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RBP5 / Retinol-binding protein 5 · IHC design guide
Design Immunohistochemistry for RBP5
Plan RBP5 staining in paraffin sections using hepatocytes and glomerular cells as high-staining reference populations (HPA tissue IHC). The catalog antibody lists an IHC dilution of 1:100–1:300 (datasheet).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RBP5 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) | |
| Staining pattern | Variable cytoplasmic staining across tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Kidney+4 more · see all | |
| Negative control | Smooth muscle |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A30467) | |
| Caveat | Antibody staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Reduced in hepatocellular carcinoma (UniProt) | |
| Isoform / epitope | No annotated isoforms; one chain, residues 1–135 (UniProt) |
Section 1
Recommended RBP5 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet) with the published chromogenic RBP5 IHC procedure for paraffin-embedded keloid and skin tissue (PMC11251391).
| Sample | Paraffin-embedded human lung carcinoma tissue; fixative not specified (datasheet A30467) |
| Fixation | Image fixative and duration unreported (datasheet A30467); 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-RBP5, 1:100 - 1:300 (datasheet A30467) |
| 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 | RBP5-positive staining in cells in glomeruli of kidney (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression at variable levels in all tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval; UniProt P82980: cytoplasm). The published procedure does not specify retrieval conditions (PMC11251391).
Section 2
What Is the Expected RBP5 Staining Pattern?
RBP5 is a cytoplasmic protein with no transmembrane segment (UniProt P82980). In paraffin-section IHC, expect cytoplasmic staining in glomerular cells and hepatocytes, with high staining also reported in selected lymphoid and epithelial cells (HPA tissue IHC). HPA describes variable cytoplasmic expression across tissues; its tissue IHC reliability is Enhanced, with medium consistency between staining and RNA data and external verification pending (HPA tissue IHC).
| Cytoplasmic staining in glomerular cells or hepatocytes, with recognizable tissue structure. | This matches HPA's High staining in kidney glomerular cells and liver hepatocytes (HPA tissue IHC). Score cell type, compartment and intensity together; the presence of brown signal alone does not establish a correct result (general IHC practice). |
| Predominantly nuclear or sharply cell-surface staining, without a convincing cytoplasmic component. | That distribution conflicts with the cytoplasmic UniProt annotation and HPA tissue IHC profile (UniProt P82980; HPA tissue IHC). Check the counterstain and morphology, then compare a known-positive section and detection controls before interpreting it as RBP5 (general IHC practice). |
| Strong staining in smooth muscle cells, especially when expected positive cells lack staining. | HPA reports smooth muscle cells as Not detected (HPA tissue IHC). An unexpected pattern raises concern for nonspecific binding or endogenous detection activity; verify it with a reagent-omission control and the expected positive-cell pattern (general IHC practice). |
| Diffuse color across cells and surrounding tissue, with little distinction between compartments. | An indiscriminate deposit cannot be scored confidently as cytoplasmic RBP5 (UniProt P82980; general IHC practice). Review blocking, washing and chromogen development, and compare the reagent-omission control to assess background from the detection system (general IHC practice). |
| No convincing cytoplasmic staining in glomerular cells or hepatocytes. | These are HPA High cell populations, so a blank result merits a technical check before a biological interpretation (HPA tissue IHC; general IHC practice). Review the catalog antibody's IHC-P instructions and run a documented positive tissue alongside the specimen (general IHC practice). |
💡Expected RBP5 appearanceCall a result positive when distinct cytoplasmic staining is present in glomerular cells or hepatocytes at the reported High level; isolated nuclear, cell-surface or indiscriminate background color is suspect (HPA tissue IHC; UniProt P82980; general IHC practice).
| Target location and topology | RBP5 is annotated in the cytoplasm and has no transmembrane segment (UniProt P82980). Evaluate staining within cells rather than treating a cell-surface outline as the expected target distribution (general IHC practice). |
| Cell-specific tissue pattern | HPA reports High staining in glomerular cells, hepatocytes, germinal center cells, splenic white-pulp cells, keratinocytes and thyroid glandular cells; adipocytes are Medium and smooth muscle cells are Not detected (HPA tissue IHC). Compare like cell types when judging intensity. |
| Tissue IHC evidence strength | The HPA tissue IHC reliability label is Enhanced, while staining and RNA show medium consistency and external verification is pending (HPA tissue IHC). Use the reported pattern as an interpretation reference, and investigate discordant specimens before assigning a biological explanation. |
| Annotated processing and variants | UniProt lists one chain spanning residues 1–135, with no signal peptide, propeptide, annotated isoforms, glycosylation sites or modified residues (UniProt P82980). These annotations give no basis for predicting a secreted or processed staining pattern. |
| IHC versus IF localization | HPA tissue IHC describes variable cytoplasmic expression, whereas HPA ICC-IF places RBP5 mainly at the Golgi apparatus and additionally in vesicles (HPA tissue IHC; HPA subcellular). Interpret each image in its assay context; the ICC-IF observation does not define a paraffin-section protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected positive tissue shows little or no signal. | The result disagrees with HPA's High staining in glomerular cells and hepatocytes; the image alone does not identify the technical cause (HPA tissue IHC; general IHC practice). | Confirm those cell types are present, check the IHC-validated antibody's documented IHC-P conditions, and process a positive section with the specimen (general IHC practice). |
| The slide is uniformly brown or has diffuse haze. | Detection background can obscure a cell-specific cytoplasmic pattern (general IHC practice; HPA tissue IHC). | Inspect a reagent-omission control; review blocking, washes and chromogen development using the detection system's instructions (general IHC practice). |
| Strong nuclear or cell-border signal dominates. | The dominant compartment disagrees with cytoplasmic RBP5 annotation and the HPA tissue IHC pattern (UniProt P82980; HPA tissue IHC). | Check morphology and counterstain, compare a known-positive section, and investigate nonspecific or detection-related staining before scoring (general IHC practice). |
| Smooth muscle stains strongly while the expected positive cells are weak. | HPA reports smooth muscle cells as Not detected and glomerular cells and hepatocytes as High (HPA tissue IHC). | Verify cell identity and examine reagent-omission and positive-tissue controls; treat the discordant pattern as unresolved until the controls support interpretation (general IHC practice). |
| Kidney and liver show different apparent staining strengths. | HPA reports cytoplasmic expression at variable levels across tissues, even though glomerular cells and hepatocytes are both listed as High (HPA tissue IHC). | Compare the specified cell populations and assess staining against background within each section; avoid scoring an entire organ from one field (general IHC practice). |
| Why does an ICC-IF image look more Golgi-centered than the tissue IHC image? | HPA reports mainly Golgi and additional vesicular localization in ICC-IF, while its tissue IHC profile is cytoplasmic (HPA subcellular; HPA tissue IHC). | Record the assay and compartment with each observation, then interpret the paraffin-section result against the tissue IHC pattern (general IHC practice; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing RBP5 staining
A30467 · IHCImmunohistochemistry analysis of paraffin-embedded human lung carcinoma tissue, using CRBP III Antibody. The picture on the right is blocked
A30467 · IFImmunofluorescence analysis of LOVO cells, using CRBP III Antibody. The picture on the right is blocked with the synthesized peptide.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000139194-RBP5/tissue/cerebral+cortex#imid_9806988
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000139194-RBP5/tissue/colon#imid_9794106
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000139194-RBP5/tissue/liver#imid_9794099
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000139194-RBP5/tissue/kidney#imid_9794116
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA038723; green = RBP5, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA038723; green = RBP5, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RBP5 IHC & IF
🧪Run kidney first and look for staining in cells in glomeruli (High; HPA: kidney); run smooth muscle as the negative tissue (Not detected; HPA: smooth muscle). On the kidney slide, neighboring cells outside the HPA-designated positive population should remain at background signal, but do not assume a particular cell type is negative without checking it (HPA: cells in glomeruli).
Positive control tissue: Kidney (Cells in glomeruli, HPA High)
Negative control tissue: Smooth muscle (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RBP5 in A-431, Hep-G2, U2OS, with annotated localisation: Golgi apparatus (approved) (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, and a biological specificity control such as target knockout tissue or peptide competition where available (standard IHC practice; selected-SKU caption: staining blocked with synthesized peptide). For chromogenic kidney IHC, check endogenous peroxidase and, if using biotin-based detection, endogenous biotin before interpreting signal (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and the effect of antigen retrieval are unreported in the supplied evidence; compare retrieval conditions during optimization (supplied target/application evidence; standard IHC practice). The selected A30467 caption shows staining in paraffin-embedded lung carcinoma, but its fixative is unreported (selected-SKU tissue-IHC caption). The evidence does not establish whether frozen sections or IF are easier; kidney background from endogenous peroxidase or biotin warrants control checks for chromogenic detection (supplied target/application evidence; standard IHC practice).
HPA tissue IHC evidence for RBP5
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium 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 |
|---|---|---|---|---|
| Kidney | Cells in glomeruli | High | Protein (IHC) | HPA → |
| Liver | Hepatocytes | High | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | High | Protein (IHC) | HPA → |
| Skin | Keratinocytes | High | Protein (IHC) | HPA → |
| Spleen | Cells in white pulp | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RBP5 IHC Tips
Troubleshoot RBP5 staining in paraffin sections using the selected tissue image, expression references, and compartment controls (catalog caption A30467; HPA; UniProt P82980).
What retrieval should I start with for RBP5 paraffin sections?
Use citrate pH 6.0 heat-induced retrieval at 95–98 °C for 20 min as the starting condition (page retrieval rule). Keep section thickness, heating, cooling, and detection conditions matched across the test and control sections so retrieval is the variable under evaluation (standard IHC practice). Assess staining in hepatocytes or glomerular cells, which have high reported tissue-IHC expression, alongside smooth muscle cells, where staining was not detected (HPA tissue IHC). If signal remains weak, compare a longer citrate incubation on replicate sections while checking morphology and background; this is an optimisation experiment, not an established RBP5 requirement (standard IHC practice).
How should I assess whether fixation is limiting RBP5 staining?
RBP5-specific fixation sensitivity is unknown from the supplied evidence, so treat fixation as an experimental variable rather than an established cause of weak staining (supplied evidence). The selected product image shows staining in paraffin-embedded human lung carcinoma tissue, but its caption does not state the fixative (catalog caption A30467). Record the fixative, fixation duration, tissue thickness, and processing schedule for each specimen before comparing staining intensity (standard IHC practice). If comparable material is available, stain sections processed under different documented fixation conditions in the same run, using identical citrate pH 6.0 retrieval and detection (page retrieval rule; standard IHC practice).
Which RBP5 staining pattern should I accept in tissue sections?
Evaluate RBP5 primarily as an intracellular signal: UniProt assigns it to the cytoplasm and reports no transmembrane segment or signal peptide (UniProt P82980). HPA tissue IHC describes variable cytoplasmic expression across tissues, with high staining in hepatocytes and cells in glomeruli (HPA tissue IHC). HPA cell imaging places RBP5 mainly at the Golgi apparatus and additionally in vesicles, so a punctate cytoplasmic pattern may warrant closer inspection (HPA subcellular). Score diffuse membrane-only or nuclear-only staining cautiously, and compare its distribution with a no-primary control and expected positive cells before calling it specific (UniProt P82980; standard IHC practice).
How can epitope choice explain discordant RBP5 staining?
UniProt lists one RBP5 chain spanning residues 1–135 and no annotated isoforms, signal peptide, propeptide, glycosylation sites, or modified residues (UniProt P82980). Those annotations do not identify the catalog antibody’s epitope or show that it survives every paraffin-processing condition (UniProt P82980; supplied catalog caption). The selected paraffin-section image includes a synthesized-peptide blocking comparison, which provides a product-specific specificity check without establishing the peptide sequence here (catalog caption A30467). If two antibodies disagree, document their stated immunogens where available and compare cell distribution and peptide competition before assigning the difference to an RBP5 isoform (standard IHC practice; UniProt P82980).
How should I check an RBP5 IF pattern against the tissue IHC result?
For a separate IF assay, pair RBP5 with a marker that identifies the expected cells, such as a hepatocyte or glomerular-cell marker appropriate to the specimen (HPA tissue IHC; standard IF practice). Choose fluorophores after inspecting unstained tissue for autofluorescence, and assign the weaker RBP5 signal to a channel with good separation from that background (standard IF practice). Because RBP5 is intracellular and has no transmembrane segment, include permeabilisation and compare its effect on signal and morphology (UniProt P82980; standard IF practice). HPA cell images report Golgi and vesicular localisation; use that observation as a comparison, while keeping tissue IHC and cell IF findings distinct (HPA subcellular; HPA tissue IHC).
What should I adjust if RBP5 staining is widespread or muddy?
First compare a no-primary control with the test section to identify detection-system background, and inspect blank regions for uneven reagent deposition (standard IHC practice). For chromogenic detection, include an endogenous-peroxidase block before the enzyme-based detection step and check whether blocking reduces signal unrelated to primary antibody binding (standard IHC practice). Titrate the primary antibody and detection reagent on adjacent sections while holding citrate pH 6.0 retrieval constant (page retrieval rule; standard IHC practice). Widespread cytoplasmic staining alone is insufficient evidence of artefact because HPA reports variable cytoplasmic expression across tissues; compare cell types and controls before adjusting the threshold (HPA tissue IHC; standard IHC practice).
How should I score RBP5 across specimens with different cell mixtures? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because HPA reports high staining in hepatocytes, glomerular cells, and several other specific cell populations (HPA tissue IHC). Within the defined population, record the percentage of positive cells and an intensity-based H-score, or count positive cells per mm² when density is the study endpoint (standard IHC practice). Normalise counts to the sampled viable tissue area and report the fraction of each relevant cell type so differences in tissue composition remain visible (standard IHC practice). Apply one staining threshold, exposure or scan setting, and exclusion rule to every comparison group, with controls reviewed before scoring (standard IHC practice).
When is an apparent RBP5-positive focus likely to be artefactual?
A credible focus should appear within cells and fit a cytoplasmic pattern, while its cell type should be checked against the tissue context (UniProt P82980; HPA tissue IHC). High staining in hepatocytes or glomerular cells is supported by HPA, whereas smooth muscle cells were reported as not detected; these are comparison patterns, not universal cutoffs (HPA tissue IHC). Treat staining confined to section edges, necrotic regions, or the no-primary control as suspect, and check for residual endogenous-peroxidase signal in chromogenic sections (standard IHC practice). The selected lung carcinoma paraffin image includes peptide blocking, but that result alone does not establish specificity in every tissue or processing condition (catalog caption A30467; standard IHC practice).
Boster reagents
Best RBP5 / Retinol-binding protein 5 IHC Antibodies
A30467 has IHC data from human lung carcinoma paraffin sections (IHC image caption) and IF data from LOVO cells (IF image caption); listed reactivity is human, mouse and rat (catalog: reactivity).
Real IHC data
Anti-CRBP III Antibody
Cat # A30467
A30467 will render with an IHC figure from paraffin-embedded human lung carcinoma tissue, including a peptide-blocked comparison (IHC image caption). Its IF figure shows LOVO cells with a peptide-blocked comparison (IF image caption); the catalog lists IHC, IF and ICC applications and human, mouse and rat reactivity (catalog: applications and reactivity).
Which to pick: Choose A30467 for tissue IHC: its figure shows paraffin-embedded human lung carcinoma tissue (IHC image caption); the fixative is unreported (IHC image caption). Choose A30467 for IF/ICC because both applications are listed, although its pictured IF example is limited to LOVO cells (catalog: applications; IF image caption). For cross-species work, A30467 lists human, mouse and rat reactivity and is polyclonal (catalog: reactivity; dilution_raw: Polyclonal).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P82980 (RET5_HUMAN, Retinol-binding protein 5).
- Human Protein Atlas. RBP5 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. RBP5 subcellular location (ICC-IF): Mainly localized to the Golgi apparatus. In addition localized to vesicles..
- Human Protein Atlas. RBP5 antibody validation summary (1 antibodies).
- Identification of novel immune-related signatures for keloid diagnosis and treatment: insights from integrated bulk RNA-seq and scRNA-seq analysis. Human genomics 2024 — PMC11251391.
- PubMed PMID:11274389 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RBP5 (Retinol-binding protein 5). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A30467 at 1:100 - 1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Kidney (Cells in glomeruli); negative Smooth muscle. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung carcinoma tissue; fixative not specified (datasheet A30467); 2. Fixation: Image fixative and duration unreported (datasheet A30467); 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-RBP5, 1:100 - 1:300 (datasheet A30467); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A30467); Caveat: Antibody staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: No annotated isoforms; one chain, residues 1–135 (UniProt); Regulation: Reduced in hepatocellular carcinoma (UniProt). Evidence: UniProt P82980, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the Golgi apparatus. In addition localized to…, Datasheet A30467, PMC11251391. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RBP5/immunohistochemistry-ihc-guide.