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PFKFB2 / 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 · IHC design guide
Design Immunohistochemistry for PFKFB2
Plan chromogenic PFKFB2 IHC in paraffin sections using cytoplasmic staining as the expected pattern (HPA tissue IHC). Compare test sections with tissue controls, and interpret results in light of the uncertain tissue staining record (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
PFKFB2 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 staining in glandular and glial cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A05977) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A05977) | |
| Caveat | Heart specificity contrasts with undetected cardiomyocytes (UniProt; HPA tissue IHC) | |
| Regulation | Staining intensity varies by tissue (HPA tissue IHC) | |
| Isoform / epitope | 2 isoforms; confirm epitope coverage (UniProt) |
Section 1
Recommended PFKFB2 IHC & IF Protocols
The catalog antibody has an IHC-P protocol (datasheet A05977). One published PFKFB2 IHC protocol using paraffin-embedded GIST tissue is listed below (PMC10918489).
| Sample | Paraffin-embedded human cortical adenomas tissue; fixative not specified (datasheet A05977) |
| Fixation | Image fixative and duration unreported (datasheet A05977); 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 A05977); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A05977) |
| Primary antibody | Rabbit anti-PFKFB2, 2-5 μg/ml (datasheet A05977) |
| Primary incubation | Overnight at 4 °C (datasheet A05977) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A05977) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | PFKFB2-positive staining in glandular cells of appendix (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteTry heat-mediated EDTA pH 8.0 retrieval first (datasheet A05977); the published GIST excerpt does not specify retrieval conditions (PMC10918489).
Section 2
What Is the Expected PFKFB2 Staining Pattern?
In paraffin-section IHC, expect mainly cytoplasmic staining in selected glandular and glial cells; HPA describes cytoplasmic expression across most tissues (HPA tissue IHC). Interpret this pattern cautiously: the tissue IHC reliability is Uncertain, pending external verification (HPA tissue IHC). UniProt assigns no subcellular location and reports no transmembrane segment (UniProt O60825 topology). ICC-IF reports an enhanced nucleoplasmic pattern, so assay context matters (HPA subcellular ICC-IF).
| Cytoplasmic staining in appendix or colon glandular cells, or cerebral cortex glial cells. | This fits the reported Medium cell-level IHC pattern (HPA tissue IHC). Judge the stained cells against adjacent architecture and controls; a matching pattern remains provisional because the tissue IHC assessment is Uncertain (HPA tissue IHC). |
| Predominantly nuclear staining on the chromogenic tissue slide, with little matching cytoplasmic signal. | Treat this as a compartment mismatch requiring an artefact check against controls and morphology (HPA tissue IHC; standard IHC practice). Nuclear signal is not automatically false: HPA reports enhanced nucleoplasmic staining by ICC-IF, a separate assay (HPA subcellular ICC-IF). |
| Strong signal in cardiomyocytes or adipocytes, or across cell types expected to be negative. | HPA reports PFKFB2 as Not detected in cardiomyocytes and adipocytes by tissue IHC (HPA tissue IHC). Check antibody cross-reactivity and endogenous detection activity before assigning such staining to PFKFB2 (standard IHC practice). |
| Uniform brown haze over cells, stroma, and the negative-control section. | A broadly shared background pattern cannot establish cell-specific PFKFB2 staining (standard IHC practice). Inspect blocking, washing, chromogen development, and the detection-only control; HPA's reported signal is assigned to particular cells (HPA tissue IHC). |
| No visible signal in appendix glandular cells or cerebral cortex glial cells. | These are reported Medium examples, so absence warrants a run check, including control performance and detection settings (HPA tissue IHC; standard IHC practice). They are reference examples rather than guaranteed positives because HPA rates tissue IHC Uncertain (HPA tissue IHC). |
💡Expected PFKFB2 appearanceCall a result provisionally positive when distinct cytoplasmic signal is present in the reported Medium glandular or glial cells; widespread cell-independent haze or strong staining of reported negative cell types is suspect (HPA tissue IHC; standard IHC practice).
| Tissue and cell selection | Appendix and colon glandular cells and cerebral cortex glial cells are Medium IHC examples; cardiomyocytes and adipocytes are Not detected examples (HPA tissue IHC). Compare like cell types. |
| Strength of IHC evidence | The tissue profile is marked Uncertain and pending external verification; antibody HPA049975 is also IHC Uncertain (HPA tissue IHC; HPA antibodies). Treat agreement as supportive, not definitive. |
| IHC versus ICC-IF localisation | Tissue IHC reports mainly cytoplasmic expression, whereas ICC-IF reports enhanced nucleoplasmic localisation (HPA tissue IHC; HPA subcellular ICC-IF). Interpret each assay with its own controls. |
| IF/ICC Q: What compartment should be checked? | A: Check the nucleoplasm in ICC-IF; HPA lists A-431, HeLa, U2OS, and SiHa images and rates the listed antibodies ICC Enhanced (HPA subcellular ICC-IF; HPA antibodies). This does not validate nuclear tissue IHC. |
| Molecular features | UniProt reports 2 isoforms, no transmembrane segment, no signal peptide, and no propeptide (UniProt O60825). Antibody epitope coverage is unspecified, so an isoform-specific staining prediction is unsupported. |
| Heart as a reference tissue | UniProt lists heart tissue specificity, while HPA reports cardiomyocytes Not detected by tissue IHC (UniProt O60825; HPA tissue IHC). Do not assume heart muscle will be an IHC positive control. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected glandular or glial cells are unstained. | A staining-run failure is possible; these cells show Medium signal in HPA examples, with Uncertain IHC reliability (HPA tissue IHC). | Review section integrity, detection-only and run controls, antibody dilution, and chromogen development (standard IHC practice). Avoid declaring biological absence from one run. |
| Nuclei dominate the tissue-section result. | That differs from the cytoplasmic tissue IHC profile, although nucleoplasm is reported in ICC-IF (HPA tissue IHC; HPA subcellular ICC-IF). | Check counterstain and detection-only controls, then assess whether cytoplasmic signal is independently present (standard IHC practice). Report the assay-specific discrepancy. |
| Cardiomyocytes stain strongly. | This conflicts with the HPA Not detected cardiomyocyte IHC result, despite UniProt's heart tissue-specificity entry (HPA tissue IHC; UniProt O60825). | Inspect no-primary and tissue controls for endogenous activity or nonspecific detection (standard IHC practice); do not use this signal alone to validate specificity. |
| Brown staining spreads across tissue and background. | Diffuse signal can arise from nonspecific detection or excess chromogen development (standard IHC practice); it obscures HPA's cell-level pattern (HPA tissue IHC). | Compare the no-primary control, review blocking and washes, and assess chromogen timing before scoring cells (standard IHC practice). |
| A reported negative cell type appears positive. | Cross-reactivity or endogenous detection activity is possible (standard IHC practice); HPA lists adipocytes and bone-marrow hematopoietic cells as Not detected (HPA tissue IHC). | Compare morphology, a detection-only control, and reported positive cell types on the same run before attributing the signal to PFKFB2 (standard IHC practice; HPA tissue IHC). |
| Replicate sections differ in staining intensity. | Variation in section handling or development can change apparent intensity (standard IHC practice); the HPA tissue IHC assessment remains Uncertain (HPA tissue IHC). | Run comparable sections with consistent retrieval, antibody dilution, and development, then score cell type and compartment alongside intensity (standard IHC practice). |
Experiment evidence
IHC & IF images showing PFKFB2 staining
A05977 · IHCIHC analysis of PFKFB2 using anti-PFKFB2 antibody (A05977). PFKFB2 was detected in a paraffin-embedded section of human cortical adenomas ti
A05977 · IHCIHC analysis of PFKFB2 using anti-PFKFB2 antibody (A05977). PFKFB2 was detected in a paraffin-embedded section of human esophageal squamous
A05977 · IFIF analysis of PFKFB2 using anti-PFKFB2 antibody (A05977). PFKFB2 was detected in immunocytochemical section of HELA cells. Enzyme antigen r
A30440 · IHCImmunohistochemistry analysis of paraffin-embedded human colon carcinoma tissue, using PFKFB2 Antibody. The picture on the right is blocked
P30440 · IHCImmunohistochemistry analysis of paraffin-embedded human colon carcinoma, using PFKFB2 (Phospho-Ser483) Antibody. The picture on the right i
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000123836-PFKFB2/tissue/cerebral+cortex#imid_13529535
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000123836-PFKFB2/tissue/colon#imid_13529461
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000123836-PFKFB2/tissue/liver#imid_13529470
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000123836-PFKFB2/tissue/kidney#imid_13529471
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA049975; green = PFKFB2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA049975; green = PFKFB2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for PFKFB2 IHC & IF
🧪Run colon first: glandular cells should stain at a medium level (HPA: colon glandular cells, Medium). Use cervix glandular cells as a negative tissue (HPA: cervix glandular cells, Not detected); on the colon slide, unstained neighboring cells provide a background reference, but the supplied HPA row does not identify a specific internal negative cell type (HPA: colon glandular cells, Medium).
Positive control tissue: Appendix (Glandular cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show PFKFB2 in A-431, HeLa, U2OS, SiHa, with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a host- and clonality-matched isotype control; the caption identifies a rabbit primary but does not state its clonality (A05977 tissue-IHC caption). Use PFKFB2 knockout material or cognate immunizing-peptide competition, if available, as a biological specificity control; quench endogenous peroxidase and assess endogenous biotin background in colon because the reported detection uses a biotin-based complex with DAB (A05977 tissue-IHC caption).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A05977 paraffin-section caption does not state the fixative (A05977 tissue-IHC caption). The reported IHC procedure uses heat retrieval in EDTA at pH 8.0; its dependence on retrieval has not been established (A05977 tissue-IHC caption). Frozen-section or tissue IF ease cannot be ranked from the supplied evidence; the colon slide should be checked for endogenous peroxidase and biotin background with the stated biotin-based DAB detection (HPA: ICC-IF images in A-431, HeLa, U2OS and SiHa; A05977 tissue-IHC caption).
HPA tissue IHC evidence for PFKFB2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Pending external verification.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Caudate | Glial cells | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Glial cells | Medium | Protein (IHC) | HPA → |
| Colon | Glandular cells | Medium | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PFKFB2 IHC Tips
Troubleshoot PFKFB2 staining in paraffin sections by checking the documented retrieval condition, cellular pattern, controls, and scoring method.
What should I check when PFKFB2 staining is weak after antigen retrieval?
Use heat-mediated antigen retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A05977). The documented tissue image used this condition, followed by 10% goat-serum block and 2 μg/ml primary antibody overnight at 4°C (caption A05977). If staining is weak, check deparaffinization and keep heating, cooling, and washing consistent across adjacent sections (standard IHC practice). Screen another retrieval buffer only as a fallback, while holding antibody concentration and detection constant (standard IHC practice). Compare the result with a no-primary control, since the available tissue staining profile has uncertain reliability (HPA tissue IHC: Uncertain).
Could fixation explain variable PFKFB2 staining between paraffin blocks?
Target-specific sensitivity to fixation is unknown for PFKFB2 because the selected paraffin-section caption does not state a fixative (caption A05977). Record each block’s fixative and fixation time before comparing staining intensity, and process matched sections together where possible (standard IHC practice). Use the documented EDTA pH 8.0 retrieval and 2 μg/ml primary concentration as a consistent starting point (caption A05977). If blocks differ, test adjacent sections with the same retrieval, antibody incubation, and chromogen development before attributing a difference to biology (standard IHC practice). Do not infer fixation sensitivity from tissue distribution or phosphorylation annotations (HPA tissue IHC; UniProt O60825).
How should I assess cytoplasmic versus nuclear PFKFB2 staining?
Assess cytoplasmic and nuclear staining separately because the tissue profile describes cytoplasmic expression, while cell imaging reports enhanced nucleoplasmic localisation (HPA tissue IHC; HPA subcellular). UniProt provides no subcellular annotation and reports no transmembrane segment, so it does not resolve that difference (UniProt O60825). In paraffin sections, compare staining with cell morphology and a no-primary control under the same retrieval and DAB development conditions (standard IHC practice). Record the fraction of cells in each compartment rather than combining them into one score (standard IHC practice). Treat either pattern as provisional until it is reproduced with an independent antibody or another target-specific control (HPA tissue IHC: Uncertain).
Can isoforms or phosphorylation change what the antibody detects in tissue?
PFKFB2 has 2 listed isoforms, but the supplied antibody caption does not map its epitope to either one (UniProt O60825; caption A05977). Annotated modified residues include PKA-associated phosphoserine 29 and AMPK-associated phosphoserine 466; their effect on this antibody’s staining is unknown (UniProt O60825). Check any available immunogen sequence against both isoforms before interpreting a negative section as absence of all PFKFB2 (standard IHC practice). Keep EDTA pH 8.0 retrieval constant while assessing section-to-section variation (datasheet A05977). Report the result as antibody-detected PFKFB2 staining unless isoform selectivity and modification sensitivity have been established independently (standard IHC practice).
How can IF help assess a disputed PFKFB2 staining pattern?
For a separate IF experiment, multiplex PFKFB2 with a marker identifying the cell population being evaluated; tissue IHC lists glandular and glial cells among positive populations (HPA tissue IHC). Select a fluorophore channel after examining unstained tissue for autofluorescence, and include single-label controls to assess channel bleed-through (standard IF practice). Because PFKFB2 has no transmembrane segment and its antibody epitope is not mapped here, test permeabilisation for access to intracellular compartments without assuming a specific membrane side (UniProt O60825; caption A05977). Compare cytoplasmic and nucleoplasmic signal separately, since those locations differ between the available tissue and cell-image profiles (HPA tissue IHC; HPA subcellular).
What causes diffuse brown background in PFKFB2 chromogenic IHC?
Compare the affected slide with a no-primary section to identify signal produced by the detection system rather than primary-antibody binding (standard IHC practice). For a peroxidase and DAB workflow, include a peroxidase block and keep chromogen development consistent across slides (standard IHC practice). The documented A05977 image used 10% goat serum, biotinylated secondary antibody, a streptavidin-biotin complex, and DAB (caption A05977). With that detection system, a reagent-control section can help assess background unrelated to PFKFB2 binding (standard IHC practice). Recheck washing and primary concentration before interpreting diffuse brown signal as cytoplasmic expression (standard IHC practice; HPA tissue IHC: cytoplasmic profile).
How should I quantify PFKFB2 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, since reported PFKFB2 patterns differ between cytoplasmic tissue staining and nucleoplasmic cell imaging (HPA tissue IHC; HPA subcellular). For chromogenic sections, record the percentage of positive cells and staining intensity, or calculate an H-score from prespecified intensity categories (standard IHC practice). Normalise counts to evaluable cells or tissue area, and exclude folds, damaged edges, and necrotic regions using the same rules for every section (standard IHC practice). Keep retrieval, antibody concentration, and DAB development matched across samples; the documented starting conditions include EDTA pH 8.0 and 2 μg/ml primary antibody (caption A05977).
How can I distinguish PFKFB2 staining from tissue artefact?
Look for reproducible staining within intact cells and compare its compartment and cell population with appropriate controls (standard IHC practice). The tissue profile reports cytoplasmic staining in most tissues, including medium signal in selected glandular and glial cells, but assigns the profile uncertain reliability (HPA tissue IHC). A predominantly nucleoplasmic pattern is also reported in cell imaging, so nuclear signal alone does not establish an artefact or a true tissue result (HPA subcellular). Exclude edge staining, necrotic areas, and signal retained in a no-primary section before scoring (standard IHC practice). In a DAB assay, check peroxidase-block controls when brown signal could reflect endogenous enzyme activity (standard IHC practice).
Boster reagents
Best PFKFB2 / 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2 IHC Antibodies
Three antibodies have human paraffin-section IHC images (catalog IHC captions); A05977 also has HeLa-cell IF data (A05977 IF caption). All list Human, Mouse, and Rat reactivity (catalog).
Real IHC data
Anti-PFKFB2 Antibody ®
Cat # A05977
Real IHC data
Anti-PFKFB2/P Pfk 2 Car Antibody
Cat # A30440
Real IHC data
Anti-Phospho PFKFB2 (Ser483) Antibody
Cat # P30440
A05977 has IHC images from human cortical adenoma, esophageal squamous carcinoma, liver cancer, and lung cancer paraffin sections, plus IF data in HeLa cells (A05977 image captions). A30440 has a human colon carcinoma paraffin-section IHC image; P30440 has one for phospho-Ser483 PFKFB2 in human colon carcinoma (A30440 and P30440 IHC captions).
Which to pick: For tissue IHC, choose A05977 when a documented retrieval and detection workflow is useful (A05977 IHC caption); A30440 covers total PFKFB2 and P30440 targets phospho-Ser483 in paraffin-embedded human colon carcinoma, with the fixative unreported for both (A30440 and P30440 IHC captions). For IF/ICC, A05977 has a HeLa-cell IF image, while A30440 and P30440 list IF/ICC applications without IF images in this payload (catalog applications and image captions). All three list Human, Mouse, and Rat reactivity, but their supplied IHC images use human tissue; A30440 and P30440 are described as polyclonal (catalog reactivity, dilution details, and IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O60825 (F262_HUMAN, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2).
- Human Protein Atlas. PFKFB2 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. PFKFB2 subcellular location (ICC-IF): Localized to the nucleoplasm..
- Human Protein Atlas. PFKFB2 antibody validation summary (2 antibodies).
- Mutation of regulatory phosphorylation sites in PFKFB2 worsens renal fibrosis. Scientific reports 2020 — PMC7471692.
- BRAF activation by metabolic stress promotes glycolysis sensitizing NRAS(Q61)-mutated melanomas to targeted therapy. Nature communications 2022 — PMC9675737.
- Identification and validation of protein biomarkers for predicting gastrointestinal stromal tumor recurrence. Computational and structural biotechnology journal 2024 — PMC10918489.
- PubMed PMID:9652401 — UniProt-cited evidence.
- PubMed PMID:11374908 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for PFKFB2 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 2). Expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A05977). Validated IHC antibody: A05977 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human cortical adenomas tissue; fixative not specified (datasheet A05977); 2. Fixation: Image fixative and duration unreported (datasheet A05977); 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 A05977); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A05977); 8. Primary antibody: Rabbit anti-PFKFB2, 2-5 μg/ml (datasheet A05977); 9. Primary incubation: Overnight at 4 °C (datasheet A05977). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A05977); Caveat: Heart specificity contrasts with undetected cardiomyocytes (UniProt; HPA tissue IHC); Isoform / epitope: 2 isoforms; confirm epitope coverage (UniProt); Regulation: Staining intensity varies by tissue (HPA tissue IHC). Evidence: UniProt O60825, HPA tissue IHC · Uncertain, HPA ICC-IF · nucleoplasm., Datasheet A05977, PMC10918489. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PFKFB2/immunohistochemistry-ihc-guide.