC; standard IHC practice). Compare puncta only across sections processed and imaged comparably; a change in pattern alone does not measure autophagic flux (standard IHC practice).
Could isoforms or epitope masking change the staining pattern?
ATG14 has two listed isoforms and a 492-amino-acid canonical chain, but the supplied antibody caption does not map its epitope (UniProt Q6ZNE5 isoforms and processing; caption PB9481). Request the immunogen coordinates before claiming that this antibody distinguishes isoforms (standard IHC practice). Reported phosphorylation includes Ser29, Ser416, and Thr429; their effect on this antibody's staining is unknown without epitope information (UniProt Q6ZNE5 modified residues; caption PB9481). Compare adjacent sections with the same citrate pH 6 retrieval and consistent detection before interpreting differences as epitope loss (caption PB9481; standard IHC practice). If an independently validated antibody to another region is available, concordant cellular staining can strengthen an assignment, although it cannot identify an isoform by itself (standard IHC practice).
How can IF help assess ATG14 localisation alongside cell identity?
For a separate IF experiment, pair ATG14 with a marker of the expected cell type; a respiratory epithelial marker is pertinent when examining bronchus, where glandular cells show medium ATG14 staining (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and inspect unstained tissue in each channel before assigning signal, because tissue autofluorescence can obscure puncta (standard IF practice). ATG14 is intracellular and has no transmembrane segment, so use controlled permeabilisation to expose an intracellular epitope while checking that cellular structure remains intact (UniProt Q6ZNE5 subcellular location and topology; standard IF practice). HPA reports supported vesicular localisation in cell imaging, while plasma-membrane localisation is uncertain (HPA subcellular). The 1 μg/ml paraffin IHC condition is a starting reference, not an IF validation or established IF concentration (caption PB9481; standard IF practice).
How do I separate ATG14 staining from chromogenic background?
The selected image used 10% goat-serum blocking, a biotinylated secondary, a streptavidin-biotin complex, and DAB (caption PB9481). Include a no-primary section to reveal staining from detection reagents, and evaluate endogenous peroxidase and biotin where relevant to that workflow (standard IHC practice). Examine whether brown deposits follow cell boundaries or instead cluster at folds, cut edges, and damaged areas (standard IHC practice). In spleen, take particular care with weak signals: human red-pulp cells are listed as low by HPA, whereas the selected product image is rat spleen (HPA tissue IHC; caption PB9481). Reduce background through consistent washes and controlled DAB development, then compare matched sections before changing the primary concentration (standard IHC practice).
What is a defensible way to score ATG14 IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, since HPA describes both cytoplasmic and nuclear tissue staining (HPA tissue IHC). For a chosen population, record the percentage of positive viable cells and an intensity score, or calculate an H-score from 0–300 using the percentages at each intensity (standard IHC practice). If puncta are the endpoint, count them per cell or report puncta-positive cells rather than treating total DAB area as a puncta count (standard image-analysis practice). Normalise counts to the number of eligible cells, or area-based measures to mm² of viable tissue, using identical thresholds across groups (standard IHC practice). Keep retrieval, section thickness, illumination, and DAB development comparable, and report nuclear and cytoplasmic scores separately when both are analysed (standard IHC practice).
Which staining patterns support genuine ATG14 detection?
Cytoplasmic or vesicle-associated signal is biologically plausible for ATG14, and punctate localisation may accompany autophagy stimuli (UniProt Q6ZNE5 subcellular location; PubMed:19050071). Interpret a positive cell against its tissue context: HPA reports medium staining in bronchial respiratory epithelial cells but low staining in human spleen red-pulp cells (HPA tissue IHC). Nuclear staining requires careful control review because HPA tissue IHC reports it, while UniProt emphasises cytoplasmic and membrane-associated sites (HPA tissue IHC; UniProt Q6ZNE5 subcellular location). Distrust signal restricted to section edges, necrotic regions, or no-primary controls, and check endogenous enzyme activity in DAB workflows (standard IHC practice). A clean pattern still indicates antigen detection, not the rate of autophagic flux; interpreting pathway activity requires additional evidence (standard IHC interpretation).
Boster reagents
Best ATG14 / Beclin 1-associated autophagy-related key regulator IHC Antibodies
The catalog includes ATG14 antibodies with paraffin-section IHC images from rat spleen and human lung cancer tissue, plus IF images from human tissue and cells (PB9481 image captions; A03546 image caption).
Real IHC data
Anti-ATG14L Antibody ®
Cat # PB9481
Real IF data
Anti-ATG14 Antibody
Cat # A03546
PB9481 supports IHC in human and rat, with paraffin-section images from human lung cancer tissue and rat spleen (PB9481 applications, reactivity and IHC image captions). A03546 supports IF, with an image from human small intestine tissue; its listed reactivity is human, mouse and rat (A03546 applications, reactivity and IF image caption).
Which to pick: Choose PB9481 for chromogenic tissue IHC: its own captions document citrate retrieval at pH 6 and 1 μg/mL antibody on paraffin sections; the fixative is unreported (PB9481 IHC image captions). For IF/ICC, PB9481 has an ICC image in U2OS cells and IF images from paraffin tissue, while A03546 has a human small intestine IF image; both are rabbit antibodies, and clonality is unreported (PB9481 and A03546 catalog entries and image captions). For human and rat IHC, choose PB9481; A03546 lists mouse reactivity but provides no mouse IF image or IHC application (PB9481 and A03546 applications, reactivity and image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q6ZNE5 (BAKOR_HUMAN, Beclin 1-associated autophagy-related key regulator).
- Human Protein Atlas. ATG14 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ATG14 subcellular location (ICC-IF): Localized to the plasma membrane and vesicles..
- Human Protein Atlas. ATG14 antibody validation summary (2 antibodies).
- miR-25-3p promotes proliferation and inhibits autophagy of renal cells in polycystic kidney mice by regulating ATG14-Beclin 1. Renal failure 2020 — PMC7241494.
- Long non-coding RNA MLLT4 antisense RNA 1 induces autophagy to inhibit tumorigenesis of cervical cancer through modulating the myosin-9/ATG14 axis. Scientific reports 2024 — PMC10944523.
- PubMed PMID:10048485 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:12508121 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ATG14 (Beclin 1-associated autophagy-related key regulator). Expected localisation Cytoplasmic and nuclear staining in tissues (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet PB9481). Validated IHC antibody: PB9481 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded Rat Spleen tissue; fixative not specified (datasheet PB9481); 2. Fixation: Image fixative and duration unreported (datasheet PB9481); 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: Citrate pH 6 , 20 min (datasheet PB9481); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB9481); 8. Primary antibody: Rabbit anti-ATG14, 0.5-1μg/ml (datasheet PB9481); 9. Primary incubation: Overnight at 4 °C (datasheet PB9481). Watch for: Fixation: Keep fixation conditions consistent between samples. (standard IHC practice; not target-specific); Caveat: Autophagy stimuli may shift cytosolic signal to foci (UniProt); Isoform / epitope: 2 isoforms; antibody epitope coverage is unknown (UniProt); Regulation: Broad tissue expression (HPA tissue IHC). Evidence: UniProt Q6ZNE5, HPA tissue IHC · Approved, HPA ICC-IF · plasma membrane and vesicles., Datasheet PB9481, PMC7241494. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ATG14/immunohistochemistry-ihc-guide.