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ATG4B / Cysteine protease ATG4B · IHC design guide
Design Immunohistochemistry for ATG4B
Plan chromogenic IHC-P for ATG4B using appendix, colon and duodenum glandular cells as strong staining references (HPA tissue IHC). This guide covers fixation, antigen retrieval, the catalog antibody’s 5 μg/mL starting concentration (datasheet), and interpretation of nuclear staining flagged as off target (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
ATG4B 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 | Predominantly cytoplasmic in tissue IHC (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic; high in appendix, colon and duodenum 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 | 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 | Nuclear staining was disregarded as off target (HPA tissue IHC) | |
| Regulation | Expression regulation is not annotated (UniProt) | |
| Isoform / epitope | Five isoforms; antibody epitope coverage is unspecified (UniProt; datasheet) |
Section 1
Recommended ATG4B IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with four published ATG4B IHC protocols (PMC13313432; PMC9388977; PMC11416411; PMC6966594).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A02885); 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-ATG4B, 5 μg/mL (datasheet A02885) |
| 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 | ATG4B-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in a majority of tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen-retrieval rule); compare the published conditions for the relevant sample.
Section 2
What Is the Expected ATG4B Staining Pattern?
ATG4B is mainly cytoplasmic, including cytosolic, with smaller autophagosome, endoplasmic reticulum and mitochondrial pools; it has no transmembrane segment (UniProt Q9Y4P1 localization and topology). In paraffin tissue sections, expect cytoplasmic staining across many tissues, particularly glandular cells in appendix, colon and duodenum (HPA tissue IHC: High). HPA rates its tissue staining Approved, with medium agreement between staining and RNA expression and some presumed off-target binding (HPA tissue IHC).
| Glandular cells in appendix, colon or duodenum show clear cytoplasmic staining, stronger than adjacent background (HPA tissue IHC: High). | This matches the strongest supplied tissue observations and the main cytoplasmic localization (HPA tissue IHC; UniProt Q9Y4P1 localization). Assess the named cell population, because a tissue-level label alone can conceal different staining in neighboring cells. |
| Predominantly nuclear chromogenic staining appears, even when cytoplasmic staining is weak or absent. | Treat nuclear tissue IHC as suspect: HPA explicitly disregarded nuclear staining as off-target in its tissue assessment (HPA tissue IHC reliability description). HPA's separate ICC-IF assessment approves nucleoplasm and cytosol; that does not validate a nuclear paraffin-section IHC result (HPA subcellular ICC-IF). |
| Strong staining appears in adipocytes, cardiomyocytes or other cells listed as not detected. | Check whether signal follows the listed cell type rather than another cell in the section. HPA reports adipocytes in adipose tissue and cardiomyocytes in heart muscle as not detected (HPA tissue IHC). Unexpected signal may reflect cross-reactivity or detection background; those possibilities require controls before assignment. |
| A broad, even deposit covers cells and surrounding tissue without a clear cytoplasmic boundary. | This is poor evidence of ATG4B localization. HPA describes cytoplasmic expression in a majority of tissues, not uniform staining of every structure (HPA tissue IHC). Review background controls, blocking and detection conditions using standard IHC practice before scoring cells as positive. |
| The expected glandular cells in a positive-control section show no convincing cytoplasmic signal. | A negative run is inconclusive until the antibody and detection system work in the same run. Appendix, colon and duodenum glandular cells are High reference examples (HPA tissue IHC). Check section identity, reagent performance and the validated IHC-P procedure before interpreting test specimens as negative (standard IHC practice). |
💡Expected ATG4B appearanceCall an IHC result consistent with ATG4B when glandular cells in appendix, colon or duodenum show conspicuous cytoplasmic chromogen (HPA tissue IHC: High; UniProt Q9Y4P1 localization); predominantly nuclear tissue staining is a warning for off-target signal (HPA tissue IHC reliability description).
| Cell type and tissue context | HPA reports High staining in appendix, colon and duodenum glandular cells; Medium staining in hippocampal neurons, lung macrophages, lymph-node non-germinal-center cells, pancreatic exocrine cells and placental trophoblasts (HPA tissue IHC). Compare like cells, not whole-organ darkness. |
| Compartment and conditional mitochondrial localization | ATG4B is mainly cytoplasmic; a small portion localizes to mitochondria, promoted by Ser-34 phosphorylation (UniProt Q9Y4P1 localization). That conditional pool does not make diffuse nuclear tissue staining an expected positive pattern (HPA tissue IHC reliability description). |
| Assay-specific antibody validation | CAB037195 is Approved for IHC, while HPA069803 is Approved for ICC and has no listed IHC status (HPA antibody validation). Use the IHC-validated antibody's own IHC-P instructions for assay setup; ICC approval alone does not establish paraffin-section performance. |
| Low versus undetected cell populations | HPA lists low staining in cerebral-cortex neurons, thyroid and adrenal glandular cells, bronchial respiratory epithelium, gallbladder glandular cells and kidney tubule cells (HPA tissue IHC: Low). A faint result there needs more cautious scoring than a High reference cell. |
| Isoforms and epitope coverage | Five isoforms are listed, but the supplied record does not locate either antibody's epitope (UniProt Q9Y4P1 isoforms; HPA antibody validation). Do not assign an isoform to a stained cell or explain an absent signal by isoform choice without separate epitope evidence. |
| Situation | Likely cause | Next action |
|---|---|---|
| A positive-control glandular cell population remains blank. | The run may have failed, or the chosen antibody may lack demonstrated IHC performance; the slide alone cannot distinguish these causes (standard IHC practice; HPA antibody validation). | Confirm the named glandular cells and run controls, then follow the IHC-validated antibody's IHC-P instructions for retrieval and detection (HPA tissue IHC: High; standard IHC practice). |
| Nuclear staining dominates a paraffin-section slide. | HPA disregarded nuclear tissue staining as off-target, despite its separate ICC-IF nucleoplasm call (HPA tissue IHC reliability description; HPA subcellular ICC-IF). | Score cytoplasm separately from nuclei and compare with a High glandular reference and appropriate negative controls; do not count nuclear-only IHC as confirmation (HPA tissue IHC; standard IHC practice). |
| Chromogen appears in HPA-undetected cells or without primary antibody. | Signal in an undetected cell type conflicts with that HPA observation; signal without primary antibody indicates detection background (HPA tissue IHC: Not detected; standard IHC practice). | Check primary-omission and detection controls, including endogenous detection activity where relevant, before attributing signal to ATG4B (standard IHC practice). |
| Most structures carry diffuse brown background. | The pattern lacks the cellular cytoplasmic distribution described for tissue IHC; nonspecific binding or detection background is possible (HPA tissue IHC; standard IHC practice). | Inspect negative controls and optimize blocking, antibody concentration, washing and chromogen development within the assay instructions (standard IHC practice). |
| A weak signal appears in a tissue expected to stain strongly. | HPA levels apply to specified cells: a field without the listed glandular population may look weak (HPA tissue IHC: High). Run performance is another possibility (standard IHC practice). | Identify the relevant cells first, then compare a same-run High reference and review IHC-P conditions before changing interpretation (HPA tissue IHC; standard IHC practice). |
| Q: Does nuclear IF/ICC staining establish an expected nuclear IHC pattern? | A: No. HPA approves nucleoplasm and cytosol for ICC-IF, but disregards nuclear staining in tissue IHC as off-target (HPA subcellular ICC-IF; HPA tissue IHC reliability description). | Interpret each assay against its own validation and readout; use the separate IF/ICC guide for that application (HPA antibody validation; standard IHC/IF practice). |
Experiment evidence
IHC & IF images showing ATG4B staining
A02885 · IHCImmunohistochemistry of ATG4B in human spleen tissue with ATG4B antibody at 5 μg/ml.
A02885 · IFImmunofluorescence of ATG4B in human spleen tissue with ATG4B antibody at 20 μg/ml.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000168397-ATG4B/tissue/cerebral+cortex#imid_21016667
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000168397-ATG4B/tissue/colon#imid_21017449
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000168397-ATG4B/tissue/liver#imid_21017450
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000168397-ATG4B/tissue/kidney#imid_21017451
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA069803; green = ATG4B, blue = nucleus, red = microtubules.
HPA · ICC-IF MCF-7Human Protein Atlas immunofluorescence, MCF-7, antibody HPA069803; green = ATG4B, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for ATG4B IHC & IF
🧪Run colon first: glandular cells should stain (HPA: High in colon glandular cells). Use adipose tissue adipocytes as the negative comparison (HPA: Not detected in adipocytes); on the colon slide, assess non-glandular cells for background staining, but do not assume they are ATG4B-negative because the supplied HPA row characterizes only glandular cells (HPA: colon glandular cells).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ATG4B in MCF-7, RT-4, with annotated localisation: Nucleoplasm (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and clonality (standard IHC practice). Process ATG4B-knockout material alongside the samples as a biological negative; quench endogenous peroxidase in colon sections and check luminal mucus or debris for retained chromogen (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and the effect of antigen retrieval are unreported in the supplied evidence; optimize retrieval empirically for paraffin IHC (supplied evidence; standard IHC practice). The selected A02885 spleen IHC caption does not state a fixative (caption: fixative not stated). Frozen sections or IF cannot be judged easier from these data; IF/ICC images exist for MCF-7 and RT-4, while luminal mucus or debris can complicate interpretation of colon chromogenic staining (HPA subcellular; standard IHC practice).
HPA tissue IHC evidence for ATG4B
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded. Nuclear staining was considered off target and disregarded.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | Medium | Protein (IHC) | HPA → |
| Lung | Macrophages | Medium | 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 → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced ATG4B IHC Tips
Troubleshoot ATG4B staining by checking retrieval, cellular compartment, antibody specificity, and scoring against documented tissue patterns.
Which retrieval conditions should I try first for weak ATG4B staining?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval rule: cytoplasmic antigen). If signal remains weak, compare a shorter and a longer heating period on serial sections while holding antibody concentration and detection conditions constant (standard IHC practice). Use the same glandular cell region for each comparison; high staining is reported in appendix, colon, and duodenum glandular cells (HPA: tissue IHC). If citrate retrieval still fails, test EDTA at pH 9 as a fallback and assess tissue damage and background alongside signal (standard IHC practice).
Could fixation explain inconsistent ATG4B staining between paraffin blocks?
Target-specific sensitivity of ATG4B staining to fixation is unknown from the supplied evidence; the catalog antibody’s spleen caption does not state a fixative (catalog antibody A02885: spleen tissue IHC caption). Record fixative, fixation duration, block age, and processing history for each specimen before comparing staining intensity (standard IHC practice). Compare sections processed together and stained in one run, using a control block with a previously established cytoplasmic pattern (standard IHC practice; HPA: cytoplasmic tissue expression). If fixation histories differ, optimize retrieval on representative blocks and report that difference rather than assigning it a target-specific mechanism (standard IHC practice).
How should I evaluate punctate or nuclear ATG4B staining?
Expect predominantly cytoplasmic staining in tissue sections (HPA: cytoplasmic expression in a majority of tissues; UniProt Q9Y4P1: mainly cytoplasmic). ATG4B is also annotated at autophagosomes, endoplasmic reticulum, and mitochondria, with Ser-34 phosphorylation promoting mitochondrial localization (UniProt Q9Y4P1: subcellular location and modified residue). Treat puncta as a pattern requiring confirmation with an organelle marker and appropriate imaging resolution, rather than identifying every punctum as an autophagosome (standard IHC practice). Treat nuclear tissue staining cautiously: it was disregarded as presumed off-target staining in the tissue assessment, although nucleoplasmic localization was approved in separate cellular imaging (HPA: tissue IHC reliability; HPA: subcellular).
Can this stain distinguish ATG4B isoforms or modified epitopes?
ATG4B has 5 listed isoforms, but the supplied catalog caption does not identify the antibody epitope (UniProt Q9Y4P1: isoforms; catalog antibody A02885: spleen tissue IHC caption). Do not assign a paraffin-section signal to a particular isoform without epitope mapping and isoform-specific validation (standard IHC practice). The protein has no annotated transmembrane segment or propeptide, while reported modifications include Ser-34 phosphorylation and cysteine nitrosylation (UniProt Q9Y4P1: topology, processing, modified residues). If modification-dependent recognition matters, compare validated modification-specific reagents or orthogonal assays; ordinary total-protein chromogenic staining cannot establish modification state on its own (standard IHC practice).
How can I check an IHC pattern with multiplex IF?
For a separate IF experiment, pair ATG4B with a validated glandular cell marker when checking the strongly stained glandular compartments reported in tissue IHC (HPA: appendix, colon, and duodenum glandular cells). Choose a far-red channel for a dim signal when tissue autofluorescence is prominent, and include single-stain and no-primary controls for channel bleed-through and background (standard IF practice). Because ATG4B lacks a transmembrane segment and is mainly cytosolic, use mild permeabilisation to reach its expected intracellular epitope, then optimize against cell morphology (UniProt Q9Y4P1: topology and localization; standard IF practice). Interpret nuclear IF separately from chromogenic tissue staining because the two HPA assessments differ (HPA: subcellular; HPA: tissue IHC reliability).
What should I adjust when brown staining obscures cytoplasmic ATG4B?
Compare a no-primary control with the test section to identify staining from the detection system, endogenous peroxidase, or pigment (standard chromogenic IHC practice). Apply a peroxidase block before DAB development and inspect whether brown deposits persist without primary antibody (standard chromogenic IHC practice). If background follows the primary antibody, titrate concentration below the catalog image condition of 5 µg/ml while monitoring cytoplasmic signal in a matched control section (catalog antibody A02885: spleen tissue IHC caption; standard IHC practice). Score nuclear staining separately because the tissue assessment disregarded it as presumed off-target staining (HPA: tissue IHC reliability).
How should I quantify ATG4B across sections with different cellularity? ⚠ ANSWER MARKED FOR VERIFICATION
Define the relevant cell compartment before scoring, since the tissue profile emphasizes cytoplasmic expression (HPA: tissue IHC profile). For a selected cell population, report the percentage of cytoplasm-positive cells and an H-score calculated from intensity categories and their percentages; document thresholds before reviewing groups (standard IHC practice). Normalize counts to the number of eligible cells or sampled tissue area, such as cells per mm², and keep regions, exposure, and DAB development comparable (standard IHC practice). Stratify by cell type where composition differs, because high glandular staining and absent adipocyte staining are both documented (HPA: tissue IHC).
Which findings support genuine ATG4B staining rather than artefact?
Give greatest weight to reproducible cytoplasmic staining in an appropriate cell population: glandular cells in appendix, colon, and duodenum are reported as high, whereas adipocytes are not detected (HPA: tissue IHC). Check that the signal persists away from cut edges and necrotic regions, where processing and nonspecific deposition can distort chromogenic staining (standard IHC practice). Nuclear staining alone is weak evidence for tissue ATG4B because the tissue assessment disregarded presumed off-target nuclear signal (HPA: tissue IHC reliability). Compare a no-primary section and peroxidase-blocked section to exclude endogenous enzyme activity, then seek an independent antibody or orthogonal assay for a consequential finding (standard IHC practice).
Boster reagents
Best ATG4B / Cysteine protease ATG4B IHC Antibodies
A02885 has IHC and IF images from human spleen tissue (catalog image captions); its listed reactivity covers human, mouse, and rat (catalog reactivity).
Real IHC data
Anti-Cysteine protease ATG4B ATG4B Antibody
Cat # A02885
A02885 is listed for IHC-P and IF (catalog applications). Its IHC and IF images show human spleen tissue at 5 μg/mL and 20 μg/mL, respectively (catalog image captions).
Which to pick: For tissue IHC, choose A02885: it is listed for IHC-P, and its IHC image shows human spleen tissue at 5 μg/mL (catalog applications; catalog IHC image caption). For IF, A02885 has a human spleen tissue image at 20 μg/mL; ICC validation and clone type are unreported (catalog IF image caption; catalog applications; catalog clone field). A02885 also lists mouse and rat reactivity, but the supplied images show human tissue only; the fixative is unreported (catalog reactivity; catalog image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y4P1 (ATG4B_HUMAN, Cysteine protease ATG4B).
- Human Protein Atlas. ATG4B tissue IHC expression (reliability: Approved).
- Human Protein Atlas. ATG4B subcellular location (ICC-IF): Localized to the nucleoplasm and cytosol..
- Human Protein Atlas. ATG4B antibody validation summary (2 antibodies).
- Upregulated ATG4B predicts poor prognosis and correlates with angiogenesis in osteosarcoma. Journal of the Egyptian National Cancer Institute 2025 — PMC13313432.
- Nitidine chloride induces cardiac hypertrophy in mice by targeting autophagy-related 4B cysteine peptidase. Acta pharmacologica Sinica 2023 — PMC9388977.
- TDP-43 regulates LC3ylation in neural tissue through ATG4B cryptic splicing inhibition. Acta neuropathologica 2024 — PMC11416411.
- Association of ATG4B and Phosphorylated ATG4B Proteins with Tumorigenesis and Prognosis in Oral Squamous Cell Carcinoma. Cancers 2019 — PMC6966594.
- PubMed PMID:12446702 — UniProt-cited evidence.
- PubMed PMID:30808002 — UniProt-cited evidence.
- PubMed PMID:15169837 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for ATG4B (Cysteine protease ATG4B). Expected localisation Predominantly cytoplasmic in tissue IHC (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A02885 at 5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. 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 A02885); 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-ATG4B, 5 μg/mL (datasheet A02885); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Nuclear staining was disregarded as off target (HPA tissue IHC); Isoform / epitope: Five isoforms; antibody epitope coverage is unspecified (UniProt; datasheet); Regulation: Expression regulation is not annotated (UniProt). Evidence: UniProt Q9Y4P1, HPA tissue IHC · Approved, HPA ICC-IF · nucleoplasm and cytosol., Datasheet A02885, PMC13313432, PMC9388977. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/ATG4B/immunohistochemistry-ihc-guide.