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RCN2 / Reticulocalbin-2 · IHC design guide
Design Immunohistochemistry for RCN2
Plan chromogenic IHC on paraffin sections with the catalog antibody at a 1:50–1:200 starting dilution (datasheet). Compare cell type and staining intensity with the cytoplasmic tissue profile (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RCN2 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 tissue staining (HPA tissue IHC); ER lumen (UniProt) | |
| Staining pattern | Cytoplasmic signal in neurons, placenta and epididymis (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Caudate+4 more · see all | |
| 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); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A10286) | |
| Caveat | Some cell types show little or no detectable staining (HPA tissue IHC) | |
| Regulation | Tissue enhanced RNA in epididymis (HPA tissue RNA) | |
| Isoform / epitope | 2 isoforms; signal peptide cleaved, no TM split; epitope unknown (UniProt) |
Section 1
Recommended RCN2 IHC & IF Protocols
The catalog antibody protocol is paired with published RCN2 IHC methods for arterial tissue (PMC8997427) and mouse xenograft tumors (PMC10356900).
| Sample | Paraffin-embedded human gastric tissue; fixative not specified (datasheet A10286) |
| Fixation | Image fixative and duration unreported (datasheet A10286); 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-RCN2, 1:50-1:200 (datasheet A10286) |
| 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 | RCN2-positive staining in neuronal cells of caudate (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in selected tissues including neurons, seminiferous duct cells, placenta and epididymis. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval), then assess staining in the expected ER distribution (UniProt Q14257).
Section 2
What Is the Expected RCN2 Staining Pattern?
RCN2 is a soluble endoplasmic reticulum lumen protein with no transmembrane segment (UniProt Q14257 topology). In paraffin-section IHC, expect predominantly cytoplasmic staining in selected cells, especially epididymal glandular cells, neurons and pachytene spermatocytes (HPA tissue IHC: High). HPA rates the tissue pattern Enhanced because staining agrees with RNA expression, while noting that external verification is pending (HPA tissue IHC: reliability).
| Clear cytoplasmic staining in epididymal glandular cells, with little staining in adjacent negative cells. | This fits a high-expression tissue and cell population (HPA tissue IHC: Epididymis, glandular cells, High). Chromogenic IHC cannot by itself resolve the endoplasmic reticulum, so score the cell and broad compartment rather than requiring a visible reticular network (UniProt Q14257: ER lumen; general IHC practice). |
| Cytoplasmic staining in neuronal cells or pachytene spermatocytes. | This agrees with reported High staining in caudate, cerebral cortex and hippocampal neurons, and in testicular pachytene spermatocytes (HPA tissue IHC). Compare the named cell population, not overall tissue darkness; surrounding cells can have different reported levels (HPA tissue IHC: cell-specific annotations). |
| Predominantly nuclear staining or a crisp cell-surface rim without convincing cytoplasmic signal. | Treat an isolated nuclear or surface pattern as suspect: the annotated location is the ER lumen, and RCN2 has no transmembrane segment (UniProt Q14257 topology; HPA subcellular: ER). Review morphology and controls before calling it positive. A cytoplasm/membrane annotation for cerebellar granular cells does not establish exclusive cell-surface localization (HPA tissue IHC). |
| Strong stain in a cell population reported as Not detected, especially with staining in blank areas. | Adipocytes in adipose tissue and bronchial basal cells are reported Not detected (HPA tissue IHC). Such a result calls for checking nonspecific binding and, with chromogenic detection, endogenous detection activity using appropriate controls (general IHC practice). A negative HPA annotation is a comparison point, not proof that every specimen must be unstained. |
| Diffuse staining across many structures, or no stain in a known-positive control section. | Diffuse signal without cell boundaries is hard to assign to the HPA cell-specific pattern (HPA tissue IHC). Absent signal in epididymal glandular cells or a listed high-staining neuronal population fails the expected positive-control check (HPA tissue IHC: High). Investigate the assay and section before interpreting the experimental sample as RCN2 negative (general IHC practice). |
💡Expected RCN2 appearanceCall a positive result when predominantly cytoplasmic stain is readily visible in a reported High population such as epididymal glandular cells or neurons (HPA tissue IHC), consistent with ER localization (UniProt Q14257; HPA subcellular); isolated nuclear, cell-surface-only or diffuse tissue-wide stain is suspect (general IHC interpretation).
| Cell population and tissue selection | RCN2 is described as ubiquitous at the tissue level (UniProt Q14257), while HPA reports High staining in selected cell populations and Not detected staining in others (HPA tissue IHC). Select and score a named cell population; the word “ubiquitous” does not predict uniform chromogenic intensity across a section. |
| Compartment and optical resolution | The mature protein is annotated in the ER lumen, with no transmembrane segment (UniProt Q14257 topology). HPA reports mainly ER localization by ICC-IF (HPA subcellular). In paraffin-section IHC, a cytoplasmic pattern can be consistent with that location, but chromogenic staining alone does not prove ER colocalization (general IHC practice). |
| Antibody evidence | HPA030694 and HPA030695 each have Enhanced IHC validation (HPA antibodies). The tissue profile also has Enhanced reliability because staining is consistent with RNA data, with external verification pending (HPA tissue IHC: reliability). These ratings support comparison with the reported pattern; they do not establish that every specimen or assay condition will reproduce it. |
| Protein processing and isoforms | RCN2 has a signal peptide at residues 1–22, a mature chain at 23–317 and two listed isoforms (UniProt Q14257). The supplied sources give no antibody epitope or isoform-specific staining result, so they cannot justify assigning a changed IHC pattern to signal-peptide processing or one isoform. |
| Fixation and antigen retrieval | Target-specific fixation sensitivity and retrieval conditions are unreported in the supplied UniProt and HPA records. If staining fails, use the IHC-validated antibody’s documented paraffin-section procedure and its controls when available (general IHC practice); do not infer an RCN2-specific retrieval requirement from the ER annotation or tissue staining levels. |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in epididymal glandular cells on the positive-control section. | The section, detection sequence or antibody conditions may have failed; these cells are reported High (HPA tissue IHC). The supplied sources do not identify an RCN2-specific fixation or retrieval failure. | Verify the control section and IHC workflow, including retrieval, antibody incubation and chromogenic detection, against the IHC-validated antibody’s documented procedure (general IHC practice). |
| Staining appears mainly nuclear or confined to the outer cell border. | That distribution conflicts with the ER-lumen annotation and absence of a transmembrane segment (UniProt Q14257 topology; HPA subcellular). Morphology or nonspecific signal could mislead compartment assignment (general IHC practice). | Recheck focus and cellular boundaries, compare a known-positive population, and examine a no-primary control before scoring RCN2 (general IHC practice). |
| Adipocytes or bronchial basal cells stain as strongly as the intended positive cells. | Both populations are reported Not detected (HPA tissue IHC). Nonspecific antibody binding or endogenous chromogenic activity is possible (general IHC practice); the HPA comparison alone cannot identify which mechanism applies. | Check the no-primary and detection controls, then assess blocking and wash conditions using standard IHC practice. Score the intended positive and negative cell populations separately (general IHC practice). |
| Brown precipitate or diffuse color obscures cell boundaries. | Excess background or endogenous detection activity may make a cell-specific pattern unreadable (general IHC practice). HPA describes cytoplasmic expression in selected cells, not a uniform tissue-wide signal (HPA tissue IHC). | Inspect a no-primary control, review chromogen development and washes, and repeat under the documented IHC procedure if needed (general IHC practice). |
| Neuronal or testicular signal is present, but neighboring cells vary in intensity. | HPA assigns High staining to specified neuronal populations and pachytene spermatocytes, not every cell in those tissues (HPA tissue IHC). Heterogeneity alone is therefore insufficient to call assay failure. | Identify the annotated cells by morphology and score intensity within those cells; compare the same cell type across sections and include a suitable control (general IHC practice). |
| IF/ICC question: should a paraffin-section IHC pattern look reticular? | ICC-IF reports mainly ER localization and Enhanced ER assignment (HPA subcellular), whereas paraffin-section chromogenic IHC has different spatial resolution (general IHC practice). | For this IHC guide, assess cytoplasmic staining in the HPA-reported cell populations (HPA tissue IHC). Use the separate IF/ICC guide for fluorescence localization; do not treat a missing visible ER network in chromogenic IHC as failure (general IHC practice). |
Experiment evidence
IHC & IF images showing RCN2 staining
A10286 · IHCImmunohistochemistry of paraffin-embedded human gastric using RCN2 antibody at dilution of 1:100 (x40 lens).
A10286 · IHCImmunohistochemistry of paraffin-embedded rat heart using RCN2 antibody at dilution of 1:100 (x40 lens).
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000117906-RCN2/tissue/cerebral+cortex#imid_8438924
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000117906-RCN2/tissue/colon#imid_8438842
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000117906-RCN2/tissue/liver#imid_8438851
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000117906-RCN2/tissue/kidney#imid_8438852
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA030694; green = RCN2, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA030694; green = RCN2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RCN2 IHC & IF
🧪Run cerebellum first: granular cells should show cytoplasmic or membranous staining (HPA: High in cerebellar granular cells). Use adipose tissue as the negative tissue because adipocytes are listed as not detected (HPA: Not detected in adipocytes); on the cerebellar slide, nuclei and acellular spaces should remain free of specific staining (UniProt Q14257: ER lumen; standard IHC practice).
Positive control tissue: Caudate (Neuronal cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RCN2 in A-431, U-251MG, U2OS, with annotated localisation: Endoplasmic reticulum (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; an isotype control matched to the primary antibody’s host species and immunoglobulin class, or nonimmune host IgG for a polyclonal primary; and RCN2 knockout material or a peptide-block control if available (standard IHC practice). Quench endogenous peroxidase for chromogenic IHC, and account for neuronal lipofuscin autofluorescence when interpreting IF in cerebellum (standard IHC/IF practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence; the selected A10286 paraffin-section caption gives a 1:100 dilution for human gastric tissue but leaves the fixative unreported (A10286 tissue-IHC caption). Antigen-retrieval dependence is likewise unreported, so compare retrieved and unretrieved sections during IHC setup (standard IHC practice). IF/ICC has supporting ER-localization images in A-431, U-251MG and U2OS cells, but the supplied evidence does not establish that frozen sections or IF are easier; in cerebellum, small granular-cell cytoplasm and neuronal autofluorescence can complicate interpretation (HPA: ICC-IF images and cerebellar granular-cell staining; standard IHC/IF practice).
HPA tissue IHC evidence for RCN2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data. 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 |
|---|---|---|---|---|
| Caudate | Neuronal cells | High | Protein (IHC) | HPA → |
| Cerebellum | Granular cells - cytoplasm/membrane | High | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Basal cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RCN2 IHC Tips
Use RCN2’s expected endoplasmic reticulum pattern and cell type controls to troubleshoot chromogenic staining in paraffin sections (UniProt Q14257; HPA tissue IHC).
How should I optimise retrieval when RCN2 staining is weak in paraffin sections?
Start with citrate buffer at pH 6.0 for heat induced retrieval at 95–98 °C for 20 minutes (page retrieval rule). Run the same control tissue through each retrieval condition, then compare signal in the expected cells and nonspecific staining in adjacent cells (standard IHC practice; HPA tissue IHC). If staining remains weak, test an EDTA buffer at pH 9 as a fallback while keeping detection and imaging conditions constant (standard IHC practice). Judge improvement by a cellular cytoplasmic pattern compatible with RCN2’s endoplasmic reticulum location, rather than by overall chromogen intensity alone (UniProt Q14257; HPA subcellular).
Could fixation explain weak or uneven RCN2 staining?
RCN2 specific sensitivity to fixative type or fixation duration is unknown from the supplied evidence (A10286 caption: fixative unreported). The catalog image documents paraffin embedded human gastric tissue at 1:100, but its caption does not identify the fixative (A10286 caption). For a controlled comparison, process matched specimens with recorded fixation conditions and hold retrieval, antibody dilution, and detection constant (standard IHC practice). Review sections for uneven preservation, tissue folds, and loss of cellular detail before attributing patchy chromogen to RCN2 biology (standard IHC practice). Interpret any fixation effect as an observation from that comparison, rather than as an established property of RCN2 (A10286 caption: fixative unreported).
What staining location should count as plausible RCN2 signal?
RCN2 is annotated in the endoplasmic reticulum lumen, with no transmembrane segment; a cytoplasmic distribution is therefore plausible in chromogenic sections (UniProt Q14257 topology). HPA also describes mainly endoplasmic reticulum localisation and cytoplasmic tissue staining, providing independent context for pattern review (HPA subcellular; HPA tissue IHC). Assess staining within intact cell boundaries and compare its distribution with the relevant tissue compartments under the same counterstain (standard IHC practice). A predominantly nuclear precipitate or signal confined to section edges warrants investigation before it is scored as RCN2 (UniProt Q14257; standard IHC practice). Brightness alone cannot establish organelle identity at routine brightfield resolution (standard IHC practice).
Can epitope placement change how I interpret RCN2 staining?
RCN2 has 2 annotated isoforms and a precursor signal peptide at residues 1–22; the annotated mature chain spans 23–317 (UniProt Q14257). Its 6 EF hand regions occupy much of that chain, and threonine 137 is annotated as modified (UniProt Q14257). Check the antibody’s documented immunogen or epitope before claiming that tissue staining distinguishes isoforms or detects a modification (standard IHC practice; UniProt Q14257). Without such mapping, score the observed cellular staining as antibody reactivity consistent with RCN2, subject to controls (standard IHC practice). Compare any discrepant specimens using the same retrieval and detection conditions before assigning a biological explanation (standard IHC practice).
How can IF help resolve an ambiguous RCN2 IHC pattern?
Use IF as an orthogonal localisation check for ambiguous chromogenic staining, while treating it as a separate assay requiring its own controls (standard IF practice). Multiplex RCN2 with a marker for the expected cell type in the specimen, then assess whether the signal falls within those cells (HPA tissue IHC; standard IF practice). Choose fluorophores and filter sets after inspecting unstained tissue autofluorescence, and include single stain controls to assess channel bleed through (standard IF practice). Because RCN2 is in the endoplasmic reticulum lumen, establish permeabilisation that permits antibody access across cellular and organelle membranes without destroying morphology (UniProt Q14257 topology; standard IF practice). Compare the resulting intracellular pattern with HPA’s endoplasmic reticulum localisation (HPA subcellular).
How do I separate RCN2 staining from chromogenic background?
Inspect a no primary antibody control for signal arising from the detection system, and use an appropriate peroxidase block before DAB development (standard IHC practice). Titrate the catalog antibody around the documented 1:100 image condition, comparing specific cellular signal with staining in expected low signal areas (A10286 caption; standard IHC practice). HPA reports low staining in stomach glandular cells, so the gastric catalog image alone does not establish a strong positive threshold (HPA tissue IHC; A10286 caption). Check whether diffuse precipitate, section edges, or damaged areas stain more strongly than intact cells (standard IHC practice). Record exposure to chromogen consistently across comparison slides so background changes can be assessed fairly (standard IHC practice).
How should I quantify RCN2 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and scoring region before reviewing group labels, then score only intact cells with interpretable cytoplasmic staining (standard IHC practice; UniProt Q14257). An H score combines staining intensity and percentage of positive cells on a 0–300 scale; report percentage positive separately when it aids interpretation (standard IHC practice). For dispersed positive cells, positive cell density per mm² may be more informative, provided the sampled area is specified (standard IHC practice). Normalise cell based measures to the counted cells of the relevant type and area based measures to analysable tissue area (standard IHC practice). Keep retrieval, detection, counterstain, and scoring thresholds consistent across specimens (standard IHC practice).
Which findings support genuine RCN2 staining rather than artefact?
Prioritise staining in intact cells with a cytoplasmic pattern compatible with endoplasmic reticulum localisation (UniProt Q14257; HPA subcellular). HPA reports high staining in epididymal glandular cells and selected neuronal populations, while stomach glandular cells are listed as low; use cell type as context, not proof (HPA tissue IHC). Treat predominantly nuclear signal, staining restricted to tissue edges, and chromogen concentrated in necrotic areas as reasons to review the preparation (UniProt Q14257; standard IHC practice). Compare with a no primary control to identify endogenous enzyme or detection related signal, and review morphology alongside the counterstain (standard IHC practice). HPA’s enhanced tissue reliability remains pending external verification (HPA tissue IHC).
Boster reagents
Best RCN2 / Reticulocalbin-2 IHC Antibodies
A10286 has IHC images of paraffin-embedded human gastric tissue and rat heart (A10286 image captions). Its listed reactivity covers human, mouse, and rat (A10286 catalog).
Real IHC data
Anti-Reticulocalbin-2 RCN2 Antibody
Cat # A10286
A10286 lists IHC among its applications and human, mouse, and rat reactivity (A10286 catalog). Its IHC images show paraffin-embedded human gastric tissue and rat heart at 1:100 (A10286 image captions).
Which to pick: For tissue IHC, choose A10286 for paraffin sections; the image captions do not report a fixative (A10286 image captions). No listed SKU has IF/ICC application or image evidence, so there is no supported IF/ICC pick here (A10286 catalog). For cross-species IHC, A10286 lists human, mouse, and rat reactivity, while its IHC images cover human and rat; clonality is unreported (A10286 catalog; A10286 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q14257 (RCN2_HUMAN, Reticulocalbin-2).
- Human Protein Atlas. RCN2 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. RCN2 subcellular location (ICC-IF): Mainly localized to the endoplasmic reticulum..
- Human Protein Atlas. RCN2 antibody validation summary (2 antibodies).
- Reticulocalbin 2 as a Potential Biomarker and Therapeutic Target for Atherosclerosis. Cells 2022 — PMC8997427.
- Expression signature and prognostic value of CREC gene family in human colorectal cancer. BMC cancer 2023 — PMC10506189.
- RCN2 promotes Nasopharyngeal carcinoma progression by curbing Calcium flow and Mitochondrial apoptosis. Cellular oncology (Dordrecht, Netherlands) 2023 — PMC10356900.
- PubMed PMID:8034671 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:16572171 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RCN2 (Reticulocalbin-2). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC); ER lumen (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A10286 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Caudate (Neuronal cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human gastric tissue; fixative not specified (datasheet A10286); 2. Fixation: Image fixative and duration unreported (datasheet A10286); 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-RCN2, 1:50-1:200 (datasheet A10286); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A10286); Caveat: Some cell types show little or no detectable staining (HPA tissue IHC); Isoform / epitope: 2 isoforms; signal peptide cleaved, no TM split; epitope unknown (UniProt); Regulation: Tissue enhanced RNA in epididymis (HPA tissue RNA). Evidence: UniProt Q14257, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the endoplasmic reticulum., Datasheet A10286, PMC8997427, PMC10356900. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RCN2/immunohistochemistry-ihc-guide.