This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
CRHBP / Corticotropin-releasing hormone-binding protein · IHC design guide
Design Immunohistochemistry for CRHBP
Plan paraffin IHC for CRHBP using the reported cytoplasmic tissue pattern and plasma positivity (HPA tissue IHC). Round or early spermatids in testis show high staining and offer a positive reference (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CRHBP 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 with plasma positivity (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in most tissues, with plasma positivity (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Testis | |
| Negative control | Adipose tissue+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 | Secreted CRHBP may also stain plasma (HPA tissue IHC; UniProt) | |
| Regulation | No expression regulator specified (UniProt) | |
| Isoform / epitope | No annotated isoforms; signal peptide 1–24 is cleaved (UniProt) |
Section 1
Recommended CRHBP IHC & IF Protocols
The catalog antibody protocol is accompanied by published CRHBP IHC protocols for hepatocellular carcinoma tissue (PMC6096283; PMC11633302).
| Sample | Paraffin-embedded mouse kidney tissue; fixative not specified (datasheet A06764) |
| Fixation | Image fixative and duration unreported (datasheet A06764); 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 citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane 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-CRHBP, 1:100–1:500 starting range (standard) |
| 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 | CRHBP-positive staining in round or early spermatids of testis (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues, including plasma positivity. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval); the published protocols also describe high-pressure retrieval (PMC6096283; PMC11633302).
Section 2
What Is the Expected CRHBP Staining Pattern?
CRHBP is secreted and has no transmembrane segment (UniProt P24387 topology). In tissue IHC, HPA reports predominantly cytoplasmic staining across most tissues, with plasma positivity; round or early spermatids in testis are an explicitly documented High example (HPA tissue IHC). The tissue profile is rated Approved, with a caution that a secreted protein’s RNA and protein locations may differ (HPA tissue IHC).
| Strong cytoplasmic staining in round or early spermatids, with plasma positivity elsewhere on the section (HPA tissue IHC). | This matches the documented High testis cell signal and the broader cytoplasmic and plasma profile (HPA tissue IHC). Score cell staining and plasma staining separately so extracellular signal does not inflate a cell score (standard IHC practice). |
| Dominant nuclear staining or a sharp, continuous cell-membrane outline. | Neither is the reported tissue pattern (HPA tissue IHC). CRHBP is secreted and lacks a transmembrane segment (UniProt P24387 topology). Treat a compartment-only result as suspect and review controls and staining conditions (standard IHC practice). |
| Strong staining in adrenal glandular cells or adipocytes despite little signal in testis spermatids. | Those cell types are listed as Not detected, while round or early spermatids are High (HPA tissue IHC). Consider cross-reactivity or endogenous chromogenic activity; check the detection-only control before assigning the signal to CRHBP (standard IHC practice). |
| Diffuse color obscures cell boundaries and appears across the section. | HPA reports plasma positivity, so extracellular color alone is not decisive (HPA tissue IHC). Widespread haze that prevents cell-level scoring warrants a review of background controls, blocking, washes and detection conditions (standard IHC practice). |
| No visible signal in a section containing round or early spermatids. | The expected High signal is absent from the supplied positive reference (HPA tissue IHC). Confirm that those cells are present, then review the antibody and detection controls before interpreting other tissues as negative (standard IHC practice). |
💡Expected CRHBP appearanceCall the IHC positive when round or early spermatids show High cytoplasmic staining, allowing for plasma positivity (HPA tissue IHC); dominant nuclear outlines or strong staining confined to HPA Not detected cell types is suspect (HPA tissue IHC; UniProt P24387 topology).
| Secretion and processing | A signal peptide spans residues 1–24, and the annotated chain spans 25–322 (UniProt P24387 processing). Secreted localisation and plasma positivity make extracellular signal plausible (UniProt P24387; HPA tissue IHC); the record does not locate the antibody epitope. |
| Glycosylation | UniProt annotates one glycosylation site at residue 204 (UniProt P24387). With no supplied epitope map or staining comparison, its effect on this IHC antibody cannot be predicted. |
| Antibody validation | HPA lists IHC as Supported for HPA036234 and Approved for HPA046120 (HPA antibodies). Neither entry is labelled Enhanced in the supplied record; assess the chosen antibody’s controls rather than treating the statuses as identical. |
| RNA versus tissue staining | RNA is tissue enhanced in brain and liver, while HPA cautions that RNA and protein locations may differ for a secreted protein (HPA tissue IHC). Select an IHC positive control from observed protein staining, such as the documented testis cells (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Testis spermatids are unstained. | The High reference pattern is missing (HPA tissue IHC); the immediate cause is undetermined. | Verify round or early spermatids are present, then inspect antibody identity, detection reagents and a run control (standard IHC practice). Do not call the assay negative from this section alone. |
| Adrenal glandular cells or adipocytes stain strongly. | HPA lists these cells as Not detected (HPA tissue IHC); cross-reactivity or endogenous chromogenic activity is possible (standard IHC practice). | Compare with a detection-only control and the documented testis positive; reassess antibody specificity and detection blocking if the unexpected signal persists (standard IHC practice). |
| Nuclei or crisp cell borders dominate the image. | The pattern conflicts with HPA’s cytoplasmic profile and the secreted, non-transmembrane annotation (HPA tissue IHC; UniProt P24387 topology). | Review the counterstain and control sections, then score only a reproducible, cell-resolved pattern consistent with the reference (standard IHC practice). |
| Diffuse color makes cytoplasmic staining hard to score. | Plasma positivity can contribute extracellular signal (HPA tissue IHC); excess background from staining or detection remains possible (standard IHC practice). | Compare background controls, review blocking and washes, and distinguish plasma color from staining within identifiable cells (standard IHC practice). |
| Brain or liver RNA enrichment is used to predict a strong IHC control. | The RNA call is tissue enhanced, but HPA warns that secreted protein and RNA locations may differ (HPA tissue IHC). | Use the observed High round or early spermatid pattern to check IHC performance; interpret brain or liver sections by their staining, not RNA alone (HPA tissue IHC). |
| Q: Does this pattern establish what to expect in IF/ICC? | HPA calls CRHBP secreted but supplies no ICC-IF cell-line images or main subcellular location (HPA subcellular). | A: Do not transfer the tissue IHC compartment or intensity to IF/ICC as a validated result; assess that application on its own controls (HPA subcellular; standard IF practice). |
Experiment evidence
IHC & IF images showing CRHBP staining
A06764 · IHCImmunohistochemistry analysis of CRHBP in paraffin-embedded mouse kidney tissue using CRHBP pAb at a dilution of 1:300 (40x lens).
A06764 · IHCImmunohistochemistry analysis of CRHBP in paraffin-embedded rat kidney tissue using CRHBP pAb at a dilution of 1:300 (40x lens).
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000145708-CRHBP/tissue/cerebral+cortex#imid_12524049
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000145708-CRHBP/tissue/colon#imid_12523959
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000145708-CRHBP/tissue/liver#imid_12523968
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000145708-CRHBP/tissue/kidney#imid_12523977
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CRHBP IHC & IF
🧪Run testis first and look for staining in round or early spermatids (HPA: High in round or early spermatids). Use adipose tissue as a negative specimen (HPA: Not detected in adipocytes); neighboring testicular cells without specific signal can serve as internal negative cells, but their identity and staining status must be checked on the slide (standard IHC practice).
Positive control tissue: Testis (Round or early spermatids, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for CRHBP; derive a cell-line control from the positive tissue's cell type (Round or early spermatids) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control and a host- and clonality-matched isotype control, plus CRHBP knockout tissue if available as a biological specificity control (standard IHC practice). For chromogenic IHC, block endogenous peroxidase and assess background on the testis control slide; for IF, assess tissue autofluorescence with an unstained slide (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the mouse kidney paraffin-section caption for A06764 specifies 1:300 but does not report a fixative (selected IHC caption: A06764). Antigen-retrieval dependency is unreported, and the supplied evidence does not establish whether frozen sections or IF are easier (supplied application evidence). No testis-specific artefact is documented here; check background against the controls when scoring spermatids (HPA: High in round or early spermatids; standard IHC/IF practice).
HPA tissue IHC evidence for CRHBP
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Secreted protein, tissue location of RNA and protein is expected to differ.). 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 |
|---|---|---|---|---|
| Testis | Round or early spermatids | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CRHBP IHC Tips
Use compartment-aware controls and matched section processing when troubleshooting CRHBP staining in paraffin sections (UniProt P24387 topology; HPA tissue IHC).
How should I adjust retrieval when CRHBP staining is weak in paraffin sections?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting). Run a known positive section alongside the experimental section so that a weak result can be distinguished from a failed staining run (HPA tissue IHC: high staining in round or early spermatids; standard IHC practice). If signal remains weak, compare a longer citrate retrieval with a higher-pH buffer in separately processed sections while holding antibody concentration and detection constant (standard IHC practice). Watch for lost tissue, damaged morphology, or diffuse background as retrieval is intensified, and select the mildest condition that preserves interpretable cellular staining (standard IHC practice).
Could fixation explain weak CRHBP staining in my paraffin sections?
CRHBP-specific sensitivity to fixation is unknown from the supplied evidence; the selected mouse kidney image states paraffin embedding but does not report a fixative (caption: A06764). Record the actual fixative, fixation duration, tissue thickness, and processing history before comparing staining across specimens (standard IHC practice). For a controlled comparison, process matched pieces for a routine 10% neutral buffered formalin condition and an adjusted fixation duration, then apply identical retrieval and detection (standard IHC practice). Interpret any difference as an effect observed under those processing conditions, and confirm it with a positive control processed the same way (standard IHC practice).
Where should CRHBP staining appear, and how should extracellular signal be assessed?
Expect potentially cytoplasmic and extracellular staining because CRHBP is secreted and has no transmembrane segment (HPA tissue IHC: cytoplasmic expression in most tissues, including plasma positivity; UniProt P24387 topology). Its signal peptide occupies residues 1–24, while the annotated mature chain spans 25–322 (UniProt P24387 processing). Review cells and surrounding spaces separately, since secreted protein can appear away from its producing cells (HPA tissue IHC: tissue RNA and protein locations are expected to differ). Record diffuse plasma or extracellular color apart from cell-associated staining, and require preserved anatomy and matched controls before assigning a cellular source (HPA tissue IHC: plasma positivity; standard IHC practice).
Could epitope placement or processing change the CRHBP staining pattern?
Check the antibody immunogen or mapped epitope against the annotated 1–24 signal peptide and 25–322 mature chain before interpreting tissue staining (UniProt P24387 processing; standard IHC practice). An epitope restricted to the cleaved signal peptide would not be expected to report the mature secreted chain in the same way as a mature-chain epitope (UniProt P24387 processing). CRHBP has one annotated glycosylation site at residue 204 and no annotated isoforms, so a staining difference alone does not establish an isoform change (UniProt P24387 glycosylation and isoforms). Compare independently validated epitopes, where available, on matched sections and assess agreement in staining location after identical processing (standard IHC practice).
How can I use IF to examine a disputed CRHBP IHC pattern?
Treat IF as a separate assay and first check that the antibody is validated under the chosen IF conditions (standard IF practice). Multiplex CRHBP with a marker identifying the expected cell type in the specimen, then compare cell-associated signal with adjacent extracellular signal (HPA tissue IHC: secreted protein with cytoplasmic and plasma positivity; standard IF practice). Select a fluorophore whose emission avoids the specimen’s strongest autofluorescence, and include single-stain and no-primary controls when assessing bleed-through and background (standard IF practice). Because CRHBP has a signal peptide and no transmembrane segment, choose permeabilisation according to whether the validated epitope is intracellular or accessible extracellularly; do not infer its accessibility from topology alone (UniProt P24387 topology and processing; standard IF practice).
What controls help separate CRHBP signal from chromogenic background?
Include a no-primary section to reveal secondary reagent, endogenous enzyme, and tissue color contributions under the same detection conditions (standard IHC practice). Apply an appropriate peroxidase block before HRP and DAB detection, and titrate the antibody and chromogen development against a matched positive section (standard IHC practice). The selected antibody image shows staining in paraffin-embedded mouse kidney at 1:300, but its caption does not establish a universal working dilution or report fixation (caption: A06764). Score granular deposits at tissue edges, folds, and damaged regions separately from intact cellular staining, and repeat questionable patterns on a fresh section (standard IHC practice).
How should I quantify CRHBP staining when extracellular signal is present? ⚠ ANSWER MARKED FOR VERIFICATION
Define the compartment before scoring: report cell-associated staining separately from extracellular or plasma signal, which can be present for this secreted protein (UniProt P24387 subcellular location; HPA tissue IHC: plasma positivity). For a defined cell population, record the percentage of positive cells and intensity, then calculate an H-score using 0–3 intensity categories if that scale is prespecified (standard IHC practice). For spatial deposits, measure positive area or positive-cell density per mm² within a consistent anatomical region (standard IHC practice). Normalise to the number of eligible cells or analysed tissue area, and keep staining batch, threshold, and exclusion rules consistent across groups (standard IHC practice).
How can I tell genuine CRHBP staining from a misleading pattern?
First check whether staining is compatible with a secreted protein: cytoplasmic and extracellular signal may both occur, while an exclusively membrane-confined pattern needs further validation (UniProt P24387 topology; HPA tissue IHC: cytoplasmic and plasma positivity). Compare the candidate positive cells with a supported reference: HPA reports high staining in round or early spermatids and reports adipocytes as not detected (HPA tissue IHC). Exclude section edges, folds, and necrotic areas before judging intensity, and use a no-primary control to assess endogenous enzyme color in the chromogenic workflow (standard IHC practice). Reproduce the pattern in intact anatomy and distinguish deposited protein from its cell of origin when interpreting extracellular staining (HPA tissue IHC: secreted protein, with tissue RNA and protein locations expected to differ; standard IHC practice).
Boster reagents
Best CRHBP / Corticotropin-releasing hormone-binding protein IHC Antibodies
A06764 has real IHC images from paraffin-embedded mouse and rat kidney; IF/ICC is listed without an IF image (catalog: A06764 IHC captions, applications, IF image alts).
Real IHC data
Anti-CRHBP Antibody
Cat # A06764
A06764 is listed for IHC-P and IF/ICC in Human, Mouse, and Rat (catalog: A06764 applications/reactivity). Its IHC captions show paraffin-embedded mouse and rat kidney at 1:300; no IF image is supplied (catalog: A06764 IHC captions/IF image alts).
Which to pick: For tissue IHC, choose A06764 based on its own paraffin-section images; the fixative is unreported (catalog: A06764 IHC captions). For IF/ICC, A06764 lists both applications and an IF dilution of 1:50–1:200, without an IF image (catalog: A06764 applications/IF dilution/IF image alts). For cross-species work, A06764 lists Human, Mouse, and Rat reactivity and is described as a rabbit pAb (catalog: A06764 reactivity/host/IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P24387 (CRHBP_HUMAN, Corticotropin-releasing hormone-binding protein).
- Human Protein Atlas. CRHBP tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CRHBP subcellular location (ICC-IF): Secreted.
- Human Protein Atlas. CRHBP antibody validation summary (2 antibodies).
- Decreased CRHBP expression is predictive of poor prognosis in patients with hepatocellular carcinoma. Oncology letters 2018 — PMC6096283.
- Repeated exposure to neurotoxic levels of chlorpyrifos alters hippocampal expression of neurotrophins and neuropeptides. Toxicology 2016 — PMC4777610.
- Expression of genes related to the hypothalamic-pituitary-adrenal axis in murine fetal lungs in late gestation. Reproductive biology and endocrinology : RB&E 2010 — PMC2989976.
- Innovative Dual mRNA-Lipid Nanoparticle Therapy Targeting CRHBP and CFHR3 for Enhanced Treatment of Hepatocellular Carcinoma. International journal of nanomedicine 2024 — PMC11633302.
- PubMed PMID:1846945 — UniProt-cited evidence.
- PubMed PMID:8307998 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CRHBP (Corticotropin-releasing hormone-binding protein). Expected localisation Cytoplasmic staining with plasma positivity (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A06764 at 1:100–1:500 (standard starting range) (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Round or early spermatids); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded mouse kidney tissue; fixative not specified (datasheet A06764); 2. Fixation: Image fixative and duration unreported (datasheet A06764); 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 citrate buffer, pH 6.0 , 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-CRHBP, 1:100–1:500 starting range (standard); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Secreted CRHBP may also stain plasma (HPA tissue IHC; UniProt); Isoform / epitope: No annotated isoforms; signal peptide 1–24 is cleaved (UniProt); Regulation: No expression regulator specified (UniProt). Evidence: UniProt P24387, HPA tissue IHC · Approved, HPA ICC-IF · Secreted, Datasheet A06764, PMC6096283, PMC11633302. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CRHBP/immunohistochemistry-ihc-guide.