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HDAC5 / Histone deacetylase 5 · IHC design guide
Design Immunohistochemistry for HDAC5
Plan HDAC5 staining in paraffin sections using its predominantly cytoplasmic tissue pattern, with nuclear staining in some tissues (HPA tissue IHC). Compare compartments as well as intensity because HDAC5 shuttles between nucleus and cytoplasm (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HDAC5 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 in most tissues; nuclear in several (HPA tissue IHC) | |
| Staining pattern | Mostly cytoplasmic staining, with some nuclear staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A01230-6) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | None in HPA (detected in all 44 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep formalin fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Nuclear–cytoplasmic shuttling can change compartment scores (UniProt) | |
| Regulation | Differentiation promotes cytoplasmic export (UniProt) | |
| Isoform / epitope | 3 isoforms; check antibody epitope coverage (UniProt) |
Section 1
Recommended HDAC5 IHC & IF Protocols
The catalog antibody uses EDTA pH 8.0 retrieval (datasheet A01230-6). Two published IHC protocols describe HDAC5 staining in kidney and scar tissue (PMC7791055; PMC9576526).
| Sample | Paraffin-embedded mouse small intestine tissue; fixative not specified (datasheet A01230-6) |
| Fixation | Image fixative and duration unreported (datasheet A01230-6); 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 A01230-6); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A01230-6) |
| Primary antibody | Rabbit anti-HDAC5, 2-5μg/ml (datasheet A01230-6) |
| Primary incubation | Overnight at 4 °C (datasheet A01230-6) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A01230-6) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | HDAC5-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. Additional nuclear expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet A01230-6); the kidney study reports pressure-cooker retrieval without a buffer specification (PMC7791055).
Section 2
What Is the Expected HDAC5 Staining Pattern?
HDAC5 staining is predominantly cytoplasmic across tissues, with additional nuclear staining in several tissues (HPA tissue IHC). Glandular cells in appendix, colon, duodenum, salivary gland and thyroid are among the high staining examples (HPA tissue IHC). Nuclear and cytoplasmic localisation is consistent with HDAC5 shuttling between those compartments (UniProt Q9UQL6); HDAC5 has no transmembrane segment (UniProt Q9UQL6 topology). HPA rates its tissue staining Approved, with medium consistency against RNA expression (HPA tissue IHC).
| Glandular cells show clear cytoplasmic staining, with nuclear staining in some cells. | This fits the reported tissue pattern: cytoplasmic expression in most tissues and additional nuclear expression in several (HPA tissue IHC). Interpret nuclear staining alongside the tissue and cell type; HDAC5 can shuttle between nucleus and cytoplasm (UniProt Q9UQL6). |
| Signal outlines cell membranes or fills gland lumens, without convincing intracellular staining. | A membrane or luminal pattern alone does not match the reported cytoplasmic and nuclear localisation (HPA tissue IHC; UniProt Q9UQL6 topology). Check morphology and controls before treating it as HDAC5; edge staining or deposited chromogen can mislead localisation (general IHC practice). |
| Strong staining appears mainly in a cell population expected to have low staining. | For example, HPA reports low staining in cardiomyocytes, skeletal myocytes and glial cells in caudate (HPA tissue IHC). Unexpected strong signal raises the possibility of cross-reactivity or endogenous detection activity (general IHC practice); low reported staining does not mean every cell must be negative. |
| A faint, even tint covers cells and surrounding tissue. | Diffuse colour without cell boundaries or a discernible compartment is difficult to score as HDAC5 (general IHC practice). Compare with a negative reagent control and inspect washing, blocking and chromogen development before calling weak cytoplasmic staining (general IHC practice). |
| No convincing staining appears in a tissue with an HPA high staining example. | Appendix or colon glandular cells and tonsil germinal center cells are reported High (HPA tissue IHC). Absence of signal there suggests reviewing antibody and detection performance, section quality and control results (general IHC practice); HPA staining levels alone do not establish a cause. |
💡Expected HDAC5 appearanceCall a positive result when identifiable cells show clear cytoplasmic staining, with possible nuclear staining, especially in HPA High cell populations; isolated membrane, luminal or uniform background colour is suspect (HPA tissue IHC; UniProt Q9UQL6 topology; general IHC practice).
| Tissue and cell choice | HPA reports High staining in glandular cells of appendix, colon, duodenum, salivary gland and thyroid, as well as bone marrow hematopoietic cells, skin Langerhans cells and tonsil germinal center cells (HPA tissue IHC). These are useful comparison patterns, not guarantees for every section. |
| Compartment and cell state | Nuclear and cytoplasmic localisation is reported for HDAC5 (UniProt Q9UQL6). In muscle cells, differentiation can drive cytoplasmic shuttling after phosphorylation at Ser-259 and Ser-498 (UniProt Q9UQL6). Interpret a compartment shift in its biological context rather than requiring one fixed nuclear-to-cytoplasmic ratio. |
| Antibody evidence | HPA rates its tissue IHC profile Approved, describing medium consistency between antibody staining and RNA expression (HPA tissue IHC). HPA030991, HPA076420 and CAB019400 each have IHC Approved status (HPA antibodies). Approved status supports use as a reference but does not make an unexpected pattern specific by itself. |
| Protein forms and topology | UniProt lists three HDAC5 isoforms, one chain spanning residues 1–1122, no signal peptide and no transmembrane segment (UniProt Q9UQL6). Those annotations support an intracellular interpretation; they do not identify which isoform a particular antibody recognises or establish an antigen retrieval condition. |
| What should IF/ICC show? | HPA reports mainly nuclear speckles, with additional Golgi apparatus and cytosol localisation in ICC-IF (HPA subcellular). That finer pattern can inform localisation comparisons, while the tissue IHC summary remains predominantly cytoplasmic with nuclear expression in several tissues (HPA tissue IHC). |
| Target-specific fixation sensitivity | No target-specific fixation effect is established by the supplied UniProt or HPA localisation and staining records. Choose and assess routine paraffin-section preparation with appropriate controls (general IHC practice); do not infer an HDAC5 antigen retrieval requirement from tissue staining intensity. |
| Situation | Likely cause | Next action |
|---|---|---|
| High staining example gives no signal. | A failed antibody or detection step, damaged section, or unsuitable working conditions are possibilities (general IHC practice); the HPA High label does not diagnose the failure (HPA tissue IHC). | Check section morphology and a known working staining control, then verify antibody dilution, detection reagents and chromogen development (general IHC practice). |
| Signal is strong on membranes or in lumens. | The pattern conflicts with HDAC5's reported nuclear and cytoplasmic localisation and lack of a transmembrane segment (UniProt Q9UQL6; HPA tissue IHC). Artefactual deposition is possible (general IHC practice). | Inspect cell boundaries at higher magnification and compare negative reagent controls; score only convincing intracellular signal (general IHC practice). |
| Unexpected cells stain strongly. | Cell type differences may matter: HPA reports low staining in cardiomyocytes and skeletal myocytes (HPA tissue IHC). Cross-reactivity or endogenous detection activity is also possible (general IHC practice). | Compare a high staining reference cell population and an appropriate negative reagent control; assess endogenous enzyme blocking if using enzyme-based detection (general IHC practice). |
| Nuclei stain but cytoplasm appears weak. | Nuclear expression occurs in several tissues, and HDAC5 shuttles between nucleus and cytoplasm (HPA tissue IHC; UniProt Q9UQL6). Nuclear signal alone therefore does not prove a technical failure. | Confirm nuclear boundaries with the counterstain and assess the expected cell type, morphology and controls before assigning localisation (general IHC practice). |
| Colour is diffuse across the section. | Nonspecific background or excessive chromogen development can obscure intracellular boundaries (general IHC practice). HPA's cytoplasmic profile requires a cellular pattern for comparison (HPA tissue IHC). | Compare negative reagent controls; review blocking, washing, antibody concentration and chromogen development under the laboratory's routine IHC workflow (general IHC practice). |
| Muscle staining differs from a reference image. | HPA reports low staining in heart cardiomyocytes and skeletal myocytes, while UniProt describes differentiation-linked cytoplasmic shuttling in muscle cells (HPA tissue IHC; UniProt Q9UQL6). | Compare the same tissue and cell type, assess nuclear and cytoplasmic compartments separately, and verify controls before attributing a difference to biology or staining conditions (general IHC practice). |
Experiment evidence
IHC & IF images showing HDAC5 staining
A01230-6 · IHCIHC analysis of HDAC5 using anti-HDAC5 antibody (A01230-6). HDAC5 was detected in paraffin-embedded section of mouse small intestine tissue.
A01230-6 · IHCIHC analysis of HDAC5 using anti-HDAC5 antibody (A01230-6). HDAC5 was detected in paraffin-embedded section of human placenta tissue. Heat m
A01230-6 · IFIF analysis of HDAC5 using anti-HDAC5 antibody (A01230-6). HDAC5 was detected in immunocytochemical section of A431 cells. Enzyme antigen re
A01230-5 · IHCIHC analysis of HDAC5 using anti-HDAC5 antibody (A01230-5). HDAC5 was detected in paraffin-embedded section of human placenta tissue. Heat m
A01230-5 · IHCIHC analysis of HDAC5 using anti-HDAC5 antibody (A01230-5). HDAC5 was detected in paraffin-embedded section of mouse small intestine tissue.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000108840-HDAC5/tissue/cerebral+cortex#imid_10993899
HPA · CerebellumHuman Protein Atlas IHC, Cerebellum. Source: https://www.proteinatlas.org/ENSG00000108840-HDAC5/tissue/cerebellum#imid_10993910
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000108840-HDAC5/tissue/colon#imid_10993817
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000108840-HDAC5/tissue/liver#imid_10993826
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA030991; green = HDAC5, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA030991; green = HDAC5, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HDAC5 IHC & IF
🧪Run duodenum first and expect its glandular cells to stain (High; HPA: duodenum glandular cells). HPA detects HDAC5 in all 44 scored tissues, so use no-primary and isotype controls for negative staining; nonglandular cells with weaker or absent staining on the positive slide provide internal contrast, but are not confirmed HDAC5-negative cells (HPA: no negative tissue rows; HDAC5 detected in all 44 tissues).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: None in HPA: HDAC5 is detected in all 44 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 HDAC5 in A-431, U-251MG, U2OS, with annotated localisation: Nuclear speckles (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide, a concentration-matched rabbit IgG isotype control, and an HDAC5 knockout specimen if available (selected-SKU caption: rabbit primary antibody; standard IHC controls). For duodenum, check background from endogenous peroxidase and endogenous biotin, particularly if using the caption’s biotin-based detection and DAB (selected-SKU caption: biotinylated secondary, SABC and DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the exact selected-SKU paraffin-section caption does not state a fixative (selected-SKU caption: fixative not stated). That caption uses heat retrieval in EDTA, pH 8.0, but does not establish whether HDAC5 staining depends on retrieval (selected-SKU caption: EDTA retrieval). Frozen-section performance is unreported; HPA has ICC-IF images, but the supplied evidence does not show that IF/ICC is easier than paraffin IHC, and duodenal background warrants checking with the controls above (HPA: ICC-IF images in A-431, U-251MG and U2OS; selected-SKU caption: paraffin section with biotin-based DAB detection).
HPA tissue IHC evidence for HDAC5
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 → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Salivary gland | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: HDAC5 is detected in all 44 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced HDAC5 IHC Tips
Troubleshoot HDAC5 staining in paraffin sections by checking retrieval, compartment patterns, detection background, and cell-specific scoring.
What retrieval conditions should I try when HDAC5 staining is weak?
Start with heat-mediated antigen retrieval in EDTA at pH 8.0 on paraffin sections (datasheet A01230-6). The selected tissue image used this retrieval before 10% goat serum blocking and 2 µg/mL primary antibody overnight at 4°C (datasheet A01230-6). If staining is weak, check that sections reached the intended retrieval temperature and that heating and cooling were consistent across the run. Test a different heating duration on matched sections before considering citrate at pH 6.0 as a fallback; the supplied product evidence supports EDTA at pH 8.0 (datasheet A01230-6).
Could fixation explain variable HDAC5 staining between paraffin blocks?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not report its fixative or fixation time (datasheet A01230-6). Record fixative, time in fixative, processing schedule, and section age for each block, then compare sections stained in the same run. Keep the documented retrieval at EDTA pH 8.0 and the 2 µg/mL overnight primary incubation constant while assessing block-to-block variation (datasheet A01230-6). Include a consistently staining control section, and compare viable regions of the same cell type so differences in composition do not masquerade as a fixation effect.
Should HDAC5 appear in nuclei, cytoplasm, or both?
Score nuclear and cytoplasmic staining separately: HDAC5 shuttles between these compartments (UniProt Q9UQL6), while tissue IHC shows cytoplasmic expression in most tissues and additional nuclear expression in several (HPA tissue IHC). Phosphorylation at Ser-259 and Ser-498 contributes to export through interaction with a 14-3-3 chaperone (UniProt Q9UQL6). Therefore, cytoplasmic staining alone is not automatically nonspecific, and a nuclear-only acceptance rule would discard plausible signal. Compare compartment patterns within the same cell type and staining run; use matched positive and no-primary controls before assigning biological meaning to a shift.
How could isoforms or epitope accessibility alter the HDAC5 IHC pattern?
HDAC5 has 3 annotated isoforms, but the supplied antibody caption does not identify its epitope or establish which isoforms it recognizes (UniProt Q9UQL6; datasheet A01230-6). The annotated full-length chain spans residues 1–1122, with no transmembrane segment (UniProt Q9UQL6). Phosphorylation at Ser-259 and Ser-498, and acetylation at Lys-533, are documented modifications; whether they affect this antibody's binding is unknown (UniProt Q9UQL6). If compartment patterns differ between specimens, first compare retrieval and staining controls, then seek epitope mapping or an independent antibody before attributing the difference to an isoform or modification.
How should I adapt HDAC5 localisation checks to multiplex IF?
Use an independently validated IF workflow, because the supplied catalog evidence describes chromogenic staining of paraffin sections, with its fixative unreported (datasheet A01230-6). Pair HDAC5 with a marker that identifies the expected cell type, such as glandular cells in appendix, where tissue IHC reports high staining (HPA tissue IHC). Choose spectrally separated fluorophores and favor a longer-wavelength HDAC5 channel if the specimen has substantial autofluorescence; include single-stain controls for bleed-through. HDAC5 is intracellular and has no transmembrane segment (UniProt Q9UQL6), so assess permeabilisation for access to nuclear and cytoplasmic epitopes while preserving compartment boundaries.
How can I distinguish HDAC5 signal from chromogenic background?
Run a no-primary section alongside the test section and inspect diffuse deposits, tissue edges, and pigment before scoring cells. The selected workflow used 10% goat serum, a biotinylated secondary, a streptavidin-biotin complex, and DAB (datasheet A01230-6). In that detection scheme, check endogenous peroxidase and biotin-related background with appropriate blocking or detection controls as general chromogenic IHC practice. If background persists, compare a shorter DAB development or adjusted primary concentration against the documented 2 µg/mL overnight incubation, keeping retrieval at EDTA pH 8.0 constant during the comparison (datasheet A01230-6).
What is a defensible way to quantify HDAC5 IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because tissue IHC reports predominantly cytoplasmic staining with nuclear staining in several tissues (HPA tissue IHC). For each compartment, report the percentage of positive viable target cells and an H-score from 0–300 using staining intensity categories. For sparse populations, report positive-cell density per mm² of viable tissue, together with the number of target cells assessed. Normalize comparisons to the same cell type and compartment, use matched staining runs and exposure to DAB, and exclude necrotic or damaged regions so changes in tissue composition do not drive the result.
Which staining patterns support genuine HDAC5 detection rather than artefact?
A credible pattern is reproducible in viable cells and fits the broad tissue IHC profile: cytoplasmic staining is common, with nuclear staining in several tissues (HPA tissue IHC). High staining in appendix glandular cells or bone-marrow hematopoietic cells can serve as a context check, while low staining is reported in heart-muscle cardiomyocytes and skeletal-muscle myocytes (HPA tissue IHC). Neither nuclear nor cytoplasmic signal is inherently misplaced because HDAC5 shuttles between them (UniProt Q9UQL6). Treat edge-only deposits, necrotic-area staining, and signal reproduced without primary antibody as suspect; investigate endogenous enzyme background before interpreting apparent cell-specific changes.
Boster reagents
Best HDAC5 / Histone deacetylase 5 IHC Antibodies
Two anti-HDAC5 antibodies have paraffin-section IHC images from human and mouse samples (catalog IHC captions); one also has IF images from A431 and U20S cells (A01230-6 IF captions).
Real IHC data
Anti-HDAC5 Antibody ®
Cat # A01230-6
Real IHC data
Anti-HDAC5 Antibody ®
Cat # A01230-5
A01230-5 has IHC images from human placenta and mouse and rat small intestine paraffin sections (A01230-5 IHC captions). A01230-6 has IHC images from mouse small intestine, human placenta, esophageal squamous carcinoma and lung cancer paraffin sections, plus IF images from A431 and U20S cells (A01230-6 IHC and IF captions).
Which to pick: For tissue IHC across human, mouse and rat samples, choose A01230-5: its paraffin-section images cover all three species (A01230-5 IHC captions); start at 0.5–1 μg/ml (catalog IHC dilution). For IF/ICC, choose A01230-6: those applications are listed and its cell images use 5 μg/ml (catalog applications; A01230-6 IF captions). A01230-6 also has human and mouse paraffin-section IHC images at 2 μg/ml (A01230-6 IHC captions); clonality and the tissue fixative are unreported (catalog clone field; IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UQL6 (HDAC5_HUMAN, Histone deacetylase 5).
- Human Protein Atlas. HDAC5 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. HDAC5 subcellular location (ICC-IF): Mainly localized to the nuclear speckles. In addition localized to the Golgi apparatus and cytosol..
- Human Protein Atlas. HDAC5 antibody validation summary (3 antibodies).
- METTL14-regulated PI3K/Akt signaling pathway via PTEN affects HDAC5-mediated epithelial-mesenchymal transition of renal tubular cells in diabetic kidney disease. Cell death & disease 2021 — PMC7791055.
- HDAC5-mediated Smad7 silencing through MEF2A is critical for fibroblast activation and hypertrophic scar formation. International journal of biological sciences 2022 — PMC9576526.
- Gene Co-expression Analysis Identifies Histone Deacetylase 5 and 9 Expression in Midbrain Dopamine Neurons and as Regulators of Neurite Growth via Bone Morphogenetic Protein Signaling. Frontiers in cell and developmental biology 2019 — PMC6753186.
- Role of Dorsal Striatum Histone Deacetylase 5 in Incubation of Methamphetamine Craving. Biological psychiatry 2018 — PMC6026084.
- PubMed PMID:10220385 — UniProt-cited evidence.
- PubMed PMID:9628581 — UniProt-cited evidence.
- PubMed PMID:12168954 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HDAC5 (Histone deacetylase 5). Expected localisation Cytoplasmic in most tissues; nuclear in several (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A01230-6). Validated IHC antibody: A01230-6 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative None in HPA (detected in all 44 tissues); use no-primary + isotype controls. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded mouse small intestine tissue; fixative not specified (datasheet A01230-6); 2. Fixation: Image fixative and duration unreported (datasheet A01230-6); 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 A01230-6); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A01230-6); 8. Primary antibody: Rabbit anti-HDAC5, 2-5μg/ml (datasheet A01230-6); 9. Primary incubation: Overnight at 4 °C (datasheet A01230-6). Watch for: Fixation: Keep formalin fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Nuclear–cytoplasmic shuttling can change compartment scores (UniProt); Isoform / epitope: 3 isoforms; check antibody epitope coverage (UniProt); Regulation: Differentiation promotes cytoplasmic export (UniProt). Evidence: UniProt Q9UQL6, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nuclear speckles. In addition localized to…, Datasheet A01230-6, PMC7791055, PMC9576526. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HDAC5/immunohistochemistry-ihc-guide.