This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
HCAR2 / Hydroxycarboxylic acid receptor 2 · IHC design guide
Design Immunohistochemistry for HCAR2
Plan HCAR2 IHC in paraffin sections using the reported cytoplasmic staining in immune cells as a tissue reference (HPA tissue IHC). Compare that pattern with HCAR2’s annotated cell membrane location (UniProt) and account for the antibody’s potential to detect proteins from more than one gene (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HCAR2 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); cell membrane location (UniProt) | |
| Staining pattern | Cytoplasmic staining in several tissues, including immune cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, heat-mediated (datasheet A02511) | |
| Positive control | Bone marrow+4 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
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 A02511) | |
| Caveat | Antibody may detect proteins from more than one gene (HPA tissue IHC) | |
| Regulation | Expression depends on neutrophil maturity (UniProt) | |
| Isoform / epitope | No isoforms annotated; check extracellular vs cytoplasmic epitope (UniProt) |
Section 1
Recommended HCAR2 IHC & IF Protocols
The catalog antibody uses citrate pH 6.0 retrieval (datasheet A02511). One published HCAR2 IHC protocol stains paraffin sections (PMC11561673).
| Sample | Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A02511) |
| Fixation | Image fixative and duration unreported (datasheet A02511); 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 6.0 (datasheet A02511); 20 min, 95–100 °C (standard) |
| 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-HCAR2, 1:50-1:200 (datasheet A02511) |
| 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 | HCAR2-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in several tissues, including immune cells. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 heat retrieval (datasheet A02511); the published protocol also uses heated citrate but does not state its pH (PMC11561673).
Section 2
What Is the Expected HCAR2 Staining Pattern?
HCAR2 is a seven-pass cell-membrane receptor (UniProt Q8TDS4: topology and subcellular location). In paraffin-section IHC, assess staining in reported immune-cell populations, including splenic red-pulp cells and bone-marrow hematopoietic cells (HPA: Medium staining). HPA also describes cytoplasmic staining across several tissues; its IHC profile is Approved with medium consistency against RNA data and cautions that the antibody targets proteins from more than one gene (HPA: tissue IHC).
| Distinct staining in splenic red-pulp cells or bone-marrow hematopoietic cells. | These are plausible positive populations: HPA reports Medium IHC staining in both (HPA: tissue IHC). Evaluate the stained cells and their boundaries against nearby unstained cells. A membrane-associated component fits HCAR2's seven-pass topology (UniProt Q8TDS4: topology); cytoplasmic staining also appears in HPA's tissue profile (HPA: tissue IHC). |
| Predominantly nuclear staining, especially without staining at cell boundaries. | A nuclear pattern conflicts with the annotated cell-membrane location and seven transmembrane segments (UniProt Q8TDS4: subcellular location and topology). Treat it as suspect and check the counterstain, detection controls and antibody specificity before scoring it as HCAR2 (general IHC practice). |
| Strong staining in cells reported as unstained, such as adipocytes. | HPA reports adipocytes as Not detected by tissue IHC, despite UniProt's statement that expression is largely restricted to adipose tissue and spleen (HPA: tissue IHC; UniProt Q8TDS4: tissue specificity). Investigate cell identification, cross-reactivity and endogenous detection activity; this discrepancy prevents a simple tissue-wide positive or negative call. |
| Diffuse color across cells and surrounding tissue, with little distinction between structures. | That distribution is difficult to assign to the reported cell populations or receptor compartment (HPA: tissue IHC; UniProt Q8TDS4: subcellular location). Background from detection reagents or insufficient blocking is possible; compare a no-primary control and review washes and detection conditions (general IHC practice). |
| No visible signal in spleen red-pulp cells or bone-marrow hematopoietic cells. | These populations show Medium staining in HPA, so a blank result warrants a workflow check (HPA: tissue IHC). Confirm that the tissue and expected cells are present, then review primary-antibody conditions, antigen retrieval and detection with appropriate controls (general IHC practice). Medium staining is an expectation, not a guarantee for every section. |
💡Expected HCAR2 appearanceA credible IHC positive shows discernible, approximately Medium staining in reported populations such as splenic red-pulp cells, with membrane association compatible with HCAR2 and possible cytoplasmic signal (HPA: tissue IHC; UniProt Q8TDS4: topology); isolated nuclear color or uniform background is suspect (general IHC interpretation).
| Membrane annotation versus observed tissue pattern. | UniProt places HCAR2 at the cell membrane and annotates seven membrane-spanning segments (UniProt Q8TDS4: subcellular location and topology). HPA describes cytoplasmic tissue staining (HPA: tissue IHC). Record the compartment actually seen rather than rejecting every cytoplasmic signal solely on topology. |
| Antibody-validation limit. | The listed rabbit polyclonal antibody, HPA028660, has Approved IHC status, while HPA flags medium agreement with RNA data and targeting of proteins from more than one gene (HPA: antibody validation and tissue IHC). A matching location and tissue pattern support interpretation but do not establish exclusive HCAR2 detection. |
| Choice of comparison tissue and cell population. | HPA reports Medium staining in splenic red-pulp, bone-marrow hematopoietic, lymph-node non-germinal-center, tonsillar germinal-center, seminal-vesicle glandular and bladder urothelial cells (HPA: tissue IHC). It reports adipocytes and several other sampled populations as Not detected (HPA: tissue IHC). Compare identified cells, since a whole-section label can hide the relevant population. |
| Epitope and preparation information. | UniProt identifies extracellular and cytoplasmic regions but supplies no antibody epitope in this record (UniProt Q8TDS4: topology). The supplied sources do not establish HCAR2-specific retrieval requirements or fixation sensitivity; choose and document retrieval and detection conditions as general IHC workflow decisions. |
| Situation | Likely cause | Next action |
|---|---|---|
| Spleen or bone-marrow section is blank. | Expected cells may be absent from the sampled field, or the IHC workflow may have failed; HPA reports Medium staining only in specified populations (HPA: tissue IHC). | Locate red-pulp or hematopoietic cells on the counterstain, then check retrieval, primary-antibody conditions and detection using a suitable control section (general IHC practice). |
| Adipose section is blank. | HPA reports adipocytes as Not detected by IHC, although UniProt describes adipose-tissue expression (HPA: tissue IHC; UniProt Q8TDS4: tissue specificity). | Do not use an adipocyte-negative slide alone to declare assay failure. Examine an HPA-reported Medium-staining population and document the adipose discrepancy when interpreting results. |
| Color appears mainly in nuclei. | Nuclear localization does not match the annotated cell-membrane receptor (UniProt Q8TDS4: subcellular location and topology). | Compare the counterstain and no-primary control; reassess detection background and specificity before counting nuclear signal as positive (general IHC practice). |
| Many unrelated cell types stain strongly. | HPA cautions that its antibody targets proteins from more than one gene (HPA: tissue IHC); nonspecific or endogenous detection signal is also possible (general IHC practice). | Map staining by cell type against HPA's reported pattern, run no-primary and detection controls, and report unexpected populations separately from supported positives. |
| Diffuse chromogen obscures cell boundaries. | Background can arise from blocking, washing or detection conditions (general IHC practice); such color cannot reliably establish the annotated membrane location (UniProt Q8TDS4: subcellular location). | Inspect a no-primary control, review blocking and washes, and score only distinguishable cellular staining after background is resolved (general IHC practice). |
| Does an ICC-IF junction signal establish the expected paraffin IHC pattern? | HPA reports supported cell-junction localization from ICC-IF images in RT-4, with the same multi-gene-targeting caution (HPA: subcellular and antibody validation). That observation uses a different application. | Use the junction result as context for membrane-associated staining; interpret paraffin IHC against HPA's tissue-cell observations (HPA: subcellular and tissue IHC). Consult the separate IF/ICC guide for that application. |
Experiment evidence
IHC & IF images showing HCAR2 staining
A02511 · IHCImmunohistochemistry of paraffin-embedded human tonsil using GPR109A/HM74A/HCAR2 Rabbit pAb at dilution of 1:25 .Perform high pressure antig
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000182782-HCAR2/tissue/cerebral+cortex#imid_7157224
HPA · Thyroid glandHuman Protein Atlas IHC, Thyroid gland. Source: https://www.proteinatlas.org/ENSG00000182782-HCAR2/tissue/thyroid+gland#imid_7157168
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000182782-HCAR2/tissue/colon#imid_7157142
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000182782-HCAR2/tissue/liver#imid_7157151
HPA · ICC-IF RT-4Human Protein Atlas immunofluorescence, RT-4, antibody HPA028660; green = HCAR2, blue = nucleus, red = microtubules.
HPA · ICC-IF RT-4Human Protein Atlas immunofluorescence, RT-4, antibody HPA028660; green = HCAR2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HCAR2 IHC & IF
🧪Run tonsil first: germinal center cells should stain (HPA: Medium in tonsil germinal center cells; catalog antibody A02511: tonsil IHC caption). Use adrenal gland glandular cells as a negative tissue (HPA: Not detected). On the tonsil slide, morphologically unstained cells should show counterstain without specific chromogen; do not assume every cell outside a germinal center is negative.
Positive control tissue: Bone marrow (Hematopoietic cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show HCAR2 in RT-4, with annotated localisation: Cell Junctions (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a rabbit polyclonal isotype control matched to the catalog antibody (catalog antibody A02511: Rabbit pAb); use HCAR2-knockout material as a biological specificity control. For chromogenic tonsil IHC, block endogenous peroxidase and inspect control sections for residual background.
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the fixative in the selected A02511 tonsil caption is unreported. The caption specifies high-pressure retrieval in 10 mM citrate, pH 6.0, before staining at 1:25 (catalog antibody A02511: tonsil IHC caption); assess that retrieval condition during optimization. The supplied evidence does not establish that frozen sections or IF/ICC are easier than paraffin IHC; tonsil background from endogenous peroxidase warrants a no-primary control.
HPA tissue IHC evidence for HCAR2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene.). 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 |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Lymph node | Non-germinal center cells | Medium | Protein (IHC) | HPA → |
| Seminal vesicle | Glandular cells | Medium | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | Medium | Protein (IHC) | HPA → |
| Tonsil | Germinal center cells | Medium | 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 → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced HCAR2 IHC Tips
Troubleshoot HCAR2 staining in paraffin sections by checking retrieval, cell identity, and compartment before interpreting chromogenic signal.
Which retrieval condition should I start with for HCAR2 paraffin sections?
Start with heat-mediated retrieval in citrate buffer at pH 6.0 for HCAR2 paraffin sections (datasheet A02511). The selected tonsil image used high-pressure retrieval in 10 mM citrate at pH 6.0 and antibody at 1:25; those settings document that image, not an optimum for every specimen (caption A02511). If staining is weak, first check section adhesion, heating consistency, and antibody incubation against a concurrently processed tonsil control (caption A02511; standard IHC practice). Then adjust retrieval duration incrementally, recording tissue damage and background alongside signal, because excessive heating can reduce interpretable morphology (standard IHC practice).
How should I troubleshoot weak staining when fixation details are missing?
The selected HCAR2 tonsil caption identifies paraffin sections but gives no fixative, so target-specific sensitivity to fixation is unknown (caption A02511). Record each specimen’s fixative, time in fixative, and processing history before comparing staining intensity across blocks (standard IHC practice). Run a control block through the same retrieval and detection batch, and inspect whether weak staining accompanies poor morphology or broad loss of other expected immunostains (standard IHC practice). If fixation histories differ, compare results within matched processing groups and report that limitation; the caption’s 1:25 dilution and citrate pH 6.0 retrieval alone cannot establish a fixation effect (caption A02511; standard IHC practice).
Should HCAR2 appear only as a sharp membrane rim?
HCAR2 is a cell-membrane receptor with 7 transmembrane segments, so a membrane-associated pattern is biologically plausible (UniProt Q8TDS4 topology and subcellular location). HPA reports cytoplasmic staining across several tissues and supported cell-junction staining in ICC/IF, but warns that its antibodies target proteins from multiple genes (HPA tissue IHC and subcellular). Inspect whether chromogen follows cell boundaries or instead fills unrelated structures, and compare the same cell population on a no-primary control (standard IHC practice). Treat diffuse cytoplasmic staining as provisional until cell identity and antibody specificity are checked; a single compartment pattern cannot resolve the HPA cross-reactivity caution (HPA tissue IHC; standard IHC practice).
How does epitope position change interpretation of HCAR2 staining?
The supplied record lists no HCAR2 isoforms and maps extracellular residues 1–33 and cytoplasmic residues 295–363 around a 7-pass receptor (UniProt Q8TDS4 topology and isoforms). Check the antibody’s disclosed immunogen or epitope before assuming whether retrieval must expose an extracellular loop or intracellular tail (standard IHC practice). The record annotates phosphoserine at residue 328, but the selected caption does not identify an epitope or establish phosphorylation-sensitive staining (UniProt Q8TDS4 modified residues; caption A02511). Compare specificity controls and cellular distribution before attributing staining differences to an isoform, epitope masking, or phosphorylation state (standard IHC practice).
How can I check an HCAR2 IHC pattern with multiplex immunofluorescence?
Use IF/ICC as a separate validation experiment, pairing HCAR2 with a validated marker for the expected cell population rather than treating the paraffin IHC settings as an IF protocol (standard IF practice). Mature neutrophils are a biologically relevant population to assess, whereas immature neutrophils and eosinophils are reported as lacking expression (UniProt Q8TDS4 tissue specificity). Select fluorophores after examining unstained tissue autofluorescence and include single-label controls to evaluate spectral bleed-through (standard IF practice). Determine the antibody epitope before setting permeabilisation: an intracellular C-terminal epitope needs access through the membrane, while an extracellular epitope can be assessed initially without detergent (UniProt Q8TDS4 topology; standard IF practice).
What should I check when HCAR2 chromogen appears widespread?
Run no-primary and detection-only controls to distinguish antibody-associated staining from endogenous enzyme activity or nonspecific detection (standard IHC practice). For a peroxidase-based chromogenic workflow, include a peroxidase block and check whether pigment persists without primary antibody; these are general controls, not HCAR2-specific findings (standard IHC practice). Compare antibody titration and blocking conditions on adjacent sections, documenting both signal and tissue morphology rather than selecting the darkest stain (standard IHC practice). Interpret widespread staining cautiously because HPA describes cytoplasmic tissue staining yet flags antibodies that target proteins from multiple genes (HPA tissue IHC).
How should I quantify HCAR2 across paraffin specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, then use an H-score for intensity-weighted positivity or report the percentage of positive cells (standard IHC practice). For discrete immune cells, positive-cell density per mm² may be more informative; normalise to viable tissue area and report the counted cell population (standard IHC practice). Keep retrieval, detection, exposure to chromogen, and positivity thresholds consistent within a comparison, using the same control section in each staining batch (standard IHC practice). Report membrane-associated and cytoplasmic signal separately when both occur, because HCAR2 is annotated at the cell membrane while HPA reports cytoplasmic tissue staining (UniProt Q8TDS4 subcellular location; HPA tissue IHC).
When is an HCAR2-positive cell convincing rather than an artefact?
A convincing result links staining to identifiable viable cells, reproducible localisation, and a clean no-primary control (standard IHC practice). Membrane-associated signal fits HCAR2’s receptor topology, while HPA’s cytoplasmic tissue pattern requires caution because its antibodies may detect products of multiple genes (UniProt Q8TDS4 topology; HPA tissue IHC). Scrutinise strong signal at section edges, necrotic areas, and sites retaining chromogen in detection controls before counting cells as positive (standard IHC practice). Compare cell identity with the reported expression in mature neutrophils and spleen, but do not use a single positive compartment or tissue to establish antibody specificity (UniProt Q8TDS4 tissue specificity; standard IHC practice).
Boster reagents
Best HCAR2 / Hydroxycarboxylic acid receptor 2 IHC Antibodies
A02511 is listed for IHC and IF/ICC in human, mouse, and rat (catalog applications/reactivity). Its supplied figure shows IHC in paraffin-embedded human tonsil; no IF figure is supplied (IHC image caption; catalog).
Real IHC data
Anti-HCAR2 Antibody
Cat # A02511
A02511 will render with an IHC figure from paraffin-embedded human tonsil (IHC image caption). It is a rabbit polyclonal antibody listed for IHC and IF/ICC with human, mouse, and rat reactivity; the payload supplies no IF image (catalog applications/reactivity; catalog host/clonality; catalog images).
Which to pick: For tissue IHC, choose A02511: its figure shows staining in paraffin-embedded human tonsil after high-pressure retrieval in citrate buffer at pH 6.0; the fixative is unreported (IHC image caption). For IF/ICC, A02511 is listed for those applications, but no IF figure is supplied (catalog applications; catalog images). For mouse or rat samples, A02511 lists reactivity in both species, while the supplied IHC figure documents human tissue 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 Q8TDS4 (HCAR2_HUMAN, Hydroxycarboxylic acid receptor 2).
- Human Protein Atlas. HCAR2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. HCAR2 subcellular location (ICC-IF): Localized to the cell junctions. Caution: Based on antibodies targeting proteins from multiple genes..
- Human Protein Atlas. HCAR2 antibody validation summary (1 antibodies).
- Hydroxycarboxylic Acid Receptor 2 Mediates β-hydroxybutyrate's Antiseizure Effect in Mice. Annals of neurology 2026 — PMC12954166.
- β-Hydroxybutyrate enhances chondrocyte mitophagy and reduces cartilage degeneration in osteoarthritis via the HCAR2/AMPK/PINK1/Parkin pathway. Aging cell 2024 — PMC11561673.
- The niacin receptor HCAR2 modulates microglial response and limits disease progression in a mouse model of Alzheimer's disease. Science translational medicine 2022 — PMC10161396.
- PubMed PMID:12522134 — UniProt-cited evidence.
- PubMed PMID:12044878 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HCAR2 (Hydroxycarboxylic acid receptor 2). Expected localisation Cytoplasmic tissue staining (HPA tissue IHC); cell membrane location (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, heat-mediated (datasheet A02511). Validated IHC antibody: A02511 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A02511); 2. Fixation: Image fixative and duration unreported (datasheet A02511); 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 6.0 (datasheet A02511); 20 min, 95–100 °C (standard); 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-HCAR2, 1:50-1:200 (datasheet A02511); 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 A02511); Caveat: Antibody may detect proteins from more than one gene (HPA tissue IHC); Isoform / epitope: No isoforms annotated; check extracellular vs cytoplasmic epitope (UniProt); Regulation: Expression depends on neutrophil maturity (UniProt). Evidence: UniProt Q8TDS4, HPA tissue IHC · Approved, HPA ICC-IF · cell junctions. Caution: Based on antibodies targeting proteins from…, Datasheet A02511, PMC11561673. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HCAR2/immunohistochemistry-ihc-guide.