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HMGB2 / High mobility group protein B2 · IHC design guide
Design Immunohistochemistry for HMGB2
Plan chromogenic HMGB2 IHC in paraffin sections against the widespread nuclear tissue pattern (HPA tissue IHC). The catalog antibody's IHC concentration is 0.5–1 μg/mL (datasheet PB10002); interpret staining with the HPA cross-gene caution (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HMGB2 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 nuclear tissue staining (HPA tissue IHC) | |
| Staining pattern | Widespread nuclear staining across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet PB10002) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Salivary gland+1 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across samples. (standard IHC practice; not target-specific) | |
| Caveat | Staining may include proteins from other genes (HPA tissue IHC) | |
| Regulation | No stimulus-linked expression change reported (UniProt) | |
| Isoform / epitope | No isoforms annotated; no membrane-sided epitope (UniProt) |
Section 1
Recommended HMGB2 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet PB10002) with four published HMGB2 paraffin-section IHC protocols (PMC7789524; PMC7576003; PMC9769043; PMC12632091).
| Sample | Paraffin-embedded mouse intestine tissue; fixative not specified (datasheet PB10002) |
| Fixation | Image fixative and duration unreported (datasheet PB10002); 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 PB10002); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet PB10002) |
| Primary antibody | Rabbit anti-HMGB2, 0.5-1μg/ml (datasheet PB10002) |
| Primary incubation | Overnight at 4 °C (datasheet PB10002) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB10002) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | HMGB2-positive staining in endocrine cells of appendix (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet PB10002). Follow each article’s stated retrieval conditions when reproducing its protocol (PMC7789524; PMC7576003; PMC9769043; PMC12632091).
Section 2
What Is the Expected HMGB2 Staining Pattern?
HMGB2 is predominantly nuclear in its basal state, with UniProt also listing chromosome, cytoplasm and secreted locations (UniProt P26583: subcellular location). In paraffin-section IHC, expect nuclear staining across many cell types, with strong staining in selected hematopoietic, germinal center, endocrine and basal-layer cells (HPA tissue IHC: ubiquitous nuclear expression; listed High cells). HPA rates the tissue profile Enhanced but cautions that antibody staining may include proteins from more than one gene (HPA tissue IHC: reliability description).
| Distinct nuclear chromogen in hematopoietic cells of bone marrow or germinal center cells of lymph node. | This matches HPA's High staining in those cells and its ubiquitous nuclear profile (HPA tissue IHC: Bone marrow and Lymph node, High; profile). Assess cell-by-cell nuclear staining rather than treating uniform staining of the whole section as the expected result. |
| Nuclear staining in endocrine cells of appendix, colon, duodenum, rectum or small intestine, or in skin basal-layer cells. | These are reported High cell populations (HPA tissue IHC: listed High cells). Adjacent cells may differ in intensity; HPA's cell-level observations do not imply that every cell in a positive organ should stain equally. |
| Predominantly uniform cytoplasmic or stromal color with little or no discernible nuclear staining. | That distribution conflicts with the reported basal nuclear pattern (UniProt P26583: predominantly nuclear; HPA tissue IHC: ubiquitous nuclear expression). Review morphology and controls before calling it HMGB2: UniProt also lists cytoplasmic and secreted locations, so extranuclear signal alone is not proof of artefact (UniProt P26583: subcellular location). |
| Strong color in salivary-gland glandular cells or skeletal-muscle myocytes, especially without matching nuclear morphology. | HPA reports HMGB2 as Not detected in those specific cell populations (HPA tissue IHC: Salivary gland and Skeletal muscle). Check for antibody cross-reactivity or endogenous detection activity; HPA cautions that its antibody staining can include proteins from more than one gene (HPA tissue IHC: reliability description). |
| Diffuse color across cells, stroma and blank areas, or no nuclear signal in a known-positive cell population. | Diffuse, non-cell-specific color is unconvincing for a predominantly nuclear target (UniProt P26583: basal location). A blank positive control conflicts with HPA's High staining in the selected population (HPA tissue IHC: listed High cells); inspect the control and detection run before interpreting test tissue. |
💡Expected HMGB2 appearanceCall a convincing positive when chromogen is clearly nuclear and cell-resolved, with strong signal in an HPA High population such as bone-marrow hematopoietic cells (HPA tissue IHC: profile; Bone marrow, High); diffuse non-nuclear color or strong staining in an HPA Not detected cell population is suspect (HPA tissue IHC: Salivary gland and Skeletal muscle).
| Which cells are present in the section? | The High calls are cell-specific: endocrine cells in listed gastrointestinal tissues, bone-marrow hematopoietic cells, lymph-node germinal center cells and skin basal-layer cells (HPA tissue IHC: listed High cells). A section lacking the relevant population is a weak positive control, even when its organ name appears on the High list. |
| How firmly is the tissue pattern supported? | HPA labels the tissue IHC profile Enhanced, citing consistency between staining and RNA data, while warning that antibodies can detect proteins from more than one gene and that secreted variants complicate RNA–protein agreement (HPA tissue IHC: reliability description). Interpret localization and cell identity together. |
| Does extranuclear signal fit the target? | Basal HMGB2 is predominantly nuclear, but UniProt also lists chromosome, cytoplasm and secreted locations (UniProt P26583: subcellular location). Do not score a diffuse extranuclear deposit as positive solely because secretion is listed; require interpretable cellular morphology and appropriate controls (general IHC practice). |
| What should IF/ICC show? | Expect mainly nucleoplasmic fluorescence; HPA additionally reports supported nucleolar localization and approved principal-piece and annulus localization (HPA subcellular ICC-IF: locations). This answers the IF/ICC localization question; the paraffin-section IHC interpretation remains anchored to HPA's tissue nuclear profile (HPA tissue IHC: profile). |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive bone marrow or lymph node has no convincing nuclear chromogen. | The sampled field may lack the reported High population, or the IHC detection run may have failed (HPA tissue IHC: Bone marrow hematopoietic cells and Lymph node germinal center cells, High; general IHC practice). | Locate the relevant cells on the counterstain, then check a known-positive section processed in the same run. If both are blank, review primary-antibody and detection steps using the applicable IHC protocol (general IHC practice). |
| Color is diffuse across the section and obscures nuclei. | Non-cell-specific background can arise from blocking, washing or detection conditions (general IHC practice); it does not resemble the HPA ubiquitous nuclear profile (HPA tissue IHC: profile). | Inspect a no-primary control, washing and blocking conditions; compare chromogen deposition with nuclear morphology on the counterstain (general IHC practice). Reduce background before scoring cell-specific HMGB2 staining. |
| Cytoplasmic color dominates while nuclei remain pale. | This disagrees with basal predominant nuclear localization, although UniProt lists cytoplasm and secretion among possible locations (UniProt P26583: subcellular location). The image alone cannot distinguish biology from nonspecific staining. | Compare the same cell type with a known-positive nuclear control and a no-primary control; score the extranuclear pattern separately until its specificity is supported (general IHC practice). |
| Salivary-gland glandular cells or skeletal-muscle myocytes stain strongly. | These cell populations are Not detected in HPA tissue IHC; cross-reactivity or endogenous detection activity is possible (HPA tissue IHC: Salivary gland and Skeletal muscle; general IHC practice). | Verify the cell identity and inspect no-primary and detection controls. If staining persists with primary antibody only, compare with independent antibody validation; HPA warns that its staining may involve more than one gene (HPA tissue IHC: reliability description). |
| The section appears negative despite scattered nuclear-positive cells. | The expected High designation applies to named cell populations, not uniformly to every cell in the organ (HPA tissue IHC: listed High cells). A field-level impression can miss a restricted positive population. | Review multiple fields and identify the relevant cells by morphology before recording intensity and the fraction of positive nuclei (general IHC practice); use HPA's cell-level calls as the comparison (HPA tissue IHC: listed High cells). |
| Nuclear color is too strong to distinguish individual positive cells. | Overdeveloped chromogen or excessive detection can obscure cell-level localization (general IHC practice), which is needed to compare with HPA's nuclear, cell-specific observations (HPA tissue IHC: profile; listed High cells). | Review chromogen development and detection settings against the run controls, then rescore only sections with visible nuclei and interpretable cell boundaries (general IHC practice). |
Experiment evidence
IHC & IF images showing HMGB2 staining
PB10002 · IHCIHC analysis of HMGB2 using anti-HMGB2 antibody (PB10002). HMGB2 was detected in a paraffin-embedded section of mouse intestine tissue. Heat
PB10002 · IHCIHC analysis of HMGB2 using anti-HMGB2 antibody (PB10002). HMGB2 was detected in a paraffin-embedded section of rat spleen tissue. Heat medi
PB10002 · IFIF analysis of HMGB2 using anti-HMGB2 antibody (PB10002). HMGB2 was detected in immunocytochemical section of U20S cells. Enzyme antigen ret
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000164104-HMGB2/tissue/cerebral+cortex#imid_16428723
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000164104-HMGB2/tissue/colon#imid_16425137
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000164104-HMGB2/tissue/liver#imid_16425122
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000164104-HMGB2/tissue/kidney#imid_16425131
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA053314; green = HMGB2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA053314; green = HMGB2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HMGB2 IHC & IF
🧪Run colon first: its endocrine cells must show HMGB2 staining (HPA: High in colon endocrine cells). Use salivary gland glandular cells as the negative tissue (HPA: Not detected in salivary gland glandular cells); on the colon slide, cells used as internal negative comparators should lack specific nuclear DAB staining, but verify their status rather than assuming all neighboring cells are negative (UniProt P26583: predominantly nuclear in the basal state).
Positive control tissue: Appendix (Endocrine cells, HPA High)
Negative control tissue: Salivary gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show HMGB2 in A-431, U-251MG, U2OS, HEL, Rh30, hTERT-RPE1 (serum starved), Sperm, NIH 3T3, with annotated localisation: Nucleoplasm (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a host-matched rabbit IgG isotype control (selected-SKU tissue-IHC caption: rabbit primary antibody); assess biological specificity with a matched HMGB2 knockout specimen if available. Block endogenous peroxidase and check for endogenous biotin signal because the selected-SKU tissue-IHC caption uses biotin-based detection with DAB (selected-SKU tissue-IHC caption: biotinylated secondary, SABC, DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the fixative in the selected-SKU paraffin-section caption is unreported (selected-SKU tissue-IHC caption: fixative not stated). The caption reports heat retrieval in EDTA at pH 8.0, but does not establish whether retrieval is required or whether frozen sections or IF are easier (selected-SKU tissue-IHC caption: EDTA heat retrieval). In colon, identify the endocrine cells before scoring so background DAB in other cells is not mistaken for the specified positive population (HPA: High in colon endocrine cells).
HPA tissue IHC evidence for HMGB2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene. At least one protein variant secreted, tissue location of RNA and protein might differ and correlation is complex.). 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 | Endocrine cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Colon | Endocrine cells | High | Protein (IHC) | HPA → |
| Duodenum | Endocrine cells | High | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | High | Protein (IHC) | HPA → |
Section 3
Advanced HMGB2 IHC Tips
Troubleshoot HMGB2 chromogenic IHC in paraffin sections by checking retrieval, nuclear localisation, controls and cell-specific scoring.
What retrieval should I try when HMGB2 nuclear staining is weak?
Use heat-mediated retrieval in EDTA at pH 8.0 first (datasheet PB10002). The selected paraffin-section image used that condition, followed by 10% goat serum block and 1 μg/ml primary antibody overnight at 4°C (caption PB10002). If nuclei remain weak, check that sections were fully deparaffinised and compare retrieval heating and cooling across matched sections (standard IHC practice). An alternative buffer is a fallback experiment; compare its staining with EDTA pH 8.0 using identical detection and exposure conditions (standard IHC practice). Judge improvement by clearer nuclear staining within the same cell population, rather than by greater staining throughout the tissue (HPA: ubiquitous nuclear expression; standard IHC practice).
Could fixation explain absent HMGB2 staining in my paraffin sections?
The selected paraffin-section caption does not state a fixative, so HMGB2-specific fixation sensitivity is unknown (caption PB10002). Record the fixative, fixation duration and section handling for each specimen, then compare matched sections processed under documented conditions (standard IHC practice). Keep retrieval at EDTA pH 8.0 while assessing fixation as a possible variable (datasheet PB10002; standard IHC practice). Include a control section with expected nuclear staining, such as the documented intestinal context, in the same run (caption PB10002; HPA: ubiquitous nuclear expression). Weak staining alone cannot identify fixation as the cause; also assess retrieval, antibody incubation and chromogenic detection (standard IHC practice).
How should I interpret cytoplasmic staining when HMGB2 is usually nuclear?
Expect predominantly nuclear staining in basal tissue, consistent with HMGB2’s chromatin-associated role (UniProt P26583: basal localisation and function). The tissue profile describes ubiquitous nuclear expression, and cell imaging supports nucleoplasmic localisation with additional nucleolar signal (HPA: tissue profile and subcellular localisation). Cytoplasmic or extracellular signal can be biologically plausible because HMGB2 is also annotated in the cytoplasm and secreted compartment (UniProt P26583: subcellular localisation). Assess whether it occurs in intact, recognisable cells and persists across matched sections before assigning it to HMGB2 (standard IHC practice). If diffuse signal tracks damaged tissue or section edges, review controls and processing before reporting a localisation change (standard IHC practice).
Could an isoform or modified epitope cause discordant HMGB2 staining?
The supplied record lists 0 isoforms and a single 1–209 chain, so an annotated isoform does not explain discordance here (UniProt P26583: isoforms and processing). HMGB2 has no transmembrane segment, while its listed modified residues include acetyllysines, phosphoserine and an oxidised cysteine (UniProt P26583: topology and modified residues). Those annotations do not establish that this antibody recognises a modification-sensitive epitope (UniProt P26583: modified residues; caption PB10002: epitope unspecified). If staining differs between specimens, first compare retrieval, specimen processing and staining controls under identical conditions (standard IHC practice). Seek documented epitope information or an independently validated antibody before attributing the difference to epitope masking (standard IHC practice).
How can IF help check the cell identity and location of IHC staining?
Use IF as a complementary localisation check, because this page’s selected antibody evidence describes chromogenic staining in a paraffin section (caption PB10002). Pair HMGB2 with a validated marker of the expected cell population; intestinal endocrine cells are one documented high-expression population (HPA: small intestine endocrine cells, High). Choose fluorophores after examining unstained tissue autofluorescence and include single-label controls when assessing overlap (standard IF practice). For the predominantly nuclear target, optimise permeabilisation to give antibody access to intracellular and nuclear epitopes without disrupting morphology (UniProt P26583: basal nuclear localisation; standard IF practice). Compare nuclear signal and cell identity with the chromogenic section, while validating the IF workflow separately (standard IF practice).
How do I reduce diffuse DAB staining without losing nuclear signal?
Compare a no-primary control with the test section to distinguish detection background from primary-antibody-associated staining (standard IHC practice). Block endogenous peroxidase before DAB development and check whether staining follows damaged tissue or section edges (standard chromogenic IHC practice). The selected image used 10% goat serum, 1 μg/ml primary antibody overnight at 4°C, and a biotin-based DAB detection workflow (caption PB10002). If background persists, check blocking, washes, antibody concentration and development time one variable at a time (standard IHC practice). Preserve interpretable nuclear staining when adjusting the workflow, since the tissue profile describes ubiquitous nuclear expression (HPA: tissue profile).
What should I measure when HMGB2 staining varies across cell populations? ⚠ ANSWER MARKED FOR VERIFICATION
Define cell populations and regions before scoring, because the tissue profile reports high staining in selected cells alongside broadly nuclear expression (HPA: tissue profile and positive-cell table). For nuclear chromogenic signal, report the percentage of positive nuclei and an H-score based on nuclear intensity categories (standard IHC quantification practice). Normalise counts to evaluable nuclei in the same cell population, or report positive-cell density per mm² of analysable tissue (standard IHC quantification practice). Apply the same thresholds, section-quality criteria and region selection to every specimen (standard IHC quantification practice). Report cytoplasmic or extracellular staining separately from nuclear scores because HMGB2 has multiple annotated compartments (UniProt P26583: subcellular localisation).
How can I distinguish true HMGB2 staining from section artefact?
A convincing result includes nuclear signal in intact cells, consistent with HMGB2’s basal localisation and the tissue IHC profile (UniProt P26583: basal nuclear localisation; HPA: tissue profile). Cell identity matters: high staining is documented in intestinal endocrine cells, while salivary gland glandular cells and skeletal-muscle myocytes are reported as undetected (HPA: tissue positive and negative tables). Treat staining confined to edges, necrotic areas or a no-primary control as a processing or detection concern (standard IHC practice). Check endogenous peroxidase contribution when unexpected DAB deposits appear (standard chromogenic IHC practice). Cytoplasmic or extracellular staining needs separate validation, since those compartments are annotated but do not establish that every such signal is specific (UniProt P26583: subcellular localisation).
Boster reagents
Best HMGB2 / High mobility group protein B2 IHC Antibodies
PB10002 has captioned IHC images from mouse, rat and human paraffin sections, plus IF images from cells and rat intestine (PB10002 image captions).
Real IHC data
Anti-HMGB2 Antibody ®
Cat # PB10002
PB10002, the SKU with a rendered card, shows IHC staining in paraffin-embedded mouse intestine (PB10002 card caption). Its other captions show IHC in rat spleen and human intestinal cancer, and IF in U20S cells, MCF-7 cells and rat intestine (PB10002 image captions).
Which to pick: For tissue IHC, pick PB10002: its own card documents EDTA pH 8.0 retrieval and DAB detection in a paraffin-embedded section; the fixative is unreported (PB10002 card caption). For IF/ICC, PB10002 has captioned cell images and a listed IF/ICC application (PB10002 IF captions; catalog applications). For work across human, mouse and rat samples, PB10002 has listed reactivity in all three and captioned IHC images from each; M02651 also lists those species and IHC/IF applications, but has no supplied IHC or IF image caption (catalog reactivity and applications; PB10002 IHC captions; M02651 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P26583 (HMGB2_HUMAN, High mobility group protein B2).
- Human Protein Atlas. HMGB2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. HMGB2 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the nucleoli, principal piece and annulus..
- Human Protein Atlas. HMGB2 antibody validation summary (4 antibodies).
- Identification of differentially expressed proteins and clinicopathological significance of HMGB2 in cervical cancer. Clinical proteomics 2021 — PMC7789524.
- High mobility group box 2 modulates the progression of osteosarcoma and is related with poor prognosis. Annals of translational medicine 2020 — PMC7576003.
- Pivotal role of High-Mobility Group Box 2 in ovarian folliculogenesis and fertility. Journal of ovarian research 2022 — PMC9769043.
- Multi-omics analysis of the HMGB2(+) tumor epithelial cells in lactylation subgroups in colorectal cancer. Cell & bioscience 2025 — PMC12632091.
- PubMed PMID:1754403 — UniProt-cited evidence.
- PubMed PMID:1551873 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HMGB2 (High mobility group protein B2). Expected localisation Predominantly nuclear tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet PB10002). Validated IHC antibody: PB10002 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Endocrine cells); negative Salivary gland. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded mouse intestine tissue; fixative not specified (datasheet PB10002); 2. Fixation: Image fixative and duration unreported (datasheet PB10002); 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 PB10002); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet PB10002); 8. Primary antibody: Rabbit anti-HMGB2, 0.5-1μg/ml (datasheet PB10002); 9. Primary incubation: Overnight at 4 °C (datasheet PB10002). Watch for: Fixation: Keep fixation consistent across samples. (standard IHC practice; not target-specific); Caveat: Staining may include proteins from other genes (HPA tissue IHC); Isoform / epitope: No isoforms annotated; no membrane-sided epitope (UniProt); Regulation: No stimulus-linked expression change reported (UniProt). Evidence: UniProt P26583, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet PB10002, PMC7789524, PMC7576003. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HMGB2/immunohistochemistry-ihc-guide.