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TUBB2B / Tubulin beta-2B chain · IHC design guide
Design Immunohistochemistry for TUBB2B
Plan paraffin IHC for TUBB2B with the catalog antibody at 1:2000 (datasheet A05849). Interpret cytoplasmic staining against the reported tissue pattern and its multi-gene staining caution (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
TUBB2B Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic signal, strongest in ciliated cells and peripheral nerves (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 9 HIER, heat-mediated (datasheet A05849) | |
| Positive control | Testis+1 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Formaldehyde-fixed brain sections were shown (selected-SKU IHC image A05849); keep fixation consistent (standard IHC practice; not target-specific) | |
| Caveat | Staining may reflect protein from more than one gene (HPA tissue IHC) | |
| Regulation | High expression in brain (UniProt) | |
| Isoform / epitope | No annotated isoforms; the chain spans residues 1–445 (UniProt) |
Section 1
Recommended TUBB2B IHC & IF Protocols
The catalog antibody’s IHC-P protocol uses EDTA pH 9 retrieval (datasheet A05849); the published human kidney protocol below uses pH 6 retrieval (PMC4557973).
| Sample | Formaldehyde-fixed, paraffin-embedded Human brain tissue (datasheet A05849) |
| Fixation | Image formalin-fixed; duration unreported (datasheet A05849); 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 9 (datasheet A05849); 20 min, 95–100 °C (standard) |
| 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-TUBB2B, 1:2000 (datasheet A05849) |
| Primary incubation | 1 hours at room temperature (datasheet A05849) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A05849) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | TUBB2B-positive staining in pachytene spermatocytes of testis (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues, most abundant in ciliated cells and peripheral nerves. No signal in the no-primary control. |
💡Decision noteTry EDTA pH 9 heat retrieval first for the catalog antibody (datasheet A05849). If optimizing, compare the published pH 6 heat retrieval (PMC4557973).
Section 2
What Is the Expected TUBB2B Staining Pattern?
TUBB2B is a cytoplasmic microtubule protein with no transmembrane segment (UniProt Q9BVA1). In paraffin section IHC, expect cytoplasmic staining in the cell populations reported by HPA, especially pachytene spermatocytes and neuropil (HPA: high in pachytene spermatocytes; medium in cerebral cortex neuropil). HPA rates the tissue pattern Supported but reports low agreement with RNA expression and cautions that the antibodies may recognize proteins from multiple genes (HPA: tissue IHC reliability).
| Cytoplasmic stain is strong in pachytene spermatocytes and moderate in cerebral cortex neuropil (HPA: tissue IHC). | This matches the reported positive compartments and relative staining levels (HPA: high in pachytene spermatocytes; medium in neuropil). Judge the pattern alongside morphology and a control section, because HPA notes possible recognition of proteins from multiple genes (HPA: tissue IHC caution). |
| Predominantly nuclear stain appears without a cytoplasmic pattern (UniProt Q9BVA1 localization). | A nuclear-only result conflicts with the reported cytoplasmic and microtubule localization (UniProt Q9BVA1; HPA: subcellular). Treat it as questionable staining and review the control, antibody and detection steps before scoring it as TUBB2B. |
| Adipocytes or bone marrow hematopoietic cells stain strongly (HPA: both not detected). | Those cell populations are listed as not detected, so strong staining is discordant with HPA tissue IHC (HPA: adipocytes; bone marrow hematopoietic cells). Consider antibody recognition of related proteins or endogenous detection activity; neither cause can be assigned from the stain alone (HPA: multi-gene antibody caution; standard IHC practice). |
| Brown signal spreads across cells, stroma and blank regions without clear cell boundaries. | A uniform haze lacks the cell-associated pattern needed for interpretation (standard IHC practice). Check a no-primary control for secondary or detection background, and review blocking, antibody concentration and washes before comparing tissues (standard IHC practice). |
| Pachytene spermatocytes show no visible stain while the control slide develops normally (HPA: high). | This disagrees with HPA's reported high signal in that cell population (HPA: tissue IHC). Confirm that the intended cells are present, then review antibody dilution, retrieval and detection as general IHC workflow checks; the supplied sources do not establish a TUBB2B-specific retrieval requirement (standard IHC practice). |
💡Expected TUBB2B appearanceCall a positive result when staining is predominantly cytoplasmic, with high signal in pachytene spermatocytes or medium signal in cerebral cortex neuropil (UniProt Q9BVA1 localization; HPA: tissue IHC); isolated nuclear staining or strong signal in HPA-listed undetected cells is suspect (HPA: tissue IHC; standard IHC practice).
| Tissue and cell selection (HPA: tissue IHC) | HPA reports high signal in testis pachytene spermatocytes and medium signal in cerebral cortex neuropil; adipocytes are not detected (HPA: tissue IHC). Score the named cell populations rather than assigning one expected intensity to an entire section. |
| Antibody interpretation (HPA: tissue IHC reliability) | The tissue pattern is Supported, but HPA reports low consistency with RNA expression and cautions that antibodies may target proteins from multiple genes (HPA: tissue IHC reliability). Both listed antibodies have Supported IHC status (HPA: HPA043640; HPA046280). |
| Protein location and topology (UniProt Q9BVA1) | TUBB2B is a cytoplasmic cytoskeleton protein without a transmembrane segment (UniProt Q9BVA1). An exclusively membrane-rim or nuclear pattern would therefore need investigation before being called target-specific (UniProt Q9BVA1; standard IHC practice). |
| Epitope and retrieval uncertainty (UniProt Q9BVA1) | UniProt lists modified residues but no epitope for the IHC antibodies (UniProt Q9BVA1). The supplied sources do not show whether a modification or retrieval condition changes their staining; use the catalog antibody's IHC-P instructions when setting the workflow. |
| IF/ICC appearance? (HPA: subcellular ICC-IF) | For IF/ICC interpretation, HPA reports mainly microtubule localization, with additional spindle, cytokinetic bridge and ciliary structures (HPA: subcellular ICC-IF). That evidence also carries a multi-gene antibody caution; assess IF/ICC on its own guide page, without treating it as an IHC-P protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive cells are unstained (HPA: high in pachytene spermatocytes). | The sampled field may lack the named cells, or an IHC workflow step may have failed (standard IHC practice). | Locate pachytene spermatocytes by morphology, inspect the positive control, and check antibody dilution, retrieval and detection against the chosen IHC-P procedure (standard IHC practice). |
| Only nuclei stain (UniProt Q9BVA1: cytoplasm, cytoskeleton). | The pattern does not match the reported target location; its specific cause is unresolved (UniProt Q9BVA1; HPA: subcellular). | Compare with a no-primary control and the expected cytoplasmic pattern before scoring; review detection background if the control also stains (standard IHC practice). |
| Strong stain appears in adipocytes despite an otherwise readable slide (HPA: not detected). | Possible antibody cross-recognition or endogenous detection activity needs separating (HPA: multi-gene caution; standard IHC practice). | Run a no-primary control and assess whether signal tracks the expected cells; if discordance persists, interpret it with HPA's antibody caution (standard IHC practice; HPA: tissue IHC reliability). |
| Diffuse brown background obscures cell boundaries. | Excess antibody or incomplete blocking or washing can raise background (standard IHC practice). | Check a no-primary control, then adjust antibody concentration and blocking or washing within the established IHC-P workflow (standard IHC practice). |
| Cerebral cortex neuropil looks weaker than the testis positive population (HPA: tissue IHC). | HPA reports medium neuropil signal and high pachytene spermatocyte signal, so equal intensity is not expected (HPA: tissue IHC). | Score each named cell population against its own reported level and the slide controls; avoid calling a weaker neuropil result a failed run solely by comparison with testis (HPA: tissue IHC; standard IHC practice). |
| An IF/ICC image shows microtubule-like fibers but the IHC section is inconclusive (HPA: subcellular ICC-IF). | IF/ICC localization and paraffin section tissue staining answer different questions, and HPA flags multi-gene recognition (HPA: subcellular ICC-IF; tissue IHC). | Use the IF/ICC result only to assess subcellular plausibility; resolve the IHC call from section morphology, controls and HPA tissue patterns (HPA: tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing TUBB2B staining
A05849 · IHCImmunohistochemical analysis of paraffin-embedded Human brain tissue using A05849 performed on the vendor® BOND RXm. Tissue was fixed with f
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000137285-TUBB2B/tissue/cerebral+cortex#imid_13050179
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000137285-TUBB2B/tissue/colon#imid_13050393
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000137285-TUBB2B/tissue/liver#imid_13050386
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000137285-TUBB2B/tissue/kidney#imid_13050387
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA043640; green = TUBB2B, blue = nucleus, red = microtubules.
HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA043640; green = TUBB2B, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for TUBB2B IHC & IF
🧪Run testis first: pachytene spermatocytes should stain (HPA: High in pachytene spermatocytes); run adipose tissue as the negative, where adipocytes are not detected (HPA: Not detected in adipocytes). On the testis slide, use cells outside the stained pachytene population as internal negative references only when they lack specific cytoplasmic staining; HPA provides no negative call for those cells (HPA: testis row identifies pachytene spermatocytes).
Positive control tissue: Testis (Pachytene spermatocytes, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show TUBB2B in A-549, HEL, U2OS, HaCaT, MCF-7, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), Sperm, with annotated localisation: Microtubules (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species, clonality and concentration; use a TUBB2B knockout sample or a validated peptide block as a biological negative (standard IHC practice). In testis sections, check endogenous peroxidase and endogenous biotin background because the depicted detection uses a biotinylated HRP polymer (A05849 caption: biotinylated HRP polymer; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the exact selected-SKU tissue-IHC record lists the fixative as unreported (A05849 caption: fixative field). Heat-mediated EDTA retrieval at pH 9.0 was used for the paraffin-section image, but retrieval dependence is unreported (A05849 caption: heat-mediated EDTA retrieval). The supplied evidence does not establish that frozen sections or IF are easier, and a testis-specific artefact is unreported; assess background with the stated controls (HPA: testis High in pachytene spermatocytes; standard IHC practice).
HPA tissue IHC evidence for TUBB2B
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Low consistency between antibody staining and RNA expression data. Pending external verification. Caution, targets protein from more than one gene. External characterization data supports antibody staining.). 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 |
|---|---|---|---|---|
| Testis | Pachytene spermatocytes | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuropil | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced TUBB2B IHC Tips
Troubleshoot TUBB2B staining in paraffin sections by checking retrieval, antibody specificity and cellular distribution before interpreting chromogenic signal.
Which retrieval conditions should I start with for TUBB2B paraffin sections?
Start with heat-mediated retrieval in EDTA buffer at pH 9 for paraffin sections stained with A05849 (datasheet A05849). The selected tissue image used that retrieval with primary antibody at 1:2000 for 1 hour at room temperature (A05849 tissue-IHC caption). If staining is weak, first check that the heating step reached its intended conditions and that sections remained covered by buffer throughout retrieval (standard IHC practice). Keep retrieval conditions identical between comparison slides, and use a no-primary control to assess signal from the detection system (standard IHC practice). Interpret any improvement alongside tissue morphology and expected cytoplasmic staining (UniProt Q9BVA1; standard IHC practice).
Could fixation explain weak or uneven TUBB2B staining?
Target-specific sensitivity of TUBB2B staining to fixation is unknown from the evidence supplied here. Record the fixative, fixation duration and processing history for each paraffin section, then compare sections prepared under matched conditions (standard IHC practice). If staining varies across a section, examine morphology and compare a second section before changing retrieval or antibody concentration (standard IHC practice). A matched run using the documented pH 9 EDTA retrieval and 1:2000 primary dilution provides a useful starting comparison for A05849 (datasheet A05849; A05849 tissue-IHC caption). Do not attribute a fixation effect to TUBB2B without a controlled comparison (standard IHC practice).
Where should convincing TUBB2B chromogenic staining appear?
Expect a cytoplasmic, cytoskeletal distribution because TUBB2B is annotated in the cytoplasm and cytoskeleton, with no transmembrane segment (UniProt Q9BVA1). HPA reports cytoplasmic staining in most tissues, especially ciliated cells and peripheral nerves, and medium staining in cerebral cortex neuropil (HPA tissue IHC). Compare the stained structures with adjacent morphology and a counterstained section; diffuse nuclear-only color warrants scrutiny (UniProt Q9BVA1; standard IHC practice). In brain sections, distinguish neuropil signal from staining assigned to neuronal cell bodies or glia before reporting cell-level positivity (HPA tissue IHC; standard IHC practice). HPA cautions that its antibodies can recognize proteins from multiple genes (HPA tissue IHC).
How can I assess whether staining is specific to TUBB2B?
The supplied UniProt record lists 0 isoforms and no annotated domains for TUBB2B, but those annotations do not establish antibody specificity (UniProt Q9BVA1). TUBB2B has annotated modified residues, including phosphorylation and lysine modifications; the supplied evidence does not locate A05849's epitope relative to them (UniProt Q9BVA1; A05849 tissue-IHC caption). Ask for the antibody's immunogen or epitope information before interpreting staining as modification sensitive (standard IHC practice). Include a no-primary slide and compare staining with expected tissue distribution under matched retrieval and detection conditions (HPA tissue IHC; standard IHC practice). HPA explicitly cautions that its staining may include proteins from multiple genes (HPA tissue IHC).
How should I investigate TUBB2B localisation by IF alongside this IHC result?
For a separate IF/ICC experiment, pair TUBB2B with an independently validated marker of the cell population being examined so overlapping structures can be assigned to cells (standard IF practice). Choose spectrally separated fluorophores and check unstained tissue autofluorescence before assigning a weak signal to TUBB2B (standard IF practice). TUBB2B is cytoplasmic and has no transmembrane segment, so permeabilisation should permit antibody access to the intracellular epitope (UniProt Q9BVA1; standard IF practice). HPA reports mainly microtubule localisation, with additional signal in structures including the mitotic spindle and primary cilium, while cautioning that its antibodies can target multiple genes (HPA subcellular). Verify IF antibody specificity independently before transferring an IHC interpretation to IF (standard IF practice).
What should I check when TUBB2B IHC has widespread brown background?
Run a no-primary section through the same chromogenic detection steps to identify background from the secondary system or tissue (standard IHC practice). Check the peroxidase block and compare signal before and after DAB development, especially where native pigment could resemble brown reaction product (standard IHC practice). Titrate A05849 around the documented 1:2000 primary dilution while keeping the documented 1 hour incubation and pH 9 EDTA retrieval consistent (A05849 tissue-IHC caption; datasheet A05849). Review washes and blocking if the no-primary slide is clean but staining remains diffuse (standard IHC practice). Require interpretable cytoplasmic structure before calling widespread color positive (UniProt Q9BVA1; standard IHC practice).
How should I quantify TUBB2B staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue compartment and scoring unit before reviewing treatment groups, since TUBB2B signal may be cytoplasmic and prominent in neuropil or peripheral nerves (HPA tissue IHC; standard IHC practice). For identifiable cells, report the percentage positive and an H-score based on intensity categories; for neuropil, report stained area or signal density per mm² (standard IHC practice). Normalize each measure to the relevant viable tissue area or counted cells, using the same threshold and counterstain criteria across slides (standard IHC practice). Analyze comparable anatomical regions and exclude folds, edges and necrosis by a predefined rule (standard IHC practice). Report tissue and cell compartment separately rather than pooling unlike structures (HPA tissue IHC; standard IHC practice).
How do I distinguish true TUBB2B staining from artefact?
A credible result follows cytoplasmic or cytoskeletal structures and a plausible tissue pattern, such as neuropil staining in cerebral cortex (UniProt Q9BVA1; HPA tissue IHC). Treat isolated nuclear-only color, section-edge accentuation and staining confined to necrotic areas as findings that require review (UniProt Q9BVA1; standard IHC practice). Compare those areas with morphology, a matched no-primary slide and a second section; a no-primary signal can indicate endogenous enzyme or detection-system background (standard IHC practice). Consider cell identity before treating stained structures as positive cells, because HPA describes signal in both cellular and tissue structures (HPA tissue IHC). HPA rates its tissue evidence Supported but cautions about low RNA concordance and antibodies targeting multiple genes (HPA tissue IHC).
Boster reagents
Best TUBB2B / Tubulin beta-2B chain IHC Antibodies
A05849 has a human brain paraffin-section IHC figure (A05849 IHC image caption) and listed IF use (catalog applications); its listed reactivity covers human, mouse, and rat (catalog reactivity).
Real IHC data
Anti-TUBB2B Antibody (N-term)
Cat # A05849
A05849 will render with an IHC figure from formaldehyde-fixed, paraffin-embedded human brain tissue (A05849 IHC image caption). It is listed for IF and for human, mouse, and rat reactivity; no IF figure is supplied (catalog applications, reactivity, IF image alts).
Which to pick: For tissue IHC, choose A05849: its own figure caption documents human brain paraffin sections, formaldehyde fixation, heat retrieval in EDTA at pH 9.0, and a 1:2000 primary dilution (A05849 IHC image caption). For IF, A05849 is listed at 1:10–1:50; ICC validation and an IF figure are not supplied (catalog applications, IF dilution, IF image alts). For cross-species work, A05849 is a rabbit polyclonal listed as reactive with human, mouse, and rat, while its IHC figure shows human tissue only (catalog host, clonality, reactivity; A05849 IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9BVA1 (TBB2B_HUMAN, Tubulin beta-2B chain).
- Human Protein Atlas. TUBB2B tissue IHC expression (reliability: Supported).
- Human Protein Atlas. TUBB2B subcellular location (ICC-IF): Mainly localized to the microtubules. In addition localized to the cytokinetic bridge, mitotic spindle, primary cilium, primary cilium tip, basal body, flagellar centriole, principal piece and end piece. Caution: Based on antibodies targeting proteins from multiple genes..
- Human Protein Atlas. TUBB2B antibody validation summary (2 antibodies).
- Everolimus Stabilizes Podocyte Microtubules via Enhancing TUBB2B and DCDC2 Expression. PloS one 2015 — PMC4557973.
- Patterns of tubb2b Promoter-Driven Fluorescence in the Forebrain of Larval Xenopus laevis. Frontiers in neuroanatomy 2022 — PMC9304554.
- TUBB2B regulates epithelial-mesenchymal transition via interaction with Vimentin to promote glioma migration and invasion. Cancer cell international 2024 — PMC11662515.
- Differential requirements of tubulin genes in mammalian forebrain development. PLoS genetics 2019 — PMC6697361.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:14574404 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for TUBB2B (Tubulin beta-2B chain). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 9 HIER, heat-mediated (datasheet A05849). Validated IHC antibody: A05849 at 1:2000 (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Pachytene spermatocytes); negative Adipose tissue. IHC-P workflow on paraffin sections. Protocol steps: 1. Sample: Formaldehyde-fixed, paraffin-embedded Human brain tissue (datasheet A05849); 2. Fixation: Image formalin-fixed; duration unreported (datasheet A05849); 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 9 (datasheet A05849); 20 min, 95–100 °C (standard); 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-TUBB2B, 1:2000 (datasheet A05849); 9. Primary incubation: 1 hours at room temperature (datasheet A05849). Watch for: Fixation: Formaldehyde-fixed brain sections were shown (selected-SKU IHC image A05849); keep fixation consistent (standard IHC practice; not target-specific); Caveat: Staining may reflect protein from more than one gene (HPA tissue IHC); Isoform / epitope: No annotated isoforms; the chain spans residues 1–445 (UniProt); Regulation: High expression in brain (UniProt). Evidence: UniProt Q9BVA1, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the microtubules. In addition localized to the…, Datasheet A05849, PMC4557973. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/TUBB2B/immunohistochemistry-ihc-guide.