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RSPO1 / R-spondin-1 · IHC design guide
Design Immunohistochemistry for RSPO1
Plan chromogenic RSPO1 IHC on paraffin sections with the catalog antibody at 2.5–5 μg/mL (datasheet A02857-1). Assess cytoplasmic staining in glandular cells while allowing for uncertainty from secreted variants (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RSPO1 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 tissue sections (HPA tissue IHC) | |
| Staining pattern | Glandular cells show cytoplasmic staining in several tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A02857-1) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | Bone marrow+3 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Formaldehyde-fixed paraffin sections were used (selected-SKU IHC image A02857-1) | |
| Caveat | Secreted variants may make RNA and protein locations differ (HPA tissue IHC) | |
| Regulation | No specific expression inducer is reported (UniProt) | |
| Isoform / epitope | 3 isoforms; the signal peptide is cleaved, and there is no cytoplasmic domain (UniProt) |
Section 1
Recommended RSPO1 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with two published RSPO1 chromogenic IHC protocols (datasheet A02857-1; PMC4496395; PMC7065809).
| Sample | Formaldehyde-fixed, paraffin-embedded mouse spleen tissue (datasheet A02857-1) |
| Fixation | Image formalin-fixed; duration unreported (datasheet A02857-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6 (datasheet A02857-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% serum (datasheet A02857-1) |
| Primary antibody | Rabbit anti-RSPO1, 2.5-5 μg/mL (datasheet A02857-1) |
| 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 | RSPO1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several different tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate retrieval at pH 6 for the catalog antibody; the glioma paper does not report retrieval conditions (datasheet A02857-1; PMC4496395).
Section 2
What Is the Expected RSPO1 Staining Pattern?
RSPO1 is secreted, has no transmembrane segment, and may also localize to nucleoli (UniProt Q2MKA7). In paraffin sections, expect chiefly cytoplasmic staining in glandular cells and selected epithelial or tubular cells (HPA: tissue IHC). Treat this as a comparison pattern, not proof of specificity: HPA rates the tissue profile Uncertain because secreted protein can appear apart from its RNA source (HPA: reliability).
| Cytoplasmic staining in adrenal glandular cells, esophageal squamous epithelium, or kidney tubules (HPA: High). | This matches reported high tissue IHC staining in the named cell types (HPA: tissue IHC). Compare signal with an appropriate negative control; the HPA tissue profile remains Uncertain (HPA: reliability). |
| Dominant, uniform nuclear staining with little cytoplasmic signal in an otherwise positive section. | This diverges from the reported tissue IHC pattern (HPA: cytoplasmic expression). Check for artefact before scoring it as RSPO1; UniProt also notes possible nucleolar localization, so a nuclear focus alone cannot be dismissed (UniProt Q2MKA7). |
| Strong staining of marrow hematopoietic cells or smooth muscle cells (HPA: Not detected). | These are poor fits for the reported tissue pattern (HPA: tissue IHC). Cross-reactivity or endogenous detection activity is possible; compare antibody omission and detection controls before attributing the signal to RSPO1 (general IHC practice). |
| Diffuse chromogen over tissue and blank areas, with little cell-type contrast. | Interpretation is unreliable because the expected pattern has defined cellular contrast (HPA: tissue IHC). Review blocking, washing, detection background, and counterstain against controls (general IHC practice); diffuse staining does not establish secreted RSPO1 deposition (UniProt Q2MKA7). |
| No staining in adrenal glandular cells or kidney tubules (HPA: High). | The run has missed an expected positive pattern (HPA: tissue IHC). Check tissue preservation, retrieval, antibody dilution, and detection with run controls (general IHC practice); the HPA rating of Uncertain limits any single specimen as a definitive control (HPA: reliability). |
💡Expected RSPO1 appearanceCall positive when discernible cytoplasmic staining occurs in reported high-expressing glandular, squamous, or tubular cells (HPA: tissue IHC); uniform signal in reported negative cells or across blank areas warrants investigation (HPA: Not detected; general IHC practice).
| Secreted protein and location (UniProt Q2MKA7). | The signal peptide spans residues 1–20, and the mature chain begins at 21 (UniProt Q2MKA7). Extracellular distribution is biologically plausible, but HPA's observed tissue pattern is cytoplasmic (HPA: tissue IHC); do not require membrane staining. |
| Tissue and cell type (HPA: tissue IHC). | High staining is reported in adrenal, appendix, breast, colon, duodenal, and gallbladder glandular cells; esophageal squamous cells; and kidney tubules (HPA: High). Ovary follicle cells and fallopian tube glandular cells are reported low (HPA: Low). |
| Protein versus RNA location (HPA: reliability). | HPA calls tissue RNA enhanced in cervix, endometrium, and fallopian tube, yet warns that secreted protein and RNA locations can differ (HPA: RNA specificity; reliability). Select positive IHC controls from observed protein staining, not RNA enrichment alone. |
| Isoforms and molecular processing (UniProt Q2MKA7). | UniProt lists 3 isoforms and glycosylation sites at 137, 153, and 156 (UniProt Q2MKA7). Epitope dependence cannot be predicted without antibody epitope information; these annotations do not establish an antigen-retrieval or fixation effect. |
| Subcellular evidence differs by assay (HPA: tissue IHC; HPA: ICC-IF). | HPA reports cytoplasmic tissue staining but approved vesicle localization in THP-1 ICC-IF images (HPA: tissue IHC; ICC-IF). Compare each result with its own assay and specimen context; neither observation proves that all tissue staining must be vesicular. |
| Antibody evidence (HPA: antibody validation). | HPA046154 has Uncertain IHC status, whereas HPA062923 has Approved ICC status (HPA: antibodies). ICC approval does not validate an IHC result; interpret a new paraffin-section pattern alongside controls and the tissue profile. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected high-staining cells are blank (HPA: High). | The run may lack usable antigen signal or detection; HPA's tissue profile also carries uncertainty (general IHC practice; HPA: reliability). | Check a same-run positive section, detection reagents, retrieval conditions, and the antibody's documented IHC dilution (general IHC practice). Do not assign a target-specific fixation effect without evidence. |
| Marrow hematopoietic cells or splenic red-pulp cells stain strongly (HPA: Not detected). | The pattern conflicts with HPA tissue observations; cross-reactivity or endogenous detection activity is possible (HPA: tissue IHC; general IHC practice). | Run antibody-omission and detection controls, then compare staining in reported positive cells on the same run (general IHC practice; HPA: High). |
| Chromogen spreads across the section or appears outside tissue. | Background can obscure cell boundaries and the cytoplasmic pattern reported for RSPO1 (general IHC practice; HPA: tissue IHC). | Inspect wash, blocking, chromogen development, and counterstain controls before scoring; document the tissue compartment that remains interpretable (general IHC practice). |
| Colon glandular cells stain despite UniProt listing colon among tissues without abundant expression. | The sources disagree: HPA reports High colon glandular-cell IHC, while UniProt does not list colon as abundantly expressed (HPA: tissue IHC; UniProt Q2MKA7). | Record the cell type and controls, and describe the result as consistent with HPA IHC while acknowledging the source discrepancy; avoid treating either source as proof of antibody specificity. |
| Only nuclear or nucleolar signal is visible. | HPA describes cytoplasmic tissue staining, while UniProt notes possible nucleolar localization (HPA: tissue IHC; UniProt Q2MKA7). | Review positive and omission controls and score nuclear signal separately from cytoplasmic signal; do not call a nuclear-only tissue pattern confirmed from these sources. |
| Q: Should an IF/ICC vesicle image dictate paraffin IHC scoring? | HPA's approved vesicle localization comes from THP-1 ICC-IF, whereas tissue IHC is rated Uncertain and described as cytoplasmic (HPA: ICC-IF; tissue IHC). | A: No. Use the tissue IHC pattern and controls for paraffin-section interpretation (HPA: tissue IHC; general IHC practice). Consult the separate IF/ICC guide for that application. |
Experiment evidence
IHC & IF images showing RSPO1 staining
A02857-1 · IHCImmunohistochemistry Validation of RSPO1 in Human Heart Immunohistochemical analysis of paraffin-embedded mouse spleen tissue using anti-RSP
A02857-1 · IHCImmunohistochemistry Validation of RSPO1 in Human Spleen Tissue Immunohistochemical analysis of paraffin-embedded human spleen tissue using
A02857-1 · IFImmunofluorescence Validation of RSPO1 in Human Spleen Immunofluorescent analysis of 4% paraformaldehyde-fixed human spleen labeling RSPO1 w
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000169218-RSPO1/tissue/cerebral+cortex#imid_12777995
HPA · Adrenal glandHuman Protein Atlas IHC, Adrenal gland. Source: https://www.proteinatlas.org/ENSG00000169218-RSPO1/tissue/adrenal+gland#imid_12777963
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000169218-RSPO1/tissue/colon#imid_12774177
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000169218-RSPO1/tissue/liver#imid_12774178
HPA · ICC-IF THP-1Human Protein Atlas immunofluorescence, THP-1, antibody HPA062923; green = RSPO1, blue = nucleus, red = microtubules.
HPA · ICC-IF THP-1Human Protein Atlas immunofluorescence, THP-1, antibody HPA062923; green = RSPO1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RSPO1 IHC & IF
🧪Run adrenal gland first: glandular cells should stain strongly (HPA: High in adrenal gland glandular cells). Use bone marrow hematopoietic cells as the negative tissue (HPA: Not detected); on the adrenal slide, compare glandular cells with neighboring unstained cells, but confirm their identity before treating them as internal negatives.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RSPO1 in THP-1, with annotated localisation: Vesicles (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and immunoglobulin class (standard IHC practice). Use RSPO1 knockout tissue, if available, as a biological negative; for HRP detection in adrenal gland, quench endogenous peroxidase and distinguish tissue pigment from chromogen (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A02857-1 tissue-IHC caption’s fixative is unreported. The caption uses heat-mediated citrate retrieval at pH 6, but does not establish that retrieval is required for RSPO1; no supplied evidence shows frozen sections or IF to be easier than paraffin IHC (A02857-1 caption). Adrenal tissue pigment can complicate interpretation of brown chromogen, so assess morphology and the negative controls together (standard IHC practice).
HPA tissue IHC evidence for RSPO1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — At least one protein variant secreted, tissue location of RNA and protein might differ and correlation is complex. 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | High | Protein (IHC) | HPA → |
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RSPO1 IHC Tips
Troubleshoot RSPO1 staining in paraffin sections by checking tissue controls, compartment patterns, processing conditions and the reliability of each evidence source.
Which retrieval conditions should I start with for RSPO1 paraffin IHC?
Start with heat-mediated retrieval in citrate buffer at pH 6 (datasheet A02857-1). The selected antibody caption reports that condition for paraffin-embedded mouse spleen, followed by primary incubation overnight at 4°C (caption A02857-1). If staining is weak, compare a second retrieval condition on adjacent sections while holding antibody concentration, heating time and detection constant (standard IHC practice). Include a no-primary control and inspect tissue integrity, because excessive heating can damage morphology or increase diffuse staining (standard IHC practice). Record the retrieval time and heating device for each section so the comparison remains interpretable (standard IHC practice).
How should I assess fixation-related loss of RSPO1 staining?
Target-specific RSPO1 sensitivity to fixation is unknown from the supplied evidence; do not infer it from tissue patterns, topology or glycosylation (evidence scope). The selected caption reports formaldehyde treatment of paraffin-embedded mouse spleen, but provides no fixation duration or comparison with another fixative (caption A02857-1). Compare sections with documented processing histories using the same citrate pH 6 retrieval and detection settings (datasheet A02857-1; standard IHC practice). Examine morphology alongside staining, and avoid assigning a negative result to fixation alone without a positive control from the same run (standard IHC practice). Keep section thickness and primary incubation consistent during that comparison (standard IHC practice).
Which cellular compartments make an RSPO1 IHC signal plausible?
Assess cytoplasmic and extracellular-associated staining first, since RSPO1 is secreted and lacks a transmembrane segment (UniProt Q2MKA7 topology). Vesicles are the approved subcellular location in cell imaging, while the tissue IHC profile describes cytoplasmic staining across several tissues (HPA subcellular; HPA tissue IHC). UniProt also lists a nuclear location with apparent nucleolar enrichment, so isolated nuclear colour requires compartment-specific confirmation rather than automatic dismissal (UniProt Q2MKA7 subcellular). Compare the pattern with a no-primary section and a cellular counterstain, then document whether colour follows cells, extracellular material or damaged areas (standard IHC practice). Score these compartments separately before combining fields (standard IHC practice).
Could isoforms or processing explain a negative RSPO1 stain?
RSPO1 has 3 listed isoforms and a cleaved signal peptide spanning residues 1–20 (UniProt Q2MKA7 processing; UniProt Q2MKA7 isoforms). The mature chain spans residues 21–263, and listed glycosylation sites are 137, 153 and 156 (UniProt Q2MKA7 processing; UniProt Q2MKA7 glycosylation). Check the antibody's documented immunogen and epitope before attributing a negative section to isoform absence or epitope masking (standard IHC practice). If that information is unavailable, report isoform coverage as unresolved and compare independent tissue controls under identical retrieval conditions (standard IHC practice). Avoid inferring epitope accessibility from the TSP type-1 domain at 147–207 alone (UniProt Q2MKA7 domains).
How can IF help verify an ambiguous chromogenic RSPO1 pattern?
Use IF on matched material as a separate validation experiment, since the selected product evidence describes chromogenic staining in paraffin sections (caption A02857-1). Multiplex RSPO1 with a validated marker for the expected cell type, and inspect separate channels before interpreting overlap (standard IF practice). Choose a fluorophore in a channel with low tissue autofluorescence, using unstained material to assess background in that channel (standard IF practice). Match permeabilisation to the tested epitope: an intracellular epitope needs membrane access, whereas extracellular staining can be assessed without permeabilisation (standard IF practice; UniProt Q2MKA7 topology). Vesicular IF localisation offers a comparison, but does not establish an identical paraffin IHC pattern (HPA subcellular).
What should I change when RSPO1 IHC has diffuse background?
Run no-primary and secondary-only controls to separate antibody-independent chromogen deposition from primary-dependent staining (standard IHC practice). For HRP detection, include a peroxidase block and inspect blood-rich areas for residual endogenous activity before interpreting brown signal (standard IHC practice). The selected caption used 10% serum blocking for 1 hour at room temperature, primary incubation overnight at 4°C, and an HRP secondary at 1:250 (caption A02857-1). If background remains primary-dependent, titrate the primary and strengthen washing while preserving the same citrate pH 6 retrieval for comparison (datasheet A02857-1; standard IHC practice). Judge improvement against tissue morphology and control sections, not background intensity alone (standard IHC practice).
How should I quantify heterogeneous RSPO1 staining in tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and staining compartment before scoring, because RSPO1 can be secreted and tissue IHC describes cytoplasmic expression (UniProt Q2MKA7 topology; HPA tissue IHC). For cellular staining, record the percentage of positive cells and an intensity-weighted H-score using one threshold across the comparison set (standard IHC practice). For discrete deposits, report positive area or object density per mm² of viable tissue, with the segmentation rule stated (standard IHC practice). Normalise counts to the number of eligible cells or viable tissue area, and exclude folds, edges and necrotic regions consistently (standard IHC practice). Keep glandular and stromal compartments separate where both are present (standard IHC practice).
When is an RSPO1-positive IHC result convincing rather than artefactual?
A convincing result has reproducible cellular or extracellular-associated staining, appropriate control behaviour and a distribution assessed against RSPO1's secreted biology (UniProt Q2MKA7 topology; standard IHC practice). HPA reports high glandular-cell staining in adrenal gland and no detected staining in spleen red-pulp cells, but labels its tissue IHC reliability uncertain (HPA tissue IHC). The selected caption describes mouse spleen staining despite a human-heart title, so verify specimen identity before treating that image as a reference pattern (caption A02857-1). Flag staining confined to section edges, necrosis, unexpected cell populations or no-primary controls as suspect, including residual endogenous peroxidase signal (standard IHC practice). Investigate isolated nuclear colour separately because nucleolar localisation is also reported (UniProt Q2MKA7 subcellular).
Boster reagents
Best RSPO1 / R-spondin-1 IHC Antibodies
The catalog antibody A02857-1 has IHC images of paraffin-embedded human and mouse spleen and an IF image of human spleen (catalog image captions).
Real IHC data
Anti-R-spondin-1 RSPO1 Antibody
Cat # A02857-1
A02857-1 is listed for IHC-P and IF in human and mouse samples (catalog applications and reactivity). Its images show IHC in paraffin-embedded human and mouse spleen and IF in human spleen (catalog image captions).
Which to pick: For tissue IHC, choose A02857-1: its spleen images use paraffin sections fixed with formaldehyde, citrate retrieval at pH 6, and primary antibody at 2.5–5 μg/mL (A02857-1 IHC captions). For IF, the same SKU has a human spleen image at 5 μg/mL; ICC would need separate validation (A02857-1 IF caption; catalog applications). For cross-species work, A02857-1 lists human and mouse reactivity and shows IHC images from both species; the catalog describes IHC validation in human samples (catalog reactivity; A02857-1 IHC captions; catalog validation statement).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q2MKA7 (RSPO1_HUMAN, R-spondin-1).
- Human Protein Atlas. RSPO1 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. RSPO1 subcellular location (ICC-IF): Localized to vesicles..
- Human Protein Atlas. RSPO1 antibody validation summary (2 antibodies).
- Phenotypic and Molecular Alterations in the Mammary Tissue of R-Spondin1 Knock-Out Mice during Pregnancy. PloS one 2016 — PMC5017653.
- Silencing of R-Spondin1 increases radiosensitivity of glioma cells. Oncotarget 2015 — PMC4496395.
- Targeting the Wnt signaling pathway through R-spondin 3 identifies an anti-fibrosis treatment strategy for multiple organs. PloS one 2020 — PMC7065809.
- Identification and Validation of Immune Implication of R-Spondin 1 and an R-Spondin 1-Related Prognostic Signature in Esophagus Cancer. International journal of genomics 2024 — PMC11222003.
- PubMed PMID:16109882 — UniProt-cited evidence.
- PubMed PMID:17041600 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RSPO1 (R-spondin-1). Expected localisation Cytoplasmic staining in tissue sections (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A02857-1). Validated IHC antibody: A02857-1 at 2.5-5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative Bone marrow. IHC-P workflow on paraffin sections. Protocol steps: 1. Sample: Formaldehyde-fixed, paraffin-embedded mouse spleen tissue (datasheet A02857-1); 2. Fixation: Image formalin-fixed; duration unreported (datasheet A02857-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: Citrate pH 6 (datasheet A02857-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% serum (datasheet A02857-1); 8. Primary antibody: Rabbit anti-RSPO1, 2.5-5 μg/mL (datasheet A02857-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Formaldehyde-fixed paraffin sections were used (selected-SKU IHC image A02857-1); Caveat: Secreted variants may make RNA and protein locations differ (HPA tissue IHC); Isoform / epitope: 3 isoforms; the signal peptide is cleaved, and there is no cytoplasmic domain (UniProt); Regulation: No specific expression inducer is reported (UniProt). Evidence: UniProt Q2MKA7, HPA tissue IHC · Uncertain, HPA ICC-IF · vesicles., Datasheet A02857-1, PMC4496395, PMC7065809. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RSPO1/immunohistochemistry-ihc-guide.