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HTR3A / 5-hydroxytryptamine receptor 3A · IHC design guide
Design Immunohistochemistry for HTR3A
Plan chromogenic IHC for HTR3A in paraffin sections using the reported immune-cell staining pattern (HPA tissue IHC). The catalog antibody was shown on human tonsil at 1:200 with overnight primary incubation at 4°C (datasheet A02391-1).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HTR3A 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 | Immune-cell cytoplasm and membrane (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic and membranous staining mainly in immune cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A02391-1) | |
| Positive control | Lymph node+1 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 paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Whole-tissue scoring can obscure cell-specific staining (HPA tissue IHC) | |
| Regulation | Expression regulation is unreported (UniProt) | |
| Isoform / epitope | 5 isoforms; check whether the epitope is extracellular or cytoplasmic (UniProt) |
Section 1
Recommended HTR3A IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published HTR3A IHC methods for lung adenocarcinoma, human brain, and rat intestine (PMC7540989; PMC10233771; PMC11250020).
| Sample | Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A02391-1) |
| Fixation | Image fixative and duration unreported (datasheet A02391-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 9.0 (datasheet A02391-1); 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-HTR3A, 1:100-1:300 (datasheet A02391-1) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | HTR3A-positive staining in non-germinal center cells of lymph node (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic and membranous expression mainly in immune cells. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 heat retrieval for the catalog antibody (datasheet A02391-1). The rat study reports heat conditioning but does not specify its buffer composition (PMC11250020).
Section 2
What Is the Expected HTR3A Staining Pattern?
HTR3A is a cell membrane receptor with 4 transmembrane segments and a postsynaptic location (UniProt P46098 topology; UniProt P46098 subcellular location). In paraffin tissue sections, expect cytoplasmic and membranous staining mainly in immune cells, especially non-germinal center cells in lymph node and tonsil (HPA tissue IHC). HPA rates the tissue pattern Supported, with medium consistency between staining and RNA expression (HPA tissue IHC).
| Membranous staining, with some cytoplasmic signal, in non-germinal center cells of lymph node or tonsil. | This matches HPA's immune-cell profile and its Medium staining level in those cells (HPA tissue IHC). Membrane signal also fits the receptor's topology (UniProt P46098 topology). Cytoplasmic signal can fit the observed HPA pattern, so its presence alone is not grounds to reject a slide (HPA tissue IHC). |
| Predominantly nuclear staining, with little staining at the cell edge. | A nuclear-dominant pattern conflicts with the reported membrane and cytoplasmic tissue pattern and membrane topology (HPA tissue IHC; UniProt P46098 topology). Treat it as suspect and compare it with a no-primary control and the expected cells on the same slide (general IHC practice). |
| Strong staining in cells HPA lists as not detected, such as adipocytes in adipose tissue. | That distribution conflicts with HPA's cell-specific observation (HPA tissue IHC). Cross-reactivity or endogenous detection activity is possible; check a no-primary control and, if available, an independent antibody before assigning the signal to HTR3A (general IHC practice). A negative HPA call applies to the named cell type, not automatically to every cell in that tissue (HPA tissue IHC). |
| Broad, diffuse color obscures cell boundaries across the section. | This prevents a reliable comparison with HPA's cell-restricted cytoplasmic and membranous pattern (HPA tissue IHC). Check background in the no-primary control, then review blocking, washing and detection conditions as general IHC troubleshooting steps (general IHC practice). |
| No convincing staining in non-germinal center cells of lymph node or tonsil. | Those cells show Medium staining in the HPA tissue record, so a blank result merits a workflow check (HPA tissue IHC). Confirm that the expected cells are present and review the antibody, antigen retrieval and detection controls (general IHC practice). A single negative run does not overturn the HPA observation (HPA tissue IHC). |
💡Expected HTR3A appearanceCall a positive result when non-germinal center cells in lymph node or tonsil show Medium cytoplasmic and membranous staining (HPA tissue IHC); nuclear-dominant or widespread cell-independent color is suspect (HPA tissue IHC; UniProt P46098 topology; general IHC practice).
| Topology and epitope location | HTR3A has extracellular and cytoplasmic regions around 4 transmembrane segments (UniProt P46098 topology). Interpret the cell pattern with that structure in mind; the supplied record gives no antibody epitope, so it cannot predict which region this stain detects. |
| Isoforms and processing | UniProt lists 5 isoforms and a signal peptide at residues 1–23, followed by the 24–478 chain (UniProt P46098 isoforms; UniProt P46098 processing). Without an antibody epitope, the record cannot establish isoform coverage or a processing-related staining difference. |
| Glycosylation | Four glycosylation sites are annotated at residues 28, 104, 170 and 186 (UniProt P46098 glycosylation). The supplied evidence does not show whether these modifications affect this antibody's staining or antigen retrieval. |
| Strength and scope of tissue evidence | HPA calls tissue IHC Supported and reports medium staining in specified lymph node and tonsil cells (HPA tissue IHC). The listed antibody, HPA069442, is IHC Supported (HPA antibodies); this does not establish the identity of every stained immune cell or validate an IF pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| The expected lymph node or tonsil cells appear negative. | A failed staining run or absent expected cells is possible; HPA reports Medium signal in non-germinal center cells there (HPA tissue IHC). | Verify the cells are present, inspect a run control, and review antigen retrieval, antibody dilution and detection steps (general IHC practice). |
| Nuclear staining dominates the slide. | That pattern disagrees with HPA's cytoplasmic and membranous profile and UniProt's membrane location (HPA tissue IHC; UniProt P46098 subcellular location). | Compare a no-primary control and the distribution in the expected cells before scoring it as HTR3A (general IHC practice). |
| Color appears in many cells across a nominally negative area. | Background or an off-target signal is possible; HPA's negative observations name particular cell types, including adipocytes in adipose tissue (HPA tissue IHC). | Compare the named cells and a no-primary control; review blocking, washing and endogenous detection activity (general IHC practice). |
| Membrane outlines are weak, but immune-cell cytoplasm stains. | HPA describes both cytoplasmic and membranous expression, mainly in immune cells (HPA tissue IHC). Cytoplasmic staining alone is therefore inconclusive. | Assess whether the signal remains cell restricted and whether lymph node or tonsil non-germinal center cells show the expected pattern (HPA tissue IHC). |
| Cerebral cortex appears negative in the cells being assessed. | UniProt reports expression in cerebral cortex, while HPA specifically lists cortical endothelial cells as not detected (UniProt P46098 tissue specificity; HPA tissue IHC). | Identify the cell type before interpreting the result; do not use endothelial-cell negativity to infer that every cortical cell is negative (HPA tissue IHC). |
| Can an IF/ICC image be scored against this IHC pattern? | HPA summarizes the subcellular location as membrane but provides no ICC-IF images or ICC validation for the listed antibody (HPA subcellular; HPA antibodies). | Use the membrane annotation as context, but assess IF/ICC with controls and validation appropriate to that assay (HPA subcellular; general IF practice). |
Experiment evidence
IHC & IF images showing HTR3A staining
A02391-1 · IHCImmunohistochemical analysis of paraffin-embedded human tonsil. 1, Antibody was diluted at 1:200 (4° overnight). 2, Tris-EDTA, pH9.0 was use
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000166736-HTR3A/tissue/cerebral+cortex#imid_20127221
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000166736-HTR3A/tissue/colon#imid_20129787
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000166736-HTR3A/tissue/liver#imid_20129812
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000166736-HTR3A/tissue/kidney#imid_20129765
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HTR3A IHC & IF
🧪Run tonsil first: non-germinal center cells should stain (HPA: Medium). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected), and compare staining in the tonsil cells with neighboring cells lacking specific signal as an internal background reference; HPA does not identify a specific internal negative cell type in tonsil (HPA: tonsil row).
Positive control tissue: Lymph node (Non-germinal center cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for HTR3A; derive a cell-line control from the positive tissue's cell type (Non-germinal center cells) and confirm it by RNA or western blot first.
Technical controls: Include no-primary (secondary-only) and host-species-matched isotype or normal-IgG controls, plus HTR3A knockout tissue or an antibody-specific peptide-block control when available (standard IHC practice). Check endogenous peroxidase background in tonsil for chromogenic IHC and autofluorescence if examining it by IF (standard IHC/IF practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected paraffin-section caption does not state a fixative (A02391-1 caption: fixative not stated). Start IHC-P optimization with Tris-EDTA retrieval at pH 9.0 and primary antibody at 1:200, 4°C overnight; compare with an omitted-retrieval section to assess retrieval dependence (A02391-1 caption: retrieval and primary incubation; standard IHC practice). No supplied evidence establishes that frozen sections or IF/ICC are easier than paraffin IHC; check tonsil autofluorescence if using IF (standard IF practice).
HPA tissue IHC evidence for HTR3A
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Medium 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 |
|---|---|---|---|---|
| Lymph node | Non-germinal center cells | Medium | Protein (IHC) | HPA → |
| Tonsil | Non-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 → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced HTR3A IHC Tips
Use the catalog antibody’s paraffin-section IHC conditions as a starting point, then judge staining against HTR3A localisation and tissue controls (datasheet A02391-1; UniProt P46098; HPA tissue IHC).
Which retrieval conditions should I try first for HTR3A in paraffin sections?
Use heat-mediated Tris-EDTA at pH 9.0 for antigen retrieval on paraffin sections (datasheet A02391-1). The catalog antibody’s human tonsil image used this retrieval with primary antibody at 1:200, incubated overnight at 4°C (datasheet A02391-1). Run a matched tonsil section when adjusting heating duration, and compare signal with tissue damage and background in the same staining run (standard IHC practice; datasheet A02391-1). If staining remains weak, test an alternative retrieval condition on adjacent sections while keeping detection and imaging conditions fixed; treat any improvement as a local optimisation rather than a documented HTR3A requirement (standard IHC practice).
How should I troubleshoot fixation-related loss of HTR3A staining?
Target-specific sensitivity to fixation is unknown: the catalog image identifies paraffin-embedded tonsil but does not state its fixative (datasheet A02391-1). Compare sections with documented fixation histories using the same pH 9.0 retrieval and 1:200 primary dilution before changing multiple variables (datasheet A02391-1; standard IHC practice). In routine paraffin IHC, variable fixation can alter antigen accessibility and tissue morphology, so inspect both alongside staining intensity (standard IHC practice). Include a tonsil control in each run and record fixation time when available; a weak specimen with a well-stained control warrants investigation of specimen processing before a claim about HTR3A abundance (HPA tissue IHC; standard IHC practice).
Where should convincing HTR3A staining appear in a tissue section?
HTR3A is annotated at the cell and postsynaptic membranes, with 4 transmembrane segments (UniProt P46098). Tissue IHC also reports cytoplasmic and membranous staining mainly in immune cells, so assess whether the observed compartment fits the cell population being scored (HPA tissue IHC). In tonsil, examine non-germinal center cells and compare their staining with neighboring compartments on the same section; HPA reports medium staining in those cells (HPA tissue IHC). Do not require a perfectly continuous membrane outline in chromogenic sections, but investigate diffuse staining across unrelated cells with an omission control and a matched positive tissue control (standard IHC practice; HPA tissue IHC).
Could isoforms or epitope accessibility explain inconsistent HTR3A staining?
HTR3A has 5 annotated isoforms, so isoform coverage cannot be assumed without an epitope or immunogen sequence for this antibody (UniProt P46098; standard antibody validation practice). The processed chain spans residues 24–478, and its extracellular region includes residues 24–241 (UniProt P46098 topology). Four glycosylation sites occur at residues 28, 104, 170, and 186; their presence alone does not establish that this antibody’s binding changes in sections (UniProt P46098). Obtain the antibody’s epitope information where available, then compare adjacent sections under the documented pH 9.0 retrieval condition before attributing a negative result to an isoform or modification (datasheet A02391-1; standard IHC practice).
How can I evaluate HTR3A by multiplex IF after using this IHC guide?
Treat IF as a separate validation experiment: the supplied catalog image documents paraffin-section chromogenic IHC, while HPA lists no cell-line ICC/IF images (datasheet A02391-1; HPA subcellular). Pair HTR3A with a validated marker for the cell population under study, such as an immune-cell marker when examining tonsil, and check each channel separately (HPA tissue IHC; standard IF practice). Choose a fluorophore channel with low measured tissue autofluorescence and include single-stain and no-primary controls to assess bleed-through and background (standard IF practice). Set permeabilisation only after establishing whether this antibody recognises an extracellular or cytoplasmic epitope; HTR3A contains both extracellular and cytoplasmic regions (UniProt P46098 topology; standard IF practice).
What should I change when tonsil sections show widespread chromogenic background?
First compare the stained section with a no-primary control and check whether background follows tissue edges, damaged areas, or endogenous enzyme activity (standard IHC practice). For peroxidase detection, verify the peroxidase block and limit DAB development using matched sections; these are general workflow controls, not HTR3A-specific conditions (standard IHC practice). The catalog tonsil image used primary antibody at 1:200 overnight at 4°C and secondary antibody at 1:200 for 45 minutes at room temperature (datasheet A02391-1). If controls remain clean but staining is diffuse, titrate the primary around that documented setting while retaining a positive tonsil section for comparison (datasheet A02391-1; standard IHC practice).
How should I score HTR3A staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population before reading intensity: HPA reports medium staining in tonsil and lymph-node non-germinal center cells (HPA tissue IHC). Record the percentage of positive cells and staining intensity for an H-score, or count positive cells per mm² within a consistently defined tissue compartment (standard IHC quantification practice). Normalise positive-cell counts to the number of eligible cells or sampled tissue area, and exclude necrotic, folded, or edge-damaged regions using the same rules across specimens (standard IHC practice). Keep retrieval, antibody dilution, chromogen development, counterstaining, and scoring thresholds consistent; HPA rates tissue staining as Supported with medium RNA–protein consistency (datasheet A02391-1; HPA tissue IHC; standard IHC practice).
How can I distinguish true HTR3A signal from staining artefact?
Look for staining in plausible cells and compartments: HPA reports cytoplasmic and membranous staining mainly in immune cells, while UniProt places HTR3A at the cell membrane (HPA tissue IHC; UniProt P46098). Medium staining in tonsil or lymph-node non-germinal center cells offers a reference, but it does not validate every positive cell in another specimen (HPA tissue IHC; standard IHC practice). Treat signal confined to section edges, necrotic regions, or a no-primary control as suspect, and assess endogenous peroxidase when using DAB (standard IHC practice). Interpret weak or absent staining alongside the positive control and tissue morphology because HPA assigns Supported reliability with medium consistency between staining and RNA data (HPA tissue IHC; standard IHC practice).
Boster reagents
Best HTR3A / 5-hydroxytryptamine receptor 3A IHC Antibodies
A02391-1 is a human-reactive HTR3A antibody listed for IHC and IF (catalog: applications and reactivity), with a paraffin-section IHC figure from human tonsil (catalog: IHC image caption).
Real IHC data
Anti-SR-3A HTR3A Antibody
Cat # A02391-1
A02391-1 will render with an IHC figure from paraffin-embedded human tonsil (catalog: IHC image caption). The catalog lists human reactivity and IHC and IF applications for A02391-1, but provides no IF figure (catalog: reactivity, applications and image captions).
Which to pick: Choose A02391-1 for human tissue IHC: its own figure shows paraffin-embedded tonsil, Tris-EDTA retrieval at pH 9.0, and primary antibody at 1:200 overnight at 4°C; the fixative is unreported (catalog: A02391-1 IHC image caption). A02391-1 is also listed for IF at 1:200–1:1000, but ICC validation is unreported (catalog: A02391-1 applications and dilution). No cross-species option is supported because A02391-1 lists human reactivity only (catalog: A02391-1 reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P46098 (5HT3A_HUMAN, 5-hydroxytryptamine receptor 3A).
- Human Protein Atlas. HTR3A tissue IHC expression (reliability: Supported).
- Human Protein Atlas. HTR3A subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. HTR3A antibody validation summary (1 antibodies).
- HTR3A is correlated with unfavorable histology and promotes proliferation through ERK phosphorylation in lung adenocarcinoma. Cancer science 2020 — PMC7540989.
- Inhibiting 5-hydroxytryptamine receptor 3 alleviates pathological changes of a mouse model of Alzheimer's disease. Neural regeneration research 2023 — PMC10233771.
- Serotonin Receptor 5-HT3A Affects Development of Bladder Innervation and Urinary Bladder Function. Frontiers in neuroscience 2017 — PMC5732969.
- Evaluating Small Intestinal Motility in a Rat Model of Adolescent Irritable Bowel Syndrome. Juntendo Iji zasshi = Juntendo medical journal 2022 — PMC11250020.
- PubMed PMID:7565620 — UniProt-cited evidence.
- PubMed PMID:8848005 — UniProt-cited evidence.
- PubMed PMID:9928262 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HTR3A (5-hydroxytryptamine receptor 3A). Expected localisation Immune-cell cytoplasm and membrane (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A02391-1). Validated IHC antibody: A02391-1 at 1:100-1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Lymph node (Non-germinal center cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissue; fixative not specified (datasheet A02391-1); 2. Fixation: Image fixative and duration unreported (datasheet A02391-1); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: Tris-EDTA pH 9.0 (datasheet A02391-1); 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-HTR3A, 1:100-1:300 (datasheet A02391-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Whole-tissue scoring can obscure cell-specific staining (HPA tissue IHC); Isoform / epitope: 5 isoforms; check whether the epitope is extracellular or cytoplasmic (UniProt); Regulation: Expression regulation is unreported (UniProt). Evidence: UniProt P46098, HPA tissue IHC · Supported, HPA ICC-IF · Membrane, Datasheet A02391-1, PMC7540989, PMC10233771. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HTR3A/immunohistochemistry-ihc-guide.