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TBC1D5 / TBC1 domain family member 5 · IHC design guide
Design Immunohistochemistry for TBC1D5
Plan paraffin-section TBC1D5 IHC using the catalog antibody’s documented conditions (datasheet A09263). Compare cytoplasmic staining in glandular cells with appropriate controls, since tissue staining has uncertain reliability (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
TBC1D5 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 | General cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Glandular cells show cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A09263) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Adipose tissue |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Tissue staining has uncertain reliability (HPA tissue IHC) | |
| Regulation | Starvation shifts compartmental signal (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unknown (UniProt) |
Section 1
Recommended TBC1D5 IHC & IF Protocols
The catalog antibody has an IHC-P protocol (datasheet: A09263); the published protocol below describes chromogenic TBC1D5 staining in renal carcinoma samples (PMC10900628).
| Sample | Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A09263) |
| Fixation | Image fixative and duration unreported (datasheet A09263); 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 A09263); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A09263) |
| Primary antibody | Rabbit anti-TBC1D5, 1:50 recommended; image 1:200 (datasheet A09263) |
| Primary incubation | Overnight at 4 °C (datasheet A09263) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A09263) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | TBC1D5-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet: A09263); the published protocol reports 120 °C for 5 min (PMC10900628).
Section 2
What Is the Expected TBC1D5 Staining Pattern?
TBC1D5 is associated with endosome membranes and cytoplasmic vesicles, including autophagosomes; it has no transmembrane segment (UniProt Q92609 topology and subcellular annotation). In paraffin-section IHC, expect chiefly cytoplasmic staining in epithelial or glandular cells where HPA reports expression, including colon glandular cells (HPA: High). Treat this as a provisional pattern: HPA describes general cytoplasmic expression, but rates its tissue IHC reliability Uncertain (HPA: tissue IHC).
| Cytoplasmic staining in colon or duodenal glandular cells, with little nuclear signal. | This fits HPA’s High glandular-cell staining in both tissues and its general cytoplasmic profile (HPA: tissue IHC). Vesicle-scale puncta may be hard to resolve with chromogenic IHC; assess the predominant compartment before scoring intensity. |
| Predominantly nuclear, extracellular, or sharply surface-membrane staining. | This conflicts with the reported vesicle/endosome association and general cytoplasmic IHC profile (UniProt Q92609 subcellular annotation; HPA: tissue IHC). Treat an isolated wrong-compartment pattern as possible nonspecific staining; compare controls and antibody validation before interpreting it as TBC1D5. |
| Strong adipocyte staining, especially when adjacent expected-positive cells are weak. | HPA reports TBC1D5 as Not detected in adipocytes, although its tissue IHC reliability is Uncertain (HPA: adipose tissue; HPA: tissue IHC). Consider cross-reactivity or endogenous chromogen activity; compare a no-primary control and examine the cellular distribution. |
| Broad, hazy chromogen deposits across cells and surrounding tissue. | Diffuse background obscures the cytoplasmic pattern HPA describes (HPA: tissue IHC). In general IHC practice, incomplete blocking or washing, excessive antibody concentration, and detection background can contribute; use controls to locate the source before assigning a positive score. |
| No signal in a section selected because HPA reports High staining. | A negative result in colon glandular cells, for example, conflicts with HPA’s observed High level but does not prove biological absence or assay failure (HPA: colon glandular cells; HPA: IHC reliability Uncertain). Check tissue preservation, antigen retrieval, detection controls, and antibody performance. |
💡Expected TBC1D5 appearanceCall a section provisionally positive when glandular or epithelial-cell cytoplasm stains clearly in an HPA High tissue, such as colon, while isolated nuclear or strong adipocyte staining prompts specificity checks (HPA: colon glandular cells High; HPA: adipocytes Not detected; HPA: IHC reliability Uncertain).
| Which compartment should guide IHC scoring? | Use HPA’s general cytoplasmic tissue pattern as the slide-level reference (HPA: tissue IHC). Endosome membrane and autophagosome association supports a vesicular interpretation (UniProt Q92609 subcellular annotation), but chromogenic resolution may not show individual vesicles. |
| Can cellular state change the pattern? | UniProt reports relocalization from endosomes to LC3-positive autophagosomes during starvation-induced autophagy (UniProt Q92609 subcellular annotation). A change in cytoplasmic puncta could therefore be biologically plausible, but the supplied HPA tissue images do not establish an IHC scoring rule for that state. |
| How strong is the tissue reference? | HPA reports High staining in several glandular-cell populations and low RNA tissue specificity, yet rates tissue IHC Uncertain because antibody staining and RNA data have low consistency (HPA: tissue IHC). Its two listed antibodies also have Uncertain IHC validation (HPA: HPA035125 and HPA035126). |
| Do isoforms or processing predict a different IHC pattern? | UniProt lists two isoforms, no signal peptide or propeptide, and a full-length chain of residues 1–795 (UniProt Q92609 isoforms and processing). The payload gives no antibody epitope or isoform-specific staining data, so it does not support separate IHC patterns. |
| What should IF/ICC show? | HPA places TBC1D5 mainly in vesicles, with additional Golgi localization, and marks the vesicle location Supported (HPA: subcellular ICC-IF). This answers the localization question for IF/ICC; the separate IF/ICC guide covers its experimental design. |
| Situation | Likely cause | Next action |
|---|---|---|
| A selected High-staining tissue has no visible cytoplasmic signal. | The HPA reference is uncertain; the run may also lack adequate antigen exposure or detection (HPA: tissue IHC reliability Uncertain). | Review morphology and positive detection controls, then optimize retrieval and antibody dilution as general IHC variables; use a second HPA High tissue to check whether the loss is section-specific (HPA: colon and duodenum glandular cells High). |
| Nuclei dominate the stain. | The distribution conflicts with HPA’s general cytoplasmic IHC pattern and UniProt’s vesicle/endosome localization (HPA: tissue IHC; UniProt Q92609 subcellular annotation). | Check a no-primary control and reassess antibody dilution, blocking, and detection background. Score TBC1D5 only after a credible cytoplasmic pattern is recovered. |
| Adipocytes stain strongly. | This differs from HPA’s Not detected adipocyte observation, although that IHC reference remains Uncertain (HPA: adipose tissue; HPA: tissue IHC). | Compare adjacent cell types and a no-primary control. Investigate endogenous detection activity or cross-reactivity before treating adipocyte staining as target expression. |
| Chromogen is diffuse across tissue or pools at section edges. | General IHC background or staining deposits can conceal the expected cytoplasmic distribution (HPA: general cytoplasmic expression). | Inspect no-primary and detection-only controls; adjust washing, blocking, or detection exposure as indicated. Interpret only well-preserved cells away from obvious deposits. |
| Two antibodies give different cell-type patterns. | HPA lists both antibodies as Uncertain for IHC and reports low consistency with RNA expression data (HPA: HPA035125, HPA035126; HPA: tissue IHC). | Record each antibody’s compartment and cell-type pattern separately. Favor a reproducible cytoplasmic result supported by controls; do not average discordant stains into a definitive tissue score. |
| Punctate staining differs between experimental conditions. | TBC1D5 can move from endosomes to LC3-positive autophagosomes during starvation-induced autophagy (UniProt Q92609 subcellular annotation). | Compare matched conditions and cytoplasmic morphology. Treat a shift as a hypothesis about vesicle localization, then seek an appropriate compartment marker before assigning it to autophagosomes. |
Experiment evidence
IHC & IF images showing TBC1D5 staining
A09263 · IHCIHC analysis of TBC1D5 using anti-TBC1D5 antibody (A09263). TBC1D5 was detected in a paraffin-embedded section of human colon cancer tissue.
A09263 · IHCIHC analysis of TBC1D5 using anti-TBC1D5 antibody (A09263). TBC1D5 was detected in a paraffin-embedded section of human colon cancer tissue.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000131374-TBC1D5/tissue/cerebral+cortex#imid_8923084
HPA · Thyroid glandHuman Protein Atlas IHC, Thyroid gland. Source: https://www.proteinatlas.org/ENSG00000131374-TBC1D5/tissue/thyroid+gland#imid_8923036
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000131374-TBC1D5/tissue/colon#imid_8923010
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000131374-TBC1D5/tissue/liver#imid_8922995
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA035125; green = TBC1D5, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA035125; green = TBC1D5, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for TBC1D5 IHC & IF
🧪Run colon first and look for TBC1D5 staining in glandular cells (HPA: High in colon glandular cells). Use adipose tissue as the negative tissue, focusing on adipocytes (HPA: Not detected in adipocytes); on the colon slide, use neighboring nonglandular areas to judge background without assuming those cells are target-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 ICC-IF images show TBC1D5 in A-431, U-251MG, U2OS, with annotated localisation: Vesicles (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and a rabbit IgG isotype control matched to the primary antibody where possible (caption: rabbit primary; standard IHC practice). A validated TBC1D5 knockout specimen or verified immunizing-peptide block can test specificity; block endogenous peroxidase before HRP/DAB detection in colon tissue (standard IHC practice; caption: HRP/DAB detection).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A09263 paraffin-section caption does not state a fixative (caption: fixative unreported). Start IHC with the caption’s heat-mediated EDTA retrieval at pH 8.0; that caption documents retrieval use, not a comparison of retrieval conditions (caption: EDTA retrieval, pH 8.0). For IF/ICC, vesicular signal with additional Golgi localization is an expected pattern, but the supplied evidence does not establish whether IF/ICC or frozen sections are easier than paraffin IHC (HPA: vesicles supported; Golgi approved).
HPA tissue IHC evidence for TBC1D5
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low consistency between antibody staining and RNA expression data. Pending external verification.). 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 → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Cervix | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TBC1D5 IHC Tips
Troubleshoot TBC1D5 staining in paraffin sections by checking retrieval, cellular pattern, controls and scoring before interpreting chromogenic signal.
What retrieval should I try first when TBC1D5 staining is weak?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A09263). The documented tissue image used this retrieval before 1:200 primary antibody incubation overnight at 4°C (datasheet A09263). If staining remains weak, vary heating duration on adjacent sections while keeping antibody dilution, detection and exposure to DAB constant (standard IHC practice). Compare signal in glandular cells with background on each slide, because colon glandular cells showed high staining in the tissue survey, although its overall IHC reliability is uncertain (HPA: colon glandular cells High; HPA: reliability Uncertain).
How should I assess whether fixation is affecting TBC1D5 staining?
Target-specific sensitivity to fixation is unknown; the selected paraffin-section caption does not report a fixative (datasheet A09263: fixative not stated). For a new specimen, compare matched samples fixed for 6 and 24 hours in 10% neutral buffered formalin as a local optimization, then process and stain them together (standard IHC practice). Apply the same EDTA pH 8.0 heat retrieval and 1:200 primary dilution to both conditions (datasheet A09263). Judge preservation of tissue structure alongside cytoplasmic signal and background; neither the tissue survey nor the protein annotation establishes a TBC1D5-specific fixation effect (HPA: tissue IHC; UniProt Q92609).
Where should convincing TBC1D5 chromogenic staining appear?
Assess cytoplasmic staining, including a vesicular or punctate component, in morphologically intact cells (HPA: general cytoplasmic tissue expression; UniProt Q92609: endosome and autophagosome association). Endosome-associated TBC1D5 can redistribute to LC3-positive autophagosomes during starvation-induced autophagy, so compartment patterns may depend on biological state (UniProt Q92609). Vesicles are the supported main location in the separate cell-imaging survey, with an additional Golgi location; use those findings as context for IHC, not as proof of individual DAB puncta (HPA: subcellular). Review a no-primary control and adjacent morphology before assigning dense deposits to vesicles (standard IHC practice).
Could isoforms or epitope masking explain discrepant staining?
TBC1D5 has 2 annotated isoforms and a Rab-GAP TBC domain spanning residues 81–359 (UniProt Q92609). The supplied caption gives no antibody epitope, so it cannot establish recognition of both isoforms or predict which fixation or retrieval condition might mask binding (datasheet A09263: epitope not stated). Phosphorylation is annotated at several residues, including 42, 43, 44, 460 and 522; these annotations alone do not show that staining depends on phosphorylation (UniProt Q92609). If patterns conflict, obtain the epitope information and assess specificity with an independently validated antibody or a target-depletion control processed identically (standard IHC validation practice).
How can IF help resolve an ambiguous IHC pattern?
Use IF/ICC as a separate follow-up to an ambiguous chromogenic result, retaining the IHC section as the reference for tissue architecture (standard IHC/IF practice). In colon, multiplex TBC1D5 with an epithelial cell marker to identify the glandular cells reported as highly stained in tissue IHC (HPA: colon glandular cells High). Choose a far-red fluorophore and include unstained and single-channel controls to distinguish specific fluorescence from tissue autofluorescence and channel bleed-through (standard IF practice). For an epitope established to face the cytosol, test mild detergent permeabilisation after fixation; TBC1D5 has no transmembrane segment, but the supplied record does not locate this antibody’s epitope (UniProt Q92609; datasheet A09263: epitope not stated).
How do I reduce diffuse or granular DAB background?
Begin with a no-primary section and inspect whether deposits follow tissue edges, damaged areas or the expected cytoplasmic distribution (standard IHC practice; HPA: general cytoplasmic expression). The documented section used 10% goat serum blocking, a 1:200 rabbit primary overnight at 4°C, and an HRP-based DAB readout (datasheet A09263). If background persists, compare a shorter DAB development time and a primary-antibody titration on adjacent sections while keeping retrieval fixed (standard IHC practice). Include an endogenous peroxidase block, such as 3% hydrogen peroxide for 10 minutes, as a general chromogenic IHC step rather than TBC1D5-specific evidence (standard IHC practice).
How should I score heterogeneous TBC1D5 staining across a section? ⚠ ANSWER MARKED FOR VERIFICATION
Define cell compartments and regions before scoring, and exclude folds, necrosis and section edges using the counterstained morphology (standard IHC practice). For glandular regions, report the percentage of positive cells and an H-score from 0–300, calculated as the percentage at each intensity multiplied by its 0–3 intensity grade (standard IHC scoring practice). Normalize positive-cell counts to the number of evaluable cells in the same annotated compartment, or report puncta density per mm² of viable tissue when puncta can be resolved reliably (standard IHC practice). Keep retrieval, primary dilution, DAB development and scoring thresholds identical across compared sections (standard IHC practice; datasheet A09263: EDTA pH 8.0 retrieval).
What distinguishes credible TBC1D5 staining from artefact?
A credible result should track intact-cell cytoplasm, with any punctate signal assessed against the protein’s endosomal and autophagosomal association (HPA: general cytoplasmic expression; UniProt Q92609). In colon, glandular cells offer a reported high-staining context, but the tissue survey rates its antibody staining versus RNA consistency as uncertain (HPA: colon glandular cells High; HPA: reliability Uncertain). Treat isolated nuclear deposits, strong section-edge signal, staining confined to necrotic debris, or signal retained in a no-primary control as reasons to investigate artefact (standard IHC practice). Check endogenous peroxidase blocking and repeat the comparison on intact regions before assigning DAB deposits to TBC1D5 (standard chromogenic IHC practice).
Boster reagents
Best TBC1D5 / TBC1 domain family member 5 IHC Antibodies
A09263 has IHC images of human colon, human colon cancer, and rat testis paraffin sections; IF/ICC are listed applications, but no IF image is supplied (catalog: A09263 captions and applications).
Real IHC data
Anti-TBC1D5 Antibody
Cat # A09263
A09263 will render with its human colon cancer paraffin-section IHC figure (catalog: A09263 figure caption). Additional captions show human colon and rat testis IHC; the catalog lists human, mouse, and rat reactivity and IF/ICC applications, without an IF image (catalog: A09263 captions, reactivity, and applications).
Which to pick: Choose A09263 for tissue IHC: its own caption documents heat retrieval in EDTA at pH 8.0 and antibody incubation at 1:200 overnight at 4°C in a paraffin section; the fixative is unreported (catalog: A09263 IHC caption). For IF/ICC or cross-species work, A09263 is the listed polyclonal option with human, mouse, and rat reactivity, though the supplied images document IHC in human and rat tissue only (catalog: A09263 dilution_raw, applications, reactivity, and captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q92609 (TBCD5_HUMAN, TBC1 domain family member 5).
- Human Protein Atlas. TBC1D5 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. TBC1D5 subcellular location (ICC-IF): Mainly localized to vesicles. In addition localized to the Golgi apparatus..
- Human Protein Atlas. TBC1D5 antibody validation summary (2 antibodies).
- TBC1D5 reverses the capability of HIF-2α in tumor progression and lipid metabolism in clear cell renal cell carcinoma by regulating the autophagy. Journal of translational medicine 2024 — PMC10900628.
- TBK1 Facilitates GLUT1-Dependent Glucose Consumption by suppressing mTORC1 Signaling in Colorectal Cancer Progression. International journal of biological sciences 2022 — PMC9134896.
- PubMed PMID:9039502 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16641997 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for TBC1D5 (TBC1 domain family member 5). Expected localisation General cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A09263). Validated IHC antibody: A09263 at 1:50 recommended; image 1:200 (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 colon cancer tissue; fixative not specified (datasheet A09263); 2. Fixation: Image fixative and duration unreported (datasheet A09263); 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 A09263); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A09263); 8. Primary antibody: Rabbit anti-TBC1D5, 1:50 recommended; image 1:200 (datasheet A09263); 9. Primary incubation: Overnight at 4 °C (datasheet A09263). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Tissue staining has uncertain reliability (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unknown (UniProt); Regulation: Starvation shifts compartmental signal (UniProt). Evidence: UniProt Q92609, HPA tissue IHC · Uncertain, HPA ICC-IF · Mainly localized to vesicles. In addition localized to the Golgi…, Datasheet A09263, PMC10900628. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/TBC1D5/immunohistochemistry-ihc-guide.