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CREB1 / Cyclic AMP-responsive element-binding protein 1 · IHC design guide
Design Immunohistochemistry for CREB1
Plan chromogenic IHC in paraffin sections around CREB1’s widespread nuclear staining (HPA tissue IHC). This guide covers fixation, antibody selection and interpretation of total versus phospho-CREB1 staining (UniProt; datasheet A00577).
Evidence assembled Sep 2026 · For research use; verify linked source records and product datasheet before use
CREB1 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 staining in tissue sections (HPA tissue IHC) | |
| Staining pattern | Widespread nuclear staining across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Adipose tissue+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Fix promptly in 10% NBF; prolonged fixation may mask epitopes (standard IHC practice) | |
| Caveat | Phospho-specific staining may differ from total CREB1 (datasheet A00577) | |
| Regulation | Phosphorylation can change phospho-CREB signal (UniProt) | |
| Isoform / epitope | 3 isoforms; check antibody epitope coverage across splice forms (UniProt) |
Section 1
Recommended CREB1 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with four published CREB1 or phospho-CREB1 IHC methods (PMC8421817; PMC3050929; PMC4496382; PMC13109986).
| Sample | FFPE tissue sections (standard) |
| Fixation | 10% neutral-buffered formalin, 24 h, standard processing (standard) |
| 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-CREB1, 1:2000-1:10000 (datasheet M00577-1) |
| 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 | CREB1-positive staining in adipocytes of adipose tissue (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 at 95–98 °C for 20 min (page retrieval; UniProt: nuclear localisation). Use each article’s stated retrieval when reproducing its method.
Section 2
What Is the Expected CREB1 Staining Pattern?
CREB1 is nuclear and has no transmembrane segment (UniProt P16220). Expect nuclear staining across many cell types, including adipocytes, glandular cells and hematopoietic cells (HPA tissue IHC: Supported; ubiquitous nuclear expression).
| Nuclei stain across many cell types (HPA tissue IHC). | Expected CREB1 pattern (HPA tissue IHC: ubiquitous nuclear expression). |
| Cytoplasm stains more strongly than nuclei (HPA tissue IHC: nuclear pattern). | Suspect nonspecific staining or an IHC artefact; check controls (standard IHC practice). |
| Signal appears only in an unexpected cell population (HPA tissue IHC: ubiquitous nuclear pattern). | Check cross-reactivity or endogenous detection activity (standard IHC practice). |
| Diffuse colour obscures nuclei (HPA tissue IHC: nuclear pattern). | Background may reflect excess antibody or incomplete blocking (standard IHC practice). |
| Adipocyte nuclei show no signal (HPA tissue IHC: High in adipocytes). | Check retrieval, antibody incubation and detection controls (standard IHC practice). |
💡Expected CREB1 appearanceCall positive when nuclei stain clearly in expected cells, including adipocytes; dominant diffuse cytoplasmic colour is suspect (HPA tissue IHC: High in adipocytes; ubiquitous nuclear expression).
| Fixation and cross-linking (standard IHC practice) | Overfixation can mask nuclear epitopes and weaken signal (standard IHC practice). |
| Antigen retrieval (standard paraffin IHC practice) | Insufficient retrieval can reduce nuclear staining (standard IHC practice). |
| Isoforms (UniProt P16220: 3 isoforms) | Coverage may vary if the antibody epitope differs among isoforms (UniProt P16220; IHC practice). |
| Protein processing (UniProt P16220: chain 1–327) | No signal peptide or propeptide is annotated (UniProt P16220). |
| Endogenous peroxidase (standard chromogenic IHC practice) | Incomplete quenching can create misleading colour (standard chromogenic IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| No nuclear signal in adipocytes (HPA tissue IHC: High). | Epitope masking after fixation (standard IHC practice). | Optimise retrieval using the catalog IHC-P protocol (standard IHC practice). |
| Nuclear signal is uniformly faint (HPA tissue IHC: nuclear pattern). | Weak antibody or detection conditions (standard IHC practice). | Check incubation and detection controls (standard IHC practice). |
| Cytoplasmic colour dominates (HPA tissue IHC: nuclear pattern). | Nonspecific staining or background (standard IHC practice). | Review blocking and antibody concentration (standard IHC practice). |
| Diffuse colour covers the section (HPA tissue IHC: nuclear pattern). | Excess antibody or incomplete washing (standard IHC practice). | Optimise antibody dilution and washes (standard IHC practice). |
| Colour persists in a no-primary control (standard IHC practice). | Endogenous peroxidase or detection background (standard chromogenic IHC practice). | Check peroxidase quenching and detection reagents (standard IHC practice). |
| IF/ICC signal is mainly nucleoplasmic (HPA subcellular ICC-IF). | Expected localisation (HPA subcellular ICC-IF: nucleoplasm enhanced). | Assess IF/ICC using its separate guide (HPA subcellular ICC-IF). |
Experiment evidence
IHC & IF images showing CREB1 staining
M00577-1 · IHCMouse brain was stained with anti-CREB rabbit antibody
M00577-1 · IHCRat brain was stained with anti-CREB rabbit antibody
A00577 · IHCImmunohistochemical analysis of paraffin-embedded Human hepatocarcinoma tissue using A00577 performed on the vendor® BOND RXm. Tissue was fi
A00577 · IHCImmunohistochemical analysis of paraffin-embedded Human colon carcinoma tissue using A00577 performed on the vendor® BOND RXm. Tissue was fi
A00577S129 · IHCImmunohistochemistry (IHC) analyzes of p-CREB (S129) pAb in paraffin-embedded human breast carcinoma tissue at 1:100.
M00577 · IHCImmunohistochemical analysis of paraffin-embedded mouse brain, using CREB Antibody.
M00577 · IFImmunofluorescent analysis of Hela cells, using CREB Antibody .
P00577-1 · IHCImmunohistochemical analysis of paraffin-embedded human gastric carcinoma, using Phospho-Creb (S133) Antibody.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000118260-CREB1/tissue/cerebral+cortex#imid_5193724
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000118260-CREB1/tissue/colon#imid_5193650
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000118260-CREB1/tissue/liver#imid_5193635
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000118260-CREB1/tissue/kidney#imid_5193644
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA019150; green = CREB1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA019150; green = CREB1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CREB1 IHC & IF
🧪Start with adipose tissue and look for nuclear staining in adipocytes (HPA: High in adipocytes; UniProt P16220: nucleus). HPA detects CREB1 in all 45 scored tissues, so there is no supported negative tissue; no-primary and isotype controls establish background, while any unstained nuclei on the positive slide are only conditional internal comparators, with no cell type assumed negative (HPA: no negative tissue).
Positive control tissue: Adipose tissue (Adipocytes, HPA High)
Negative control tissue: None in HPA: CREB1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CREB1 in A-431, U-251MG, U2OS, HAP1, SuSa, ASC52telo, RPTEC/TERT1, hTERT-RPE1 (serum starved), with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Run a no-primary (secondary-only) section, an isotype control matched to the primary antibody’s host species and subclass if monoclonal (or same-host normal IgG if polyclonal), and a CREB1-knockout sample as a biological specificity control (standard IHC practice). For chromogenic adipose IHC, quench endogenous peroxidase; for adipose IF, check autofluorescence with an unstained section (standard IHC/IF practice).
⚠️Feasibility: Fix adipose sections promptly and keep fixation time consistent; optimize heat-induced antigen retrieval for the catalog antibody because fixation can mask nuclear epitopes (standard IHC practice; UniProt P16220: nucleus). Frozen sections may need less retrieval but can compromise adipocyte morphology, while cultured-cell IF offers a localization check supported by HPA’s enhanced nucleoplasmic signal (standard IHC/IF practice; HPA: nucleoplasm enhanced). Lipid extraction leaves clear spaces in paraffin adipose sections, so score nuclear staining against the controls (standard histology practice).
HPA tissue IHC evidence for CREB1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — High 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 |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: CREB1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced CREB1 IHC Tips
Troubleshoot CREB1 staining by checking nuclear signal, matched processing and epitope specificity before comparing cells or conditions.
How should I retrieve CREB1 in paraffin sections when nuclear staining is weak?
Use Tris-EDTA at pH 9.0 for heat-induced retrieval at 95–98 °C for 20 min (page retrieval setting: nuclear antigen). Let slides cool consistently, then compare a known positive section processed in the same run; CREB1 should show nuclear staining (UniProt P16220: nucleus; HPA: ubiquitous nuclear expression). If signal remains weak, compare a shorter heating time on adjacent sections while keeping antibody concentration and detection conditions fixed (standard IHC practice). Check for tissue loss, uneven heating and weak counterstaining before changing the retrieval buffer, because those problems can mimic failed antigen exposure (standard IHC practice).
Could fixation explain uneven CREB1 staining across otherwise similar sections?
Yes: compare sections fixed in neutral buffered formalin for documented, comparable durations, then processed and retrieved together (standard IHC practice). Excessive crosslinking can mask a nuclear epitope, whereas inadequate fixation can compromise nuclear detail and tissue retention (standard IHC practice). CREB1 is primarily nuclear, so assess staining against intact nuclear morphology in the counterstain rather than total brown area (UniProt P16220: nucleus; HPA: ubiquitous nuclear expression). If fixation histories differ, run adjacent sections through the same Tris-EDTA pH 9.0, 95–98 °C, 20 min retrieval and report the histories alongside any intensity comparison (page retrieval setting: nuclear antigen; standard IHC practice).
What CREB1 staining pattern should I accept as specific in tissue?
Prioritise staining within morphologically intact nuclei: CREB1 is annotated as nuclear, and tissue IHC shows ubiquitous nuclear expression (UniProt P16220: nucleus; HPA: ubiquitous nuclear expression). Use the counterstain to confirm that chromogen sits over nuclei rather than along cell borders or in extracellular material (standard IHC practice). A diffuse cytoplasmic pattern alone needs additional validation; cell imaging places CREB1 mainly in the nucleoplasm and reports cytosolic localisation as uncertain (HPA subcellular: nucleoplasm enhanced; cytosol uncertain). CREB1 has no transmembrane segment, so a crisp membrane-only pattern is unexpected and warrants a detection-control and morphology review (UniProt P16220: topology; standard IHC practice).
How can epitope choice change the meaning of a CREB1 IHC result?
Check the catalog antibody’s stated immunogen or epitope against the 3 annotated CREB1 isoforms before treating absent staining as absent protein (UniProt P16220: isoforms 1–3; standard IHC practice). CREB1 contains a KID region at residues 87–146 and a bZIP region at 269–327; epitope position can affect which molecular state an antibody reports (UniProt P16220: domains). Ser-119, Ser-128 and Ser-257 are annotated phosphosites, so verify whether the antibody recognizes total CREB1 or a specified phosphorylated epitope (UniProt P16220: modified residues; standard IHC practice). Compare serial sections with the appropriate positive control and identical retrieval before interpreting differences between antibodies (standard IHC practice).
How should IF help resolve an ambiguous chromogenic CREB1 result?
On a separate IF preparation, multiplex CREB1 with a marker for the expected cell type, such as adipocytes in adipose tissue or hematopoietic cells in bone marrow (HPA tissue IHC: high in adipocytes and hematopoietic cells; standard IF practice). Choose a fluorophore channel after inspecting tissue autofluorescence and include single-stain controls to check spectral bleed-through (standard IF practice). Because CREB1 is nuclear and has no transmembrane segment, use permeabilisation appropriate for access to the nuclear epitope (UniProt P16220: nucleus and topology; standard IF practice). Compare nuclear overlap with the chromogenic section’s counterstain, while accounting for differences in fixation and detection chemistry between preparations (standard IHC/IF practice).
How do I distinguish CREB1 signal from chromogenic background?
First inspect a no-primary control from the same tissue and run to locate staining caused by detection reagents rather than the catalog antibody (standard IHC practice). Block endogenous peroxidase before peroxidase-based chromogenic detection, and use an appropriate protein block to reduce nonspecific antibody binding (standard IHC practice). If nuclei and surrounding stroma darken together, reassess blocking, antibody concentration, wash stringency and chromogen development time on adjacent sections (standard IHC practice). CREB1 should be predominantly nuclear, so widespread extracellular deposit or uniform cytoplasmic haze does not match the main reference pattern (UniProt P16220: nucleus; HPA tissue IHC: ubiquitous nuclear expression).
What is a defensible way to score CREB1 in paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and score only intact nuclei within a consistent tissue area, using the same counterstain and detection settings across slides (UniProt P16220: nucleus; standard IHC practice). Report either the percentage of positive nuclei or an H-score based on nuclear intensity and the percentage of cells at each intensity (standard IHC practice). Normalise counts to the number of evaluable nuclei in the specified cell population; if reporting positive-cell density, use a measured area in mm² (standard IHC practice). Exclude necrosis and damaged edges by a prespecified rule, and keep total CREB1 measurements distinct from phospho-epitope measurements (UniProt P16220: modified residues; standard IHC practice).
When does a positive CREB1 stain warrant skepticism?
A credible positive result aligns with intact nuclei and the expected cell population; CREB1 is nuclear and shows widespread nuclear tissue expression (UniProt P16220: nucleus; HPA tissue IHC: ubiquitous nuclear expression). Be cautious when staining is confined to cut edges, necrotic regions or extracellular deposits, because morphology and processing artefacts can produce misleading chromogen (standard IHC practice). Review a no-primary control and peroxidase blocking if diffuse colour suggests endogenous enzyme activity (standard IHC practice). Finally, do not infer CREB1 activation from total-protein staining alone: its transcriptional activity depends on phosphorylation, including the annotated Ser-119 site (UniProt P16220: function and modified residues).
Boster reagents
Best CREB1 / Cyclic AMP-responsive element-binding protein 1 IHC Antibodies
The catalog includes CREB1 IHC images from human, mouse, and rat samples (catalog IHC images), plus a HeLa IF image (catalog IF image: M00577).
Real IHC data
Anti-CREB-1 Rabbit Monoclonal Antibody
Cat # M00577-1
Real IHC data
Anti-Phospho-CREB(S133) Antibody
Cat # A00577
Real IHC data
Anti-CREB (phospho-S129) CREB1 Antibody
Cat # A00577S129
Real IHC data
Anti-CREB CREB1 Rabbit Monoclonal Antibody
Cat # M00577
Real IHC data
Anti-Phospho-Creb (S133) CREB1 Rabbit Monoclonal Antibody
Cat # P00577-1
M00577-1 has IHC images of human, mouse, and rat brain and human thyroid carcinoma (catalog IHC images); A00577 has human hepatocarcinoma and colon carcinoma IHC images (catalog IHC images); A00577S129 has a human breast carcinoma IHC image (catalog IHC image). M00577 has a mouse brain IHC image and a HeLa IF image (catalog images); P00577-1 has a human gastric carcinoma IHC image (catalog IHC image).
Which to pick: For FFPE IHC, choose M00577-1 for total CREB1 in human, mouse, or rat samples (catalog title, reactivity, and IHC images); choose P00577-1 when the target is phospho-S133 CREB1 (catalog title and IHC application). For IF/ICC, M00577 lists both applications and has a HeLa IF image (catalog applications and IF image). For IHC across species, M00577-1 is monoclonal and has brain IHC images from human, mouse, and rat (catalog description and IHC images).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P16220 (CREB1_HUMAN, Cyclic AMP-responsive element-binding protein 1).
- Human Protein Atlas. CREB1 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. CREB1 subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol, centrosome and basal body..
- Human Protein Atlas. CREB1 antibody validation summary (3 antibodies).
- Increased phosphorylated CREB1 protein correlates with poor prognosis in clear cell renal cell carcinoma. Translational andrology and urology 2021 — PMC8421817.
- cAMP response element binding protein is required for differentiation of respiratory epithelium during murine development. PloS one 2011 — PMC3050929.
- High expression of cAMP-responsive element-binding protein 1 (CREB1) is associated with metastasis, tumor stage and poor outcome in gastric cancer. Oncotarget 2015 — PMC4496382.
- Identification and Validation of Ferroptosis-Related Biomarkers and Therapeutic Targets in ARDS: A Bioinformatics and Experimental Study. Journal of inflammation research 2026 — PMC13109986.
- PubMed PMID:2142528 — UniProt-cited evidence.
- PubMed PMID:2196176 — UniProt-cited evidence.
- PubMed PMID:1966745 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CREB1 (Cyclic AMP-responsive element-binding protein 1). Expected localisation Nuclear staining in tissue sections (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: M00577-1 at 1:2000-1:10000 (the product's own IHC validation image is shown). Controls (HPA): positive Adipose tissue (Adipocytes); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. Protocol steps: 1. Sample: FFPE tissue sections (standard); 2. Fixation: 10% neutral-buffered formalin, 24 h , standard processing (standard); 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 (standard rule: nuclear antige; 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-CREB1, 1:2000-1:10000 (datasheet M00577-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Fix promptly in 10% NBF; prolonged fixation may mask epitopes (standard IHC practice); Caveat: Phospho-specific staining may differ from total CREB1 (datasheet A00577); Isoform / epitope: 3 isoforms; check antibody epitope coverage across splice forms (UniProt); Regulation: Phosphorylation can change phospho-CREB signal (UniProt). Evidence: UniProt P16220, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the nucleoplasm. In , Datasheet M00577-1, PMC8421817, PMC3050929. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CREB1/immunohistochemistry-ihc-guide.