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SPIB / Transcription factor Spi-B · IHC design guide
Design Immunohistochemistry for SPIB
Plan SPIB chromogenic IHC on paraffin sections with the catalog antibody, starting at 2.5 μg/mL (datasheet). Compare the observed cytoplasmic tissue staining (HPA tissue IHC) with SPIB’s annotated nuclear and cytoplasmic locations (UniProt), while accounting for low staining–RNA consistency (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SPIB 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 several tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in immune and other tissue cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Spleen+4 more · see all | |
| Negative control | Esophagus+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Staining and RNA show low consistency (HPA tissue IHC) | |
| Regulation | Expressed in pDCs and B cells (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope effects are unknown (UniProt) |
Section 1
Recommended SPIB IHC & IF Protocols
The catalog antibody’s IHC-P protocol accompanies three published SPIB IHC methods using lymphoma, colorectal, and gastric specimens (PMC5021043; PMC8176860; PMC13383869).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A02805-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-SPIB, 2.5 μg/mL (datasheet A02805-1) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SPIB-positive staining in cells in red pulp of spleen (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues, including immune cells. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 at 95–98 °C for 20 min for the catalog antibody (page antigen retrieval). Published methods also used low-pH or citrate retrieval (PMC5021043; PMC13383869).
Section 2
What Is the Expected SPIB Staining Pattern?
SPIB is a nuclear and cytoplasmic transcription factor with no transmembrane segment (UniProt Q01892). Expression is reported in B cells and plasmacytoid dendritic cells, but not T cells or granulocytes (UniProt Q01892). HPA reports cytoplasmic staining across several tissues; its tissue IHC reliability is Uncertain because staining and RNA data agree poorly, and it flags antibody cross-target and transcript discrepancies (HPA: tissue IHC).
| Discrete nuclear staining in a subset of immune cells, with or without cytoplasmic signal. | This is compatible with SPIB localisation and its reported B cell and plasmacytoid dendritic cell expression (UniProt Q01892). Morphology alone cannot identify those subsets. Score nuclear and cytoplasmic staining separately, then confirm cell identity with appropriate markers (general IHC practice). HPA has no ICC-IF images establishing a preferred compartment (HPA: subcellular). |
| Predominantly cytoplasmic staining in spleen red pulp cells or tonsil squamous epithelial cells. | HPA reports High staining in both populations and describes a broader cytoplasmic tissue pattern (HPA: tissue IHC). Red pulp staining does not identify the positive cell subtype. Tonsil epithelial staining should be interpreted cautiously alongside UniProt’s immune cell expression profile and HPA’s Uncertain tissue IHC reliability (UniProt Q01892; HPA: tissue IHC). |
| Sharp membrane outlining or extracellular deposits presented as SPIB positive cells. | These are discordant with the reported nuclear and cytoplasmic localisation and lack of a transmembrane segment (UniProt Q01892). Review tissue morphology and detection controls before calling them specific staining (general IHC practice). Cytoplasmic signal by itself is not a wrong compartment: both UniProt and HPA allow it (UniProt Q01892; HPA: tissue IHC). |
| Strong staining assigned to T cells or granulocytes, or broad staining across unrelated cell types. | T cells and granulocytes are reported as SPIB negative (UniProt Q01892). Confirm the stained cell identities before inferring cross-reactivity or endogenous detection activity (general IHC practice). Broad positivity also warrants caution because HPA flags cross-target staining and poor agreement with RNA expression (HPA: tissue IHC). |
| Uniform haze, or no signal in a section containing an expected positive population. | Haze that obscures cell boundaries prevents compartment scoring (general IHC practice). An absent signal may reflect the sampled cells, detection performance or antibody performance; it does not establish biological absence (general IHC practice). Compare with spleen red pulp cells, which HPA scores High, while retaining HPA’s Uncertain reliability caveat (HPA: tissue IHC). |
💡Expected SPIB appearanceCall a result compatible with SPIB when discrete nuclear and/or cytoplasmic signal occurs in appropriately identified B cells or plasmacytoid dendritic cells (UniProt Q01892), with potentially High cytoplasmic staining in HPA-reported spleen red pulp cells (HPA: tissue IHC); membrane rims, extracellular deposits and indiscriminate haze are suspect (UniProt Q01892; general IHC practice).
| Compartment and tissue evidence | UniProt places SPIB in nucleus and cytoplasm; HPA describes cytoplasmic tissue staining (UniProt Q01892; HPA: tissue IHC). Record the compartments independently. HPA lists no ICC-IF image or main subcellular location, so tissue IHC observations cannot establish a validated IF distribution (HPA: subcellular). |
| Cell selection and comparator tissue | UniProt reports expression in B cells and plasmacytoid dendritic cells, and absence in T cells and granulocytes (UniProt Q01892). HPA scores spleen red pulp cells High and skeletal muscle myocytes Not detected (HPA: tissue IHC). Use cell identity and compartment when comparing sections; a tissue label alone is insufficient (general IHC practice). |
| Antibody and transcript uncertainty | The listed HPA antibody, HPA018523, has Uncertain IHC validation; HPA flags possible staining of protein from more than one gene and a splice or transcript discrepancy (HPA: antibodies; HPA: tissue IHC). UniProt lists three SPIB isoforms (UniProt Q01892). These records do not identify which isoforms an antibody detects or resolve discordant cell staining. |
| IF/ICC question: is the expected pattern validated in cells? | No ICC-IF images or main subcellular location are available in HPA’s SPIB subcellular record, and ICC validation is unlisted for HPA018523 (HPA: subcellular; HPA: antibodies). Nuclear and cytoplasmic localisation is the available biological expectation (UniProt Q01892). Assess IF/ICC methods in its own guide; this IHC section offers no IF protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| No staining in spleen red pulp cells. | Positive cells may be absent from the sampled field, or the IHC detection may have failed (general IHC practice). | Locate intact red pulp cells, inspect a matched positive control, and check the catalog antibody’s IHC-P protocol (general IHC practice); HPA rates these cells High but tissue IHC reliability Uncertain (HPA: tissue IHC). |
| Every cell shows diffuse brown haze. | Background may arise from nonspecific binding, endogenous enzyme activity or detection reagents (general IHC practice). | Compare a primary-antibody omission control; review blocking, washes and chromogen development using general IHC practice. Do not score haze as SPIB, even where HPA reports cytoplasmic tissue expression (HPA: tissue IHC). |
| Only membrane rims or extracellular material stains. | That distribution conflicts with SPIB’s reported nucleus/cytoplasm localisation and lack of a transmembrane segment (UniProt Q01892). | Check morphology and controls, then repeat with the validated IHC conditions if needed (general IHC practice). Require cellular nuclear or cytoplasmic signal before assigning SPIB localisation (UniProt Q01892). |
| Staining appears in cells identified as T cells or granulocytes. | The cell assignment may be wrong, or the signal may be nonspecific; UniProt reports neither population expresses SPIB (UniProt Q01892). | Verify cell identity with appropriate markers and compare antibody-omission controls (general IHC practice). Treat persistent signal cautiously because HPA flags possible cross-target staining (HPA: tissue IHC). |
| Tonsil epithelium is strongly positive while expected immune cells are weak. | HPA scores tonsil squamous epithelial cells High, but its tissue IHC reliability is Uncertain (HPA: tissue IHC). | Document each cell population and compartment separately; verify cell identity and compare an independent IHC-validated antibody where available (general IHC practice). Do not use epithelial positivity alone to infer SPIB specificity. |
| IF/ICC shows a different distribution from the IHC section. | HPA supplies no SPIB ICC-IF images or assigned main subcellular location for comparison (HPA: subcellular). | Interpret IF/ICC using its own guide and controls. Keep the IHC tissue observation distinct from UniProt’s nuclear and cytoplasmic localisation annotation (HPA: tissue IHC; UniProt Q01892). |
Experiment evidence
IHC & IF images showing SPIB staining
A02805-1 · IHCImmunohistochemistry of SPIB in human prostate tissue with SPIB antibody at 2.5 μg/mL.
A02805-1 · IHCImmunohistochemistry of SPIB in human prostate tissue with SPIB antibody at 2.5 μg/ml.
A02805-1 · IFImmunofluorescence of SPIB in human prostate tissue with SPIB Antibody at 20 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000269404-SPIB/tissue/cerebral+cortex#imid_4825280
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000269404-SPIB/tissue/colon#imid_4825198
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000269404-SPIB/tissue/liver#imid_4825207
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000269404-SPIB/tissue/kidney#imid_4825200
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SPIB IHC & IF
🧪Run spleen first and look for staining in red-pulp cells (HPA: High in cells in red pulp). Use skeletal muscle myocytes as the negative tissue (HPA: Not detected in myocytes); identified T cells on the spleen section should lack SPIB-specific staining (UniProt: not expressed in T-cells).
Positive control tissue: Spleen (Cells in red pulp, HPA High)
Negative control tissue: Esophagus (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for SPIB; derive a cell-line control from the positive tissue's cell type (Cells in red pulp) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and immunoglobulin class, with clonality matched where applicable (standard IHC practice). Use a SPIB-knockout specimen as a biological negative control (standard IHC practice); on the blood-rich spleen section, block endogenous peroxidase and check for residual background before scoring chromogenic signal (standard IHC practice).
⚠️Feasibility: SPIB is reported in the nucleus and cytoplasm, so assess nuclear staining while recording any cytoplasmic signal separately (UniProt Q01892: subcellular location). A target-specific fixation window and retrieval dependency are unreported in the supplied evidence; the selected A02805-1 tissue-IHC caption also leaves the fixative unreported (A02805-1 caption: fixative not stated). Whether frozen sections or IF would be easier cannot be determined from the supplied evidence (HPA: no ICC-IF image cell lines listed).
HPA tissue IHC evidence for SPIB
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene. Caution, Splice and/or transcript discrepancy exists.). 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 |
|---|---|---|---|---|
| Spleen | Cells in red pulp | High | Protein (IHC) | HPA → |
| Tonsil | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Adipose tissue | Adipocytes | Medium | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Vagina | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced SPIB IHC Tips
Troubleshoot SPIB staining by checking retrieval, compartment, cell identity and controls before comparing signal across sections.
How should I retrieve SPIB in paraffin sections when nuclear staining is weak?
Use Tris-EDTA pH 9.0 heat-induced retrieval at 95–98 °C for 20 minutes as this page’s starting condition (page retrieval specification: nuclear antigen). Allow sections to cool in retrieval buffer, then rinse consistently before blocking so differences in handling do not confound a staining comparison (standard IHC practice). If staining remains weak, compare a milder retrieval condition on adjacent sections while keeping antibody concentration and detection constant (standard IHC practice). Score nuclear signal in morphologically identified candidate immune cells separately from cytoplasmic signal, because SPIB is annotated in both compartments (UniProt Q01892: nucleus; cytoplasm). Include a no-primary control when judging whether stronger retrieval has also increased background (standard IHC practice).
Could fixation explain variable SPIB staining between paraffin blocks?
SPIB-specific fixation sensitivity is unknown from the supplied evidence, so record each block’s fixative and processing history before attributing differences to the antigen (supplied SPIB evidence; standard IHC practice). The selected prostate-tissue caption reports 2.5 µg/mL antibody but does not state its fixative (catalog antibody A02805-1 tissue-IHC caption). Compare blocks using the same Tris-EDTA pH 9.0, 95–98 °C, 20-minute retrieval and matched detection settings (page retrieval specification; standard IHC practice). Review tissue preservation and nuclear morphology alongside staining, since poor morphology can make compartment assignment unreliable (standard IHC practice). Run a documented reference section with each batch to distinguish a block-specific issue from a staining-run issue (standard IHC practice).
Should SPIB staining be nuclear, cytoplasmic, or both?
Assess nuclear and cytoplasmic staining separately: SPIB is annotated in both compartments and functions as a sequence-specific transcriptional activator (UniProt Q01892: subcellular location; function). Its lack of a transmembrane segment gives no basis for expecting a membrane-rim pattern (UniProt Q01892: topology). HPA describes cytoplasmic staining across several tissues, including immune cells, but rates its tissue-IHC reliability uncertain because staining and RNA data have low consistency and the antibody may recognize more than one gene (HPA: tissue IHC). Use a nuclear counterstain and inspect individual, intact cells before assigning a compartment (standard IHC practice). Report the compartment and cell type together instead of treating all positive pixels as equivalent SPIB evidence (UniProt Q01892: function; HPA: reliability uncertain).
How can isoforms or epitope accessibility affect SPIB IHC?
SPIB has 3 annotated isoforms, but the supplied record does not map the catalog antibody’s epitope to their sequences (UniProt Q01892: isoforms; supplied antibody evidence). Check the antibody’s documented immunogen or epitope before claiming that staining represents every isoform (standard IHC interpretation practice). The record annotates no glycosylation sites or modified residues, which does not establish that fixation leaves this antibody’s epitope accessible (UniProt Q01892: annotations; standard IHC practice). Compare adjacent sections at the specified Tris-EDTA pH 9.0, 95–98 °C, 20-minute retrieval before changing antibody concentration (page retrieval specification; standard IHC practice). If compartment or cell-type patterns shift with retrieval, document that dependence and avoid assigning an isoform from staining alone (standard IHC interpretation practice).
How should I adapt SPIB assessment to multiplex immunofluorescence?
For a separate IF experiment, pair SPIB with a validated marker of B cells or plasmacytoid dendritic cells, the cell types in which expression is reported (UniProt Q01892: tissue specificity; standard IF practice). Select a fluorophore channel with low measured tissue autofluorescence, and assess single-stain and no-primary controls before interpreting overlap (standard IF practice). Because SPIB is annotated in the nucleus and cytoplasm and has no transmembrane segment, use permeabilisation suitable for intracellular access, then check whether nuclear and cytoplasmic patterns remain distinguishable (UniProt Q01892: location; topology; standard IF practice). Use a nuclear counterstain to assign signal to individual cells, and keep acquisition settings consistent across compared specimens (standard IF practice). The supplied HPA subcellular record lists no ICC/IF images, so it does not validate an IF staining pattern (HPA: subcellular record).
What should I change when SPIB chromogenic staining has diffuse background?
First compare the stained section with a no-primary control to identify signal from the detection system, tissue pigment or endogenous enzyme activity (standard chromogenic IHC practice). For peroxidase-based detection, check the peroxide block and wash steps, then review whether antibody concentration or incubation has increased diffuse staining (standard chromogenic IHC practice). The selected prostate image used 2.5 µg/mL, but its caption alone does not establish an optimal concentration for another specimen (catalog antibody A02805-1 tissue-IHC caption). Check whether strong color occurs at section edges, folds or damaged areas before calling it cellular SPIB signal (standard IHC interpretation practice). Treat broad cytoplasmic staining cautiously because HPA reports that pattern with uncertain tissue-IHC reliability (HPA: tissue IHC).
What is a defensible way to quantify SPIB staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue region and cell population before counting, then score nuclear and cytoplasmic staining separately because SPIB is annotated in both compartments (UniProt Q01892: location; standard IHC practice). Record the percentage of positive candidate cells and an intensity score; an H-score sums percentages at intensities 0–3 and spans 0–300 (standard IHC scoring practice). For sparse positive cells, report positive-cell density per mm² of viable tissue alongside the number of eligible cells examined (standard IHC scoring practice). Normalize comparisons to the same eligible cell population or viable tissue area, and hold staining and image thresholds constant (standard IHC practice). Keep compartment-specific results visible because HPA’s tissue-IHC assessment is uncertain (HPA: reliability uncertain).
How can I distinguish credible SPIB staining from artefact?
A credible result has cell-resolved signal in an expected compartment and a plausible cell population: SPIB is nuclear or cytoplasmic and is reported in B cells and plasmacytoid dendritic cells (UniProt Q01892: location; tissue specificity). Membrane-only rims warrant scrutiny because SPIB has no transmembrane segment (UniProt Q01892: topology). Check staining at section edges, folds and necrotic areas against intact tissue, and use a no-primary control to assess endogenous enzyme or detection background (standard chromogenic IHC practice). Do not treat cytoplasmic signal alone as confirmation, since HPA reports cytoplasmic tissue staining with uncertain reliability and warns of possible recognition of more than one gene (HPA: tissue IHC). Document the cell type, compartment and control results before interpreting differences between specimens (standard IHC interpretation practice).
Boster reagents
Best SPIB / Transcription factor Spi-B IHC Antibodies
A02805-1 has IHC and IF figures from human prostate tissue (IHC/IF image captions). Its listed reactivity covers human, mouse, and rat (catalog reactivity).
Real IHC data
Anti-Transcription factor Spi-B SPIB Antibody
Cat # A02805-1
A02805-1 will render with human prostate tissue IHC at 2.5 μg/mL (IHC image caption). Its IF figure shows human prostate tissue at 20 μg/mL (IF image caption).
Which to pick: Choose A02805-1 for paraffin-section tissue IHC (catalog applications: IHC-P); its own IHC caption shows human prostate tissue, but does not report the fixative (IHC image caption). For IF, A02805-1 has a human prostate tissue figure; ICC is not separately documented (IF image caption; catalog applications). For mouse or rat samples, A02805-1 lists reactivity with both species, while the supplied IHC and IF figures show human tissue (catalog reactivity; IHC/IF image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q01892 (SPIB_HUMAN, Transcription factor Spi-B).
- Human Protein Atlas. SPIB tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. SPIB subcellular location (ICC-IF): Highest expression in HDLM-2: 145.4 nTPM.
- Human Protein Atlas. SPIB antibody validation summary (1 antibodies).
- SPIB is a novel prognostic factor in diffuse large B-cell lymphoma that mediates apoptosis via the PI3K-AKT pathway. Cancer science 2016 — PMC5021043.
- A Dynamic Transcription Factor Signature Along the Colorectal Adenoma-Carcinoma Sequence in Patients With Co-Occurrent Adenoma and Carcinoma. Frontiers in oncology 2021 — PMC8176860.
- Multiomics identifies a prognostic signature and SPIB as a potential regulator of gastric cancer lymph node metastasis. iScience 2026 — PMC13383869.
- Ubiquitin D Promotes Lung Metastasis by Stabilizing MMP3 in Triple-Negative Breast Cancer. Research (Washington, D.C.) 2026 — PMC12828796.
- PubMed PMID:1406622 — UniProt-cited evidence.
- PubMed PMID:8670269 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SPIB (Transcription factor Spi-B). Expected localisation Cytoplasmic staining in several tissues (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A02805-1 at 2.5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Spleen (Cells in red pulp); negative Esophagus. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A02805-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-SPIB, 2.5 μg/mL (datasheet A02805-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA show low consistency (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope effects are unknown (UniProt); Regulation: Expressed in pDCs and B cells (UniProt). Evidence: UniProt Q01892, HPA tissue IHC · Uncertain, HPA cell-line RNA · Highest expression in HDLM-2: 145.4 nTPM, Datasheet A02805-1, PMC5021043, PMC8176860. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SPIB/immunohistochemistry-ihc-guide.