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RHCG / Ammonium transporter Rh type C · IHC design guide
Design Immunohistochemistry for RHCG
Use kidney distal tubules as a high-staining reference and adipose adipocytes as a no-detection comparator (HPA tissue IHC). This guide covers expected renal and squamous staining patterns (HPA tissue IHC) and the catalog antibody’s 1:100 IHC dilution (datasheet).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RHCG 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 | Renal basolateral membrane; squamous membrane and cytoplasm (HPA tissue IHC) | |
| Staining pattern | Distal tubules: basolateral rim; squamous cells: membrane and cytoplasm (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Cervix+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 paraffin fixation consistent (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 A03655) | |
| Caveat | Renal staining is basolateral despite apical annotation (HPA tissue IHC; UniProt) | |
| Regulation | Regulation unreported (UniProt) | |
| Isoform / epitope | No annotated isoforms; epitope side varies across 12 TM spans (UniProt) |
Section 1
Recommended RHCG IHC & IF Protocols
The catalog antibody protocol is followed by three published RHCG immunoperoxidase protocols for mouse brain, rat kidney, and human and rat kidney sections (PMC6106833; PMC4321814; PMC2692438).
| Sample | Paraffin-embedded human kidney tissue; fixative not specified (datasheet A03655) |
| Fixation | Image fixative and duration unreported (datasheet A03655); 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-RHCG, 1:100 (datasheet A03655) |
| 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 | RHCG-positive staining in squamous epithelial cells of cervix (HPA tissue IHC: High). HPA tissue profile: Selective basolateral membrane expression in renal tubules with membranous and cytoplasmic expression in squamous epithelia. No signal in the no-primary control. |
💡Decision noteFor paraffin sections, start with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval rule); adjust using the published pressure cooker conditions (PMC6106833; PMC2692438).
Section 2
What Is the Expected RHCG Staining Pattern?
RHCG is a 12-transmembrane protein found at the cell membrane, including apical and basolateral sites (UniProt Q9UBD6 topology and subcellular location). In tissue IHC, expect selective basolateral staining in renal tubules and membranous plus cytoplasmic staining in squamous epithelia (HPA: tissue IHC profile; Enhanced reliability). Interpret location within the identified cell type; a membrane signal alone does not establish a correct result.
| Distinct basolateral staining in kidney distal tubules (HPA: kidney distal tubules, High; tissue IHC profile). | This matches the observed renal IHC pattern (HPA: selective basolateral membrane expression in renal tubules). UniProt also lists apical membrane, basolateral membrane, and subapical vesicles; use the HPA tissue pattern when judging this paraffin-section result (UniProt Q9UBD6 subcellular location). |
| Membranous staining with some cytoplasmic signal in squamous epithelial cells (HPA: tissue IHC profile). | This can be the expected tissue pattern in cervix, esophagus, oral mucosa, tonsil, or vagina, where HPA reports High staining in squamous epithelial cells (HPA: tissue IHC). Do not reject the cytoplasmic component solely because RHCG is a membrane protein (HPA: tissue IHC profile; UniProt Q9UBD6 subcellular location). |
| Predominantly nuclear staining in an otherwise positive tissue (HPA: tissue IHC profile; UniProt Q9UBD6 subcellular location). | A nuclear pattern is unsupported by the supplied RHCG localisation evidence and raises concern for nonspecific staining (HPA: tissue IHC profile; UniProt Q9UBD6 subcellular location). Review the cell outlines and a no-primary control before scoring it as RHCG (standard IHC practice). |
| Strong signal in adipocytes or adrenal glandular cells (HPA: Not detected in these cells). | These cell types are poor matches for the HPA tissue pattern; cross-reactivity or endogenous chromogenic activity is possible (HPA: tissue IHC; standard IHC practice). Check whether the signal persists without primary antibody, then compare its compartment and morphology with a positive control (standard IHC practice). |
| No signal in kidney distal tubules or a listed squamous epithelium (HPA: High in these cell types). | A negative result in a known-positive control makes the run difficult to interpret (HPA: kidney and squamous epithelia, High; standard IHC practice). Check tissue identity, the IHC-validated antibody, retrieval, and detection controls before calling the study tissue negative (standard IHC practice). |
💡Expected RHCG appearanceCall RHCG positive when renal distal tubules show High, chiefly basolateral membrane staining, or listed squamous epithelia show High membranous and cytoplasmic staining (HPA: tissue IHC); isolated nuclear signal or staining confined to HPA-negative cell types is suspect (HPA: tissue IHC; UniProt Q9UBD6 subcellular location).
| Tissue and cell selection (HPA: tissue IHC). | HPA reports High staining in kidney distal tubules and squamous epithelial cells of cervix, esophagus, oral mucosa, tonsil, and vagina (HPA: tissue IHC). Salivary glandular cells are Low, so they provide a weaker visual benchmark (HPA: tissue IHC). |
| Compartment differs by tissue (HPA: tissue IHC profile). | Kidney staining is described as selective basolateral membrane expression, whereas squamous epithelia show membranous and cytoplasmic expression (HPA: tissue IHC profile). Apply the appropriate compartment expectation to each cell type rather than requiring an identical pattern across tissues. |
| Membrane topology (UniProt Q9UBD6 topology). | RHCG has 12 annotated transmembrane segments, with cytoplasmic and extracellular regions (UniProt Q9UBD6 topology). An antibody's epitope location is not supplied here; topology alone cannot establish which retrieval condition will work in paraffin sections. |
| Glycosylation and processing (UniProt Q9UBD6). | UniProt lists one glycosylation site at residue 48 and one chain spanning residues 1–479, with no signal peptide or propeptide annotated (UniProt Q9UBD6). These annotations do not establish a distinct shed-fragment staining pattern or a fixation effect. |
| IHC validation (HPA: antibody validation). | Two rabbit polyclonal antibodies, HPA041874 and HPA043317, carry Enhanced IHC validation (HPA: antibodies). HPA describes the tissue profile as consistent with RNA expression; this supports the reported IHC pattern but does not validate every staining result in a new run (HPA: Enhanced reliability). |
| IF/ICC evidence (HPA: subcellular and antibody records). | For the IF/ICC question, HPA's subcellular summary says “Membrane,” but lists no ICC-IF image cell lines or main location, and neither listed antibody has an ICC validation entry (HPA: subcellular; HPA: antibodies). Treat an IF/ICC localisation result as provisional; the supplied evidence supports an IHC tissue comparison, not an IF/ICC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| Kidney control shows no distal-tubule signal (HPA: kidney distal tubules, High). | The run may have a tissue-identification, primary-antibody, retrieval, or detection problem; the supplied sources do not identify a RHCG-specific fixation sensitivity (standard IHC practice; HPA: tissue IHC). | Confirm distal tubules on the section, verify that the antibody is IHC-validated, and inspect the run's positive and detection controls (standard IHC practice). Reassess the study tissue only after the expected kidney control pattern appears (HPA: tissue IHC profile). |
| Squamous epithelium looks cytoplasmic as well as membranous (HPA: tissue IHC profile). | That combination is reported for squamous epithelia and need not represent background by itself (HPA: tissue IHC profile). | Check whether signal tracks the squamous epithelial cells and whether a no-primary section stays clear (HPA: squamous epithelial cells, High; standard IHC practice). Judge the result against the squamous pattern, not the kidney basolateral pattern (HPA: tissue IHC profile). |
| Most cells have an even brown haze (standard chromogenic IHC practice). | Diffuse background can obscure the selective cell and compartment pattern; it does not match HPA's described RHCG tissue distribution (HPA: tissue IHC profile). | Inspect a no-primary control, blocking and wash steps, and chromogen development (standard IHC practice). Score RHCG only where identifiable cells show the expected distribution above background (HPA: tissue IHC profile; standard IHC practice). |
| Adipocytes or adrenal glandular cells stain strongly (HPA: Not detected). | A mismatch with HPA-negative cell types raises concern for cross-reactivity or endogenous detection activity; staining alone cannot distinguish them (HPA: tissue IHC; standard IHC practice). | Compare a no-primary section and a known-positive tissue; investigate endogenous enzyme activity if signal persists without primary antibody (standard chromogenic IHC practice). Recheck cell identity and localisation before assigning RHCG positivity (HPA: tissue IHC profile). |
| Staining is mainly nuclear (UniProt Q9UBD6 subcellular location). | Nuclear localisation is absent from the supplied RHCG locations and from the observed HPA tissue profile (UniProt Q9UBD6 subcellular location; HPA: tissue IHC profile). | Do not score nuclear-only staining as the expected RHCG pattern. Inspect the no-primary control and compare membrane and cytoplasmic signal in a known-positive section (standard IHC practice; HPA: tissue IHC profile). |
| An IF/ICC image shows a membrane signal (HPA: subcellular summary, Membrane). | A membrane signal is compatible with the HPA summary, but the supplied HPA record has no ICC-IF image cell lines and no ICC validation entry for either listed antibody (HPA: subcellular; HPA: antibodies). | Use the separate IF/ICC guide for assay design. In this IHC guide, base the interpretation on the validated tissue IHC patterns and do not infer IF/ICC performance from them (HPA: tissue IHC; HPA: antibodies). |
Experiment evidence
IHC & IF images showing RHCG staining
A03655 · IHCImmunohistochemical analysis of paraffin-embedded human-kidney, antibody was diluted at 1:100
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000140519-RHCG/tissue/cerebral+cortex#imid_10561225
HPA · EsophagusHuman Protein Atlas IHC, Esophagus. Source: https://www.proteinatlas.org/ENSG00000140519-RHCG/tissue/esophagus#imid_10561197
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000140519-RHCG/tissue/colon#imid_10559855
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000140519-RHCG/tissue/liver#imid_10559840
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RHCG IHC & IF
🧪Run kidney first: distal tubule cells should stain (HPA: High in kidney distal tubules). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes); cells outside the distal tubules on the kidney slide should show only background staining, serving as an internal comparison (HPA: High in kidney distal tubules).
Positive control tissue: Cervix (Squamous epithelial cells, 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 RHCG; derive a cell-line control from the positive tissue's cell type (Squamous epithelial cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control and a host-species- and immunoglobulin-class-matched isotype control for a monoclonal antibody, or matched nonimmune immunoglobulin for a polyclonal antibody (standard IHC practice). Confirm specificity with RHCG knockout tissue or a peptide block if the immunizing peptide is available, and quench endogenous peroxidase in kidney sections before chromogenic detection (standard IHC practice).
⚠️Feasibility: The A03655 paraffin kidney caption reports a 1:100 dilution but no fixative; a target-specific fixation window or fixation effect is unreported (A03655 caption: fixative not stated). Retrieval dependence is unreported, so optimise antigen retrieval empirically for paraffin sections (A03655 caption: no retrieval condition). The supplied evidence does not establish whether frozen sections or IF are easier; renal endogenous peroxidase can contribute chromogenic background (HPA: High in kidney distal tubules; standard IHC practice).
HPA tissue IHC evidence for RHCG
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Cervix | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Kidney | Distal tubules | High | Protein (IHC) | HPA → |
| Oral mucosa | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Skin | Arrector pili muscle cells | Low | 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 RHCG IHC Tips
Troubleshoot RHCG staining in paraffin sections by checking tubular membrane localisation, tissue controls, and the limits of the antibody’s documented IHC evidence.
How should I optimise retrieval when RHCG 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). Use the A03655 human-kidney section at the captioned 1:100 dilution as a staining reference; its fixative was not reported (caption A03655). Judge retrieval by preserved tubular morphology and a distinct membrane pattern, since RHCG has 12 transmembrane segments and renal tubular staining is expected (UniProt Q9UBD6 topology; HPA tissue IHC). If signal remains weak, evaluate a stronger retrieval condition as a fallback while keeping detection and section handling constant (standard IHC practice). Reject conditions that lift tissue or erase membrane detail (standard IHC practice).
Could fixation explain weak or diffuse RHCG staining?
Target-specific sensitivity of RHCG staining to fixation is unknown from the supplied evidence; the A03655 paraffin-section caption does not name a fixative (caption A03655). Record the fixative, fixation duration, processing history, and section age for each specimen before comparing staining across cases (standard IHC practice). If specimens were processed differently, stain representative sections together with the same citrate pH 6.0, 95–98 °C, 20 min retrieval and identical detection steps (page retrieval setting; standard IHC practice). Assess epithelial morphology alongside membrane contrast, and use a consistently processed positive-control section to detect run variation (standard IHC practice). Do not assign a fixation mechanism from staining pattern alone (standard IHC practice).
What RHCG pattern should count as convincing tissue staining?
Look first for tubular membrane staining in kidney, especially distal tubules, where HPA reports high expression and a selective basolateral pattern (HPA tissue IHC). Apical membrane, basolateral membrane, and subapical vesicle localisation are all described for RHCG, so document the compartment actually resolved by the assay (UniProt Q9UBD6 subcellular). Squamous epithelia may show membranous and cytoplasmic staining, including high expression in cervix and esophagus (HPA tissue IHC). Compare stained structures with a counterstained adjacent section and a no-primary control before calling faint cytoplasm positive (standard IHC practice). Avoid treating a uniformly brown field as evidence of a specific membrane transporter (standard IHC practice).
How can epitope position change the appearance of RHCG staining?
Check the catalog antibody’s stated immunogen or epitope before interpreting an unusual pattern; the supplied A03655 caption gives no epitope position (caption A03655). RHCG has 12 transmembrane segments, an extracellular glycosylation site at residue 48, and a cytoplasmic C-terminal region at residues 416–479 (UniProt Q9UBD6 topology and glycosylation). Those features make epitope accessibility a practical variable in fixed sections, but they do not establish the antibody’s binding site (UniProt Q9UBD6 topology; standard IHC practice). No isoforms are annotated in the supplied record, so do not explain discrepant staining by a specific RHCG isoform without independent evidence (UniProt Q9UBD6 isoform record). Compare controlled retrieval conditions before assigning a biological explanation (standard IHC practice).
How should I investigate RHCG localisation by multiplex IF?
For a separate IF experiment, pair RHCG with a validated marker of the expected distal tubular cell population in kidney, using HPA’s distal-tubule expression as the tissue reference (HPA tissue IHC; standard IF practice). Choose a fluorophore channel with low tissue autofluorescence and inspect unstained and single-label sections before interpreting overlap (standard IF practice). Set permeabilisation according to the antibody’s documented epitope: a cytoplasmic epitope requires access across the plasma membrane, whereas extracellular access may not (UniProt Q9UBD6 topology; standard IF practice). The supplied A03655 evidence documents paraffin-section IHC at 1:100, not an IF dilution or fixation condition, so optimise IF independently (caption A03655).
What should I check when DAB obscures RHCG membrane staining?
Inspect a no-primary control and the tissue margins to distinguish reagent background or edge staining from a structured tubular membrane signal (standard IHC practice). Block endogenous peroxidase before HRP and DAB detection, and check whether precipitate or excessive development is obscuring cell boundaries (standard IHC practice). Titrate the catalog antibody around its documented paraffin-section use at 1:100 without treating that single caption as a universal optimum (caption A03655; standard IHC practice). Compare signal in kidney distal tubules with compartments reported as not detected, such as adipocytes in adipose tissue, while accounting for tissue-specific background (HPA tissue IHC; standard IHC practice). Score only interpretable, well-preserved regions (standard IHC practice).
How should I quantify RHCG staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the analysis compartment before scoring: distal tubular epithelium in kidney or squamous epithelial cells in the relevant tissue (HPA tissue IHC). Report the percentage of positive target cells and an H-score for membrane staining, with cytoplasmic staining scored separately when present (HPA tissue IHC; standard IHC practice). Normalise counts or stained area to the number of eligible epithelial cells or epithelial area, rather than total section area, and record sampling rules (standard IHC practice). Keep retrieval at citrate pH 6.0, 95–98 °C, 20 min and detection settings consistent across the comparison (page retrieval setting; standard IHC practice). Exclude damaged and folded regions before analysis (standard IHC practice).
When is apparent RHCG positivity more likely to be artefact?
Give greatest weight to staining in expected cells and compartments: kidney distal tubules with a selective basolateral pattern, or squamous epithelium with membranous and cytoplasmic signal (HPA tissue IHC). A diffuse signal in unexpected cells, isolated section edges, folds, or necrotic areas needs review against morphology and a no-primary control (standard IHC practice). If DAB remains in the no-primary control, investigate endogenous enzyme activity or detection background before attributing it to RHCG (standard IHC practice). UniProt also describes apical membrane and subapical vesicles, so compartment differences alone require careful resolution and context rather than an automatic artefact call (UniProt Q9UBD6 subcellular). Confirm disputed patterns with an independently validated approach (standard IHC practice).
Boster reagents
Best RHCG / Ammonium transporter Rh type C IHC Antibodies
A03655 has an IHC image from paraffin-embedded human kidney (IHC image caption). IF is listed, but no IF image is supplied (catalog: applications; IF image alts).
Real IHC data
Anti-Ammonium transporter Rh type C RhCG Antibody
Cat # A03655
A03655 is listed for IHC and IF, with Human, Mouse, and Rat reactivity (catalog: applications; reactivity). Its IHC image shows paraffin-embedded human kidney at 1:100; no IF image is supplied (IHC image caption; IF image alts).
Which to pick: For tissue IHC, choose A03655 based on its paraffin-embedded human kidney image at 1:100; the fixative is unreported (IHC image caption). For IF/ICC, A03655 lists IF at 1:50, but supplies no IF image or ICC application claim; its host is Rabbit and clonality is unreported (catalog: applications, IF dilution, IF image alts, host, clone). For cross-species work, A03655 lists Human, Mouse, and Rat reactivity, while its pictured IHC sample is human kidney only (catalog: reactivity; IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9UBD6 (RHCG_HUMAN, Ammonium transporter Rh type C).
- Human Protein Atlas. RHCG tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. RHCG subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. RHCG antibody validation summary (2 antibodies).
- Effect of tumor necrosis factor-α on the expression of the ammonia transporter Rhcg in the brain in mice with acute liver failure. Journal of neuroinflammation 2018 — PMC6106833.
- Expression of ammonia transporters, Rhbg and Rhcg, in chronic cyclosporine nephropathy in rats. Nephron. Experimental nephrology 2008 — PMC4321814.
- Regulation of Rhcg, an ammonia transporter, by aldosterone in the kidney. The Journal of endocrinology 2021 — PMC9428946.
- RhCG is the major putative ammonia transporter expressed in the human kidney, and RhBG is not expressed at detectable levels. American journal of physiology. Renal physiology 2009 — PMC2692438.
- PubMed PMID:10852913 — UniProt-cited evidence.
- PubMed PMID:12204676 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RHCG (Ammonium transporter Rh type C). Expected localisation Renal basolateral membrane; squamous membrane and cytoplasm (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A03655 at 1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Cervix (Squamous epithelial cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human kidney tissue; fixative not specified (datasheet A03655); 2. Fixation: Image fixative and duration unreported (datasheet A03655); 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-RHCG, 1:100 (datasheet A03655); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep paraffin fixation consistent (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 A03655); Caveat: Renal staining is basolateral despite apical annotation (HPA tissue IHC; UniProt); Isoform / epitope: No annotated isoforms; epitope side varies across 12 TM spans (UniProt); Regulation: Regulation unreported (UniProt). Evidence: UniProt Q9UBD6, HPA tissue IHC · Enhanced, HPA ICC-IF · Membrane, Datasheet A03655, PMC6106833, PMC4321814. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RHCG/immunohistochemistry-ihc-guide.