SF3B1 / Splicing factor 3B subunit 1 · IHC design guide

Design Immunohistochemistry for SF3B1

Plan SF3B1 chromogenic IHC on paraffin sections using the catalog antibody at a starting dilution of 1:50 (datasheet M00507-1). Assess nuclear and cytoplasmic staining in the tissue context, accounting for transient redistribution during mitosis (HPA tissue IHC; UniProt).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for SF3B1 (IHC for SF3B1): expected localisation Nuclear and cytoplasmic tissue staining (HPA tissue IHC), antibody M00507-1, validated IHC image, and IHC protocol steps — Printable **SF3B1** IHC protocol sheet — expected localisation Nuclear and cytoplasmic tissue staining (HPA tissue IHC), antibody M00507-1, controls and protocol steps. Open the full SF3B1 IHC guide →

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SF3B1 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	Nuclear and cytoplasmic tissue staining (HPA tissue IHC)
	Staining pattern	Nuclear and cytoplasmic staining in most tissues (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet M00507-1)
	Positive control ⓘ	Bone marrow+4 more · see all
	Negative control ⓘ	Adipose tissue

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific)
	Caveat	Mitosis can disperse SF3B1 into the cytoplasm (UniProt)
	Regulation	No specific expression regulator annotated (UniProt)
	Isoform / epitope	2 isoforms; epitope coverage is unspecified (UniProt)

Section 1

Recommended SF3B1 IHC & IF Protocols

The catalog antibody protocol is supplemented by published SF3B1 IHC methods for ovarian cancer and mouse tissue (PMC10682409; PMC6025833).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human colon tissue; fixative not specified (datasheet M00507-1)
Fixation	Image fixative and duration unreported (datasheet M00507-1); 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 M00507-1); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet M00507-1)
Primary antibody	Rabbit monoclonal (clone 18S60) anti-SF3B1, 1:50 (datasheet M00507-1)
Primary incubation	Overnight at 4 °C (datasheet M00507-1)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet M00507-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	SF3B1-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nuclear expression in most tissues. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet M00507-1); the published methods do not specify a retrieval buffer (PMC10682409; PMC6025833).

Section 2

What Is the Expected SF3B1 Staining Pattern?

SF3B1 is a nuclear spliceosome protein enriched in nuclear speckles and has no transmembrane segment (UniProt O75533). Tissue IHC shows nuclear and cytoplasmic staining in most tissues, including strong staining in selected hematopoietic, neuronal, and glandular cells (HPA: tissue IHC). HPA rates the tissue pattern Approved, with medium consistency between staining and RNA expression (HPA: reliability).

What am I looking at on my slide?
Clear nuclear staining in hematopoietic cells, neurons, or glandular cells.	This fits high staining reported in bone marrow hematopoietic cells, cerebral cortex and hippocampal neurons, and several glandular tissues (HPA: tissue IHC). Nuclear staining also fits SF3B1 localisation (UniProt O75533).
Staining is predominantly membranous, with little nuclear signal.	Treat this as a suspect pattern: SF3B1 has no transmembrane segment and is assigned to the nucleus (UniProt O75533). Review morphology and controls before scoring it as SF3B1.
Strong staining appears in adipose tissue adipocytes.	This conflicts with the HPA adipose tissue result, where adipocytes were not detected (HPA: adipose tissue IHC). Investigate cross-reactivity or endogenous detection activity; breast adipocytes are a separate low-staining observation (HPA: breast IHC).
Color spreads across tissue, including areas without clear cellular boundaries.	Diffuse background cannot establish cellular localisation. Check the no-primary control, blocking, washing, and detection chemistry before interpreting the slide (general IHC practice).
No signal appears in a known high-staining tissue.	First check whether the expected cells are present; HPA reports high staining in bone marrow hematopoietic cells and several neuronal and glandular populations (HPA: tissue IHC). Then assess the assay controls (general IHC practice).

💡Expected SF3B1 appearanceCall a positive result when defined cells show convincing nuclear staining, with possible cytoplasmic staining; high intensity is documented in selected hematopoietic, neuronal, and glandular cells (HPA: tissue IHC). Predominantly membranous staining is suspect (UniProt O75533 topology).

How each factor affects the staining
Nuclear localisation and topology	SF3B1 localises to the nucleus and nuclear speckles and lacks a transmembrane segment (UniProt O75533). Use cell boundaries and a nuclear counterstain to judge compartment; a membrane-only pattern needs investigation (general IHC practice).
Cell cycle	During mitosis, SF3B1 transiently disperses from nuclear speckles into the cytoplasm (UniProt O75533). Interpret cytoplasmic signal in mitotic cells in that context; HPA also reports cytoplasmic and nuclear tissue IHC staining (HPA: tissue IHC).
Strength of tissue evidence	HPA rates tissue IHC Approved but describes only medium consistency with RNA data (HPA: reliability). Its antibody HPA050275 is Approved for IHC and Enhanced for ICC (HPA: antibody validation); those ratings do not validate every new assay condition.
IF/ICC: what pattern is expected?	Enhanced nuclear speckles are the main location in HPA cell images (HPA: subcellular ICC-IF). UniProt also assigns SF3B1 to nuclear speckles (UniProt O75533). This is an IF/ICC localisation reference, not an IHC protocol.
Isoforms and modifications	UniProt lists two isoforms and multiple modified residues (UniProt O75533). Their effects on staining cannot be predicted from these records; do not assign a compartment or tissue pattern to either isoform without further evidence.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Positive tissue has no visible nuclear stain.	The expected cell population may be absent, or the assay may have failed; high staining is cell-specific (HPA: tissue IHC).	Confirm the relevant cells on the section, then review positive and no-primary controls, retrieval, antibody dilution, and detection steps (general IHC practice).
Only membrane outlines stain.	That pattern conflicts with nuclear localisation and the lack of a transmembrane segment (UniProt O75533).	Check the no-primary control and cell morphology; reassess specificity before reporting SF3B1-positive cells (general IHC practice).
Adipose tissue adipocytes stain strongly.	HPA reports no detected staining in adipose tissue adipocytes (HPA: adipose tissue IHC); cross-reactivity or detection background is possible.	Compare with a no-primary control and a known high-staining tissue in the same run (general IHC practice).
Diffuse brown signal obscures nuclei.	Excess background or endogenous enzyme activity can obscure compartment calls (general IHC practice).	Review blocking, washing, detection controls, and counterstain intensity before scoring localisation (general IHC practice).
Some cells show cytoplasmic signal.	HPA reports cytoplasmic as well as nuclear tissue staining; mitotic dispersion is also described (HPA: tissue IHC; UniProt O75533).	Check cell morphology and the accompanying nuclear pattern. Do not label all cytoplasmic signal an artifact solely because SF3B1 is nuclear (HPA: tissue IHC).
Different tissues have unequal staining intensity.	HPA reports high staining in selected cell populations, medium staining in skin keratinocytes, and low staining in thyroid glandular cells (HPA: tissue IHC).	Score the specified cell population and compartment in each tissue; compare with HPA levels as context, not a universal intensity cutoff (HPA: tissue IHC).

Source: UniProt O75533 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing SF3B1 staining

IHC analysis of SF3B1 using anti-SF3B1 antibody (M00507-1). SF3B1 was detected in a paraffin-embedded section of human colon tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:50 rabbit anti-SF3B1 Antibody (M00507-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

M00507-1 · IHCIHC analysis of SF3B1 using anti-SF3B1 antibody (M00507-1). SF3B1 was detected in a paraffin-embedded section of human colon tissue. Heat me

IHC analysis of SF3B1 using anti-SF3B1 antibody (M00507-1). SF3B1 was detected in a paraffin-embedded section of human colon cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1:50 rabbit anti-SF3B1 Antibody (M00507-1) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

M00507-1 · IHCIHC analysis of SF3B1 using anti-SF3B1 antibody (M00507-1). SF3B1 was detected in a paraffin-embedded section of human colon cancer tissue.

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000115524-SF3B1/tissue/cerebral+cortex#imid_14206768

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000115524-SF3B1/tissue/colon#imid_14206686

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000115524-SF3B1/tissue/liver#imid_14206695

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000115524-SF3B1/tissue/kidney#imid_14206680

HPA · ICC-IF A-549Human Protein Atlas immunofluorescence, A-549, antibody HPA050275; green = SF3B1, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for SF3B1 IHC & IF

🧪Run bone marrow first and score hematopoietic cells for nuclear staining (HPA: High in bone marrow hematopoietic cells; UniProt O75533: nucleus). Use adipose tissue as the negative tissue and score adipocytes, which HPA lists as not detected (HPA: adipose tissue, adipocytes, Not detected); anucleate erythrocytes within the bone marrow section, if present, should show no nuclear staining (standard IHC practice: anucleate erythrocytes).

Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show SF3B1 in A-549, HeLa, U2OS, U2OS, siRNA 2 (10x), U2OS, siRNA 2 (40x), U2OS, scrambled (10x), U2OS, scrambled (40x), U2OS, siRNA 1 (10x), U2OS, siRNA 1 (40x), NIH 3T3, with annotated localisation: Nuclear speckles (enhanced) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only slide, a host-species-matched nonimmune rabbit IgG control, and an SF3B1-depleted biological control processed in parallel (caption: rabbit primary; standard IHC control practice). Quench endogenous peroxidase and assess background in bone marrow before HRP/DAB detection (caption: HRP/DAB detection; standard IHC practice for marrow).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected SKU’s paraffin-section caption does not state the fixative (caption: M00507-1, fixative not stated). The reported IHC example used heat-mediated EDTA retrieval at pH 8.0 and a 1:50 primary dilution; retrieval dependence beyond that example is unreported (caption: M00507-1). HPA ICC-IF images show nuclear speckles, but the supplied evidence does not establish that frozen sections or IF are easier than paraffin IHC; endogenous peroxidase is a potential bone marrow artifact with HRP/DAB detection (HPA: nuclear speckles; caption: HRP/DAB; standard IHC practice for marrow).

HPA tissue IHC evidence for SF3B1

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
Bone marrow	Hematopoietic cells	High	Protein (IHC)	HPA →
Cerebral cortex	Neuronal cells	High	Protein (IHC)	HPA →
Cervix	Glandular cells	High	Protein (IHC)	HPA →
Gallbladder	Glandular cells	High	Protein (IHC)	HPA →
Hippocampus	Neuronal cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →

Section 3

Advanced SF3B1 IHC Tips

Troubleshoot SF3B1 staining by checking retrieval, nuclear localisation, controls and cell level scoring before interpreting chromogenic IHC (UniProt O75533 localisation; standard IHC practice).

What retrieval should I try first when SF3B1 staining is weak?

Start with heat mediated antigen retrieval in EDTA at pH 8.0 (datasheet M00507-1). The selected paraffin section image used that retrieval before overnight primary incubation at 4°C and DAB detection (datasheet M00507-1). If nuclear staining remains weak, compare retrieval heating and cooling conditions on matched sections while keeping antibody incubation and detection constant (standard IHC practice). Assess signal in intact nuclei alongside tissue damage and nonspecific staining; stronger colour alone does not establish improved specificity (standard IHC practice; UniProt O75533 localisation). The caption does not report a retrieval duration, so establish that setting experimentally (datasheet M00507-1).

Could fixation explain weak or uneven SF3B1 staining?

The selected image identifies a paraffin section but does not state its fixative, so SF3B1 specific fixation sensitivity is unknown (datasheet M00507-1). Record the fixative, fixation interval and processing history for each specimen before comparing staining across sections (standard IHC practice). For uneven signal, compare intact areas with section edges and poorly preserved regions, then check whether the pattern repeats in matched sections (standard IHC practice). Keep retrieval at EDTA pH 8.0 while investigating processing differences, since that is the documented retrieval for this antibody (datasheet M00507-1). Do not assign a target specific fixation effect without a controlled fixation comparison (standard IHC practice).

Should SF3B1 appear in nuclei, cytoplasm or both?

Expect predominantly nuclear staining, with nuclear speckles as a supported subnuclear location (UniProt O75533 localisation; HPA subcellular). UniProt reports transient dispersal from nuclear speckles to cytoplasm during mitosis, so examine cell morphology before judging an isolated cytoplasmic cell (UniProt O75533 localisation). HPA tissue IHC also describes cytoplasmic and nuclear expression in most tissues, with Approved reliability and medium consistency against RNA data (HPA tissue IHC). Score nuclear and cytoplasmic staining separately rather than pooling them into one positive call (standard IHC practice). If cytoplasmic colour dominates throughout the section, review controls, counterstain and morphology before attributing it to SF3B1 (standard IHC practice).

Can this antibody distinguish SF3B1 isoforms or modification states in tissue?

Two SF3B1 isoforms are listed, but the supplied antibody caption does not map its epitope or establish isoform selectivity (UniProt O75533 isoforms; datasheet M00507-1). SF3B1 has annotated modified residues, including phosphothreonine at 125 and phosphoserine at 129; the supplied evidence does not show whether these affect antibody binding (UniProt O75533 modified residues; datasheet M00507-1). Treat chromogenic intensity as antibody reactivity in tissue, without assigning an isoform or modification state (standard IHC interpretation). Review epitope information if it becomes available and compare staining with an independently validated reagent where that distinction matters (standard IHC practice). Keep retrieval and detection conditions matched across sections used for that comparison (standard IHC practice).

How should I assess SF3B1 by multiplex IF after observing IHC staining?

Use this IHC result to choose regions for IF, then validate the IF antibody and conditions independently; the supplied caption documents chromogenic tissue IHC (datasheet M00507-1; standard IF practice). Pair SF3B1 with a marker identifying the cell population under study, such as a neuronal cell marker when examining neuronal cells with high reported tissue staining (HPA tissue IHC; standard IF practice). Choose a fluorophore channel with low tissue autofluorescence and include an unstained section to assess that background (standard IF practice). SF3B1 has no transmembrane segment and is localised to nuclear speckles, so optimise permeabilisation for access to a nuclear epitope (UniProt O75533 topology; HPA subcellular; standard IF practice).

How can I separate SF3B1 staining from diffuse brown background?

Compare the test section with a no primary control processed through the same peroxidase and DAB steps (standard IHC practice). Apply a peroxidase block and assess whether residual endogenous enzyme activity contributes to colour in the control (standard IHC practice). The selected caption used 10% goat serum blocking, 1:50 primary antibody overnight at 4°C, and a 30 minute secondary incubation at 37°C (datasheet M00507-1). If background persists, check washing, primary concentration and chromogen development on matched sections (standard IHC practice). Judge improvement by retained nuclear signal and reduced staining in the no primary control (UniProt O75533 localisation; standard IHC practice).

What should I score when comparing SF3B1 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and tissue region before scoring, then report the percentage of positive nuclei and a nuclear H-score using consistent intensity categories (standard IHC practice; UniProt O75533 localisation). Normalise positive counts or signal summaries to the number of evaluable cells in the same region; report density per mm² only when sampled area is measured (standard IHC practice). Score cytoplasmic staining separately because HPA tissue IHC reports both compartments (HPA tissue IHC). Exclude folds, damaged edges and necrotic regions using the same rules for every section (standard IHC practice). Keep counterstaining, imaging thresholds and scoring rules consistent across comparison groups (standard IHC practice).

When is an apparent SF3B1 positive cell likely to be an artefact?

A credible call has staining in preserved cells, especially their nuclei, consistent with SF3B1 nuclear and speckle localisation (UniProt O75533 localisation; HPA subcellular). Interpret cell identity against the section: HPA reports high staining in bone marrow hematopoietic cells and cerebral cortex neuronal cells, but adipocytes in adipose tissue as not detected (HPA tissue IHC). Brown colour restricted to edges, folds or necrotic tissue warrants review against intact areas (standard IHC practice). Colour persisting in a no primary control suggests detection background or endogenous enzyme activity (standard IHC practice). Diffuse cytoplasmic staining alone needs corroboration, although HPA tissue IHC does report cytoplasmic expression (HPA tissue IHC; standard IHC practice).

Boster reagents

Best SF3B1 / Splicing factor 3B subunit 1 IHC Antibodies

M00507-1 has human paraffin-section IHC images (catalog IHC captions) and lists IF/ICC and human, mouse and rat reactivity (catalog applications/reactivity); no IF image is supplied (catalog IF images).

Real IHC data

Anti-SF3B1 Rabbit Monoclonal Antibody

Cat # M00507-1

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M00507-1 has IHC images from paraffin-embedded human colon, colon cancer and liver sections (M00507-1 IHC captions). IF/ICC is listed for M00507-1, with human, mouse and rat reactivity, but no IF image is supplied (catalog applications/reactivity and IF images).

Which to pick: For tissue IHC, choose the rabbit monoclonal M00507-1: its own captions show human paraffin sections stained after EDTA pH 8.0 retrieval at 1:50 (catalog host/clone; M00507-1 IHC captions). The captions do not report the fixative (M00507-1 IHC captions). For IF/ICC or mouse and rat samples, M00507-1 lists those applications and species, although the supplied images document human IHC only (catalog applications/reactivity; M00507-1 IHC captions and IF images).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

UniProt Consortium. UniProt entry O75533 (SF3B1_HUMAN, Splicing factor 3B subunit 1).
Human Protein Atlas. SF3B1 tissue IHC expression (reliability: Approved).
Human Protein Atlas. SF3B1 subcellular location (ICC-IF): Localized to the nuclear speckles..
Human Protein Atlas. SF3B1 antibody validation summary (1 antibodies).
Exploring Splicing Modulation as an Innovative Approach to Combat Pancreatic Cancer: SF3B1 Emerges as a Prognostic Indicator and Therapeutic Target. International journal of biological sciences 2024 — PMC11186358.
Allele Loss and Reduced Expression of CYCLOPS Genes is a Characteristic Feature of Chromophobe Renal Cell Carcinoma. Translational oncology 2019 — PMC6563336.
Inhibition of SF3B1 improves the immune microenvironment through pyroptosis and synergizes with αPDL1 in ovarian cancer. Cell death & disease 2023 — PMC10682409.
Identification of anti-SF3B1 autoantibody as a diagnostic marker in patients with hepatocellular carcinoma. Journal of translational medicine 2018 — PMC6025833.
PubMed PMID:9585501 — UniProt-cited evidence.
PubMed PMID:15815621 — UniProt-cited evidence.
PubMed PMID:10882114 — UniProt-cited evidence.