This website uses cookies to ensure you get the best experience on our website.
- Table of Contents
GJA3 / Gap junction alpha-3 protein · IHC design guide
Design Immunohistochemistry for GJA3
Plan GJA3 IHC in paraffin sections using lens fiber cells as a high-staining reference (HPA tissue IHC). The guide covers fixation, detection and interpretation of the membrane and gap-junction localisation expected from UniProt, alongside HPA’s off-target staining caveat.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
GJA3 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 | Cell membrane and gap junctions (UniProt) | |
| Staining pattern | High staining in lens fiber cells; subcellular pattern unspecified (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Eye | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Presumed off-target staining was disregarded (HPA tissue IHC) | |
| Regulation | Expression regulation is not annotated (UniProt) | |
| Isoform / epitope | No isoforms annotated; extracellular vs cytoplasmic epitope matters (UniProt) |
Section 1
Recommended GJA3 IHC & IF Protocols
The catalog antibody protocol (datasheet) is accompanied by two published GJA3 IHC protocols (PMC13543546; PMC4453443).
| Sample | Paraffin-embedded human lung carcinoma tissue; fixative not specified (datasheet A30696) |
| Fixation | Image fixative and duration unreported (datasheet A30696); 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-GJA3, 1:100 (datasheet A30696) |
| 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 | GJA3-positive staining in lens fiber cells of eye (HPA tissue IHC: High). HPA tissue profile: Expression in lens. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval rule); use each paper’s conditions when reproducing its staining (PMC13543546; PMC4453443).
Section 2
What Is the Expected GJA3 Staining Pattern?
GJA3 is a four-pass membrane protein at gap junctions (UniProt Q9Y6H8 topology and subcellular location). In paraffin-section IHC, expect staining in lens fiber cells, where HPA reports High expression (HPA: eye tissue IHC). HPA rates the tissue staining Supported, while noting presumed off-target binding that was disregarded and no internal RNA data for correlation (HPA: tissue IHC reliability).
| Strong staining outlines lens fiber cells, with signal concentrated at cell contacts. | This fits High lens fiber staining (HPA: eye tissue IHC) and membrane, gap-junction localization (UniProt Q9Y6H8). Assess the cell boundaries and tissue architecture together; a contact-enriched pattern is consistent with connexons joining adjacent cells (UniProt Q9Y6H8 function). |
| Signal is predominantly nuclear or diffusely cytoplasmic, without a convincing cell-boundary pattern. | That compartment differs from the annotated membrane and gap-junction location (UniProt Q9Y6H8). Treat it as a possible staining artefact until controls and the IHC-validated antibody's documented pattern support it; location alone cannot identify the artefact's cause. |
| Strong staining appears in unrelated cells, including adipocytes or adrenal glandular cells. | HPA reports GJA3 as Not detected in those cell types (HPA: adipose tissue and adrenal gland IHC). Investigate antibody cross-reactivity or endogenous chromogenic activity (general IHC practice); HPA also notes presumed off-target binding in its reliability assessment (HPA: tissue IHC). |
| A broad haze covers cells and surrounding tissue, obscuring boundaries. | A uniform haze does not show the membrane and junctional localization annotated for GJA3 (UniProt Q9Y6H8). Check background in a control processed without primary antibody, then review blocking, washes and detection conditions (general IHC practice). |
| Lens fiber cells show no signal despite adequate tissue morphology. | This conflicts with the High lens fiber staining reported by HPA (HPA: eye tissue IHC). First check the run's controls and the catalog antibody's IHC-P instructions; absence of signal alone cannot distinguish a technical failure from differences between specimens (general IHC practice). |
💡Expected GJA3 appearanceCall the result positive when lens fiber cells show High, membrane-associated staining enriched at cell contacts (HPA: eye tissue IHC; UniProt Q9Y6H8); isolated nuclear signal or strong staining in HPA-negative cell types warrants review (UniProt Q9Y6H8; HPA: tissue IHC).
| Membrane topology | GJA3 has 4 transmembrane segments and both extracellular and cytoplasmic regions (UniProt Q9Y6H8 topology). Interpret a localized boundary signal against this anatomy; the supplied record does not identify the antibody epitope, so it cannot predict epitope-specific retrieval needs. |
| Junction assembly | Six connexins form a hemichannel; two opposing hemichannels form a gap junction (UniProt Q9Y6H8 subunit and function). Contact-enriched staining is therefore biologically plausible, but chromogenic IHC cannot by itself show that a stained junction conducts ions or small molecules. |
| Tissue and cell selection | HPA observes High staining in eye lens fiber cells and Not detected staining in several sampled cell types, including adipocytes and adrenal glandular cells (HPA: tissue IHC). Use the named cells when comparing patterns; do not extend these observations to unsampled tissues. |
| Strength of IHC evidence | The HPA tissue profile is Supported, and antibody HPA014821 is marked Supported for IHC (HPA: tissue IHC reliability; HPA: antibody validation). HPA notes presumed off-target binding and lacks internal RNA correlation, so unexpected staining needs independent control-based review. |
| RNA and protein readouts | HPA lists tissue-enhanced RNA in heart muscle, parathyroid gland and placenta, while its reported protein staining profile is expression in lens (HPA: tissue IHC and RNA specificity). The RNA list does not establish an expected positive IHC result in those tissues. |
| Processing annotations | UniProt lists no signal peptide, propeptide, annotated glycosylation sites, modified residues or isoforms for this record (UniProt Q9Y6H8 processing and annotations). These entries do not establish how fixation or antigen retrieval affects GJA3 staining; target-specific fixation sensitivity is unreported here. |
| Situation | Likely cause | Next action |
|---|---|---|
| Lens fiber cells are negative. | The result disagrees with HPA's High lens fiber staining, but the slide alone does not identify why (HPA: eye tissue IHC). | Check run controls, tissue preservation and the catalog antibody's IHC-P instructions, including its specified retrieval and dilution if supplied (general IHC practice). Do not assign a GJA3-specific fixation effect from this result. |
| Signal fills nuclei or cytoplasm more than cell borders. | The dominant compartment conflicts with membrane and gap-junction annotation (UniProt Q9Y6H8 subcellular location). | Compare with lens fiber cell boundaries, inspect a no-primary control and reassess detection background (general IHC practice). Record the discordant compartment rather than scoring it as junctional GJA3. |
| Strong staining occurs in adipocytes or adrenal glandular cells. | These cells are reported as Not detected by HPA; HPA also notes presumed off-target binding (HPA: tissue IHC and reliability). | Compare the same run's controls and the validated lens pattern. Check for antibody-dependent staining and endogenous chromogenic activity before interpreting the unexpected cells (general IHC practice). |
| Diffuse brown background obscures cell contacts. | Background can interfere with judging the annotated junctional membrane location (UniProt Q9Y6H8; general IHC practice). | Inspect a no-primary control and review blocking, washes and detection conditions (general IHC practice). Score localization only where cell boundaries remain interpretable. |
| A heart, parathyroid or placenta sample is weak or negative. | HPA's tissue-enhanced RNA category names those tissues, but its supplied IHC profile reports expression in lens (HPA: RNA specificity and tissue IHC). | Avoid treating RNA enrichment as an IHC positive-control requirement. Use the reported lens fiber pattern to assess staining performance (HPA: eye tissue IHC). |
| Can an IF/ICC image confirm the IHC pattern? | HPA summarizes the subcellular location as membrane but provides no ICC-IF image cell lines or main location here; HPA014821 has no ICC status listed (HPA: subcellular and antibody records). | Treat membrane localization as a biological expectation from UniProt and HPA, while keeping IF/ICC validation separate from this paraffin-section IHC interpretation (UniProt Q9Y6H8; HPA: subcellular record). |
Experiment evidence
IHC & IF images showing GJA3 staining
A30696 · IHCImmunohistochemistry analysis of paraffin-embedded human lung carcinoma tissue, using GJA3 Antibody. The picture on the right is blocked wit
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000121743-GJA3/tissue/cerebral+cortex#imid_4304896
HPA · EyeHuman Protein Atlas IHC, Eye. Source: https://www.proteinatlas.org/ENSG00000121743-GJA3/tissue/retina#imid_442
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000121743-GJA3/tissue/colon#imid_4304806
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000121743-GJA3/tissue/liver#imid_4304823
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for GJA3 IHC & IF
🧪Run eye first: lens fiber cells must show staining (HPA: High in lens fiber cells). Run adipose tissue as the negative, focusing on adipocytes (HPA: Not detected in adipocytes); on the eye slide, non-lens cells, if present, should lack convincing junctional staining, but their negative status needs experimental confirmation.
Positive control tissue: Eye (Lens fiber 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 GJA3; derive a cell-line control from the positive tissue's cell type (Lens fiber cells) and confirm it by RNA or western blot first.
Technical controls: Include no-primary (secondary-only) and host-species-, immunoglobulin-class-, and concentration-matched isotype controls, plus a GJA3-knockout specimen if available or a peptide-block control (A30696 caption: peptide block shown). Quench endogenous peroxidase for HRP-based IHC; for lens IF, assess autofluorescence on an unstained section (standard IHC/IF practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the fixative used for the paraffin-section image is unreported (A30696 caption: fixative not stated). A GJA3-specific antigen-retrieval requirement is unreported; optimize retrieval against lens fiber staining and the controls (HPA: High in lens fiber cells). The supplied evidence does not establish whether frozen sections or IF are easier than paraffin IHC; lens autofluorescence can complicate IF interpretation (standard IF practice).
HPA tissue IHC evidence for GJA3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Presumed off target binding observed and disregarded. External characterization data supports antibody staining but no internal RNA data available for correlation.). 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 |
|---|---|---|---|---|
| Eye | Lens fiber cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| 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 GJA3 IHC Tips
Troubleshoot chromogenic GJA3 IHC in paraffin sections by checking retrieval, membrane localisation, controls and cell specific scoring (UniProt Q9Y6H8; HPA tissue IHC).
What retrieval should I try when GJA3 staining is weak in paraffin sections?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval rule: cytoplasmic or membrane antigen). Let sections cool in buffer, then compare retrieved and unretrieved sections using the same antibody dilution and detection conditions (standard IHC practice). GJA3 has 4 transmembrane segments and a cytoplasmic C terminus spanning residues 223–435, so epitope accessibility can depend on the antibody’s binding site (UniProt Q9Y6H8 topology). If staining remains weak, optimise retrieval time on matched sections while monitoring tissue integrity and background; the supplied caption reports paraffin sections but no retrieval conditions (caption A30696).
How should I assess whether fixation is affecting GJA3 IHC?
The supplied GJA3 tissue caption identifies paraffin sections but does not state a fixative, and target specific fixation sensitivity is unknown (caption A30696). Record the actual fixative, fixation duration and processing history for each specimen before comparing staining across samples (standard IHC practice). Run a known positive control alongside the study sections and keep section thickness, retrieval and detection conditions consistent when assessing a fixation concern (HPA tissue IHC: high staining in lens fiber cells; standard IHC practice). If staining differs between processing batches, test matched sections under one retrieval series before assigning the difference to GJA3 expression; fixation sensitivity cannot be inferred from the reported tissue pattern (standard IHC practice).
Which staining pattern is credible for GJA3 in tissue sections?
Look for staining associated with cell membranes and contacts between neighbouring cells, consistent with GJA3 localisation at the cell membrane and gap junctions (UniProt Q9Y6H8 subcellular location). Lens fiber cells provide the strongest supplied tissue reference, with high staining reported in the eye (HPA tissue IHC: high in lens fiber cells). Judge a putative junctional signal against cell boundaries in the counterstain and against a positive control processed in the same run (standard IHC practice). Diffuse nuclear staining or uniform colour across unrelated compartments does not match the reported membrane location and should prompt review of antibody specificity, detection background and morphology (UniProt Q9Y6H8 subcellular location; standard IHC practice).
How does epitope position affect GJA3 staining after retrieval?
GJA3 has 4 transmembrane segments, extracellular loops at residues 41–71 and 174–201, and a cytoplasmic region at 223–435 (UniProt Q9Y6H8 topology). Check the catalog antibody’s stated immunogen or mapped epitope before choosing how aggressively to retrieve sections; no epitope sequence is supplied here (standard IHC practice; supplied product evidence). The supplied record lists 0 annotated isoforms, glycosylation sites and modified residues, which does not establish that every epitope survives tissue processing (UniProt Q9Y6H8 record). If results conflict across antibodies, compare their documented epitope regions and staining in the same positive and negative tissues under matched conditions before attributing the difference to GJA3 biology (standard IHC practice).
How can I check GJA3 localisation by IF alongside chromogenic IHC?
Use a separately validated lens fiber cell marker in another channel to identify the expected positive cell population, since high GJA3 staining is reported in lens fiber cells (HPA tissue IHC). Choose fluorophores after checking tissue autofluorescence in an unstained section, and include single channel controls to assess bleed through (standard IF practice). Match permeabilisation to the documented antibody epitope: access to the cytoplasmic C terminus at residues 223–435 can require permeabilisation, whereas extracellular loop epitopes face a different membrane compartment (UniProt Q9Y6H8 topology; standard IF practice). Confirm that any punctate signal follows cell contacts and compare it with the chromogenic pattern without assuming that the paraffin IHC caption establishes IF performance (UniProt Q9Y6H8 subcellular location; caption A30696).
What controls help distinguish GJA3 signal from chromogenic background?
Include a section processed without primary antibody to assess detection background, and inspect tissue edges and damaged areas before interpreting brown deposits (standard IHC practice). Apply a peroxidase block when using an enzyme based DAB workflow; that block and DAB are general chromogenic IHC steps, not GJA3 specific validation (standard IHC practice). The selected paraffin lung carcinoma image includes a synthesized peptide blocked comparison, but its caption does not report a fixative or prove specificity in every tissue (caption A30696). HPA reports presumed off target binding despite supported tissue staining, so evaluate background alongside a lens fiber positive reference and a tissue with no reported staining (HPA tissue IHC).
How should I quantify GJA3 staining across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the analysed cell population and tissue area before scoring, using lens fiber cells when assessing the reported eye pattern (HPA tissue IHC: high in lens fiber cells; standard IHC practice). For chromogenic sections, report the percentage of positive cells and an intensity based H-score, or junction associated positive area per mm² when cell borders can be segmented reliably (standard IHC practice). Normalise area measurements to evaluable tissue area or the number of eligible cells, and exclude folds, necrosis and blank regions using the same rules for every section (standard IHC practice). Keep retrieval, imaging and scoring thresholds fixed across batches, with a shared control section to identify technical drift (standard IHC practice).
When should apparent GJA3 positivity be treated as an artefact?
Give greatest weight to staining at cell membranes and cell contacts in an expected cell population, especially lens fiber cells (UniProt Q9Y6H8 subcellular location; HPA tissue IHC: high in lens fiber cells). Treat dominant nuclear colour, staining confined to section edges or necrotic regions, and deposits also present without primary antibody as reasons to investigate artefact (UniProt Q9Y6H8 subcellular location; standard IHC practice). Check endogenous enzyme background when using peroxidase based detection and compare any unexpected cell type with a matched negative tissue and the run’s positive control (standard IHC practice). The selected lung carcinoma caption documents a peptide blocked paraffin image, while HPA notes presumed off target binding; interpret unexpected positivity with those limits in mind (caption A30696; HPA tissue IHC).
Boster reagents
Best GJA3 / Gap junction alpha-3 protein IHC Antibodies
A30696 has IHC image data from paraffin-embedded human lung carcinoma (A30696 IHC caption). IF/ICC are listed applications, and catalog reactivity covers human, mouse, and rat (catalog: A30696 applications/reactivity).
Real IHC data
Anti-GJA3/Connexin 46 Antibody
Cat # A30696
A30696 has an IHC image of paraffin-embedded human lung carcinoma with peptide blocking shown (A30696 IHC caption). Its listed applications include IHC, IF, and ICC, but no IF image is supplied (catalog: A30696 applications/if_image_alts).
Which to pick: Choose A30696 for paraffin-section tissue IHC based on its own human lung carcinoma image (A30696 IHC caption); the fixative is unreported (A30696 IHC caption). For IF/ICC, A30696 is listed for both at 1:100, although no IF image is supplied (catalog: A30696 applications/if_dilution/if_image_alts). For cross-species work, A30696 is a rabbit polyclonal listed as reactive with human, mouse, and rat (catalog: A30696 host/dilution_raw/reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9Y6H8 (CXA3_HUMAN, Gap junction alpha-3 protein).
- Human Protein Atlas. GJA3 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. GJA3 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. GJA3 antibody validation summary (1 antibodies).
- The role of DNMT3B, GSK-3β and GJA3 in lens epithelial cell apoptosis and age-related cataract. BMC ophthalmology 2026 — PMC13543546.
- B-RAF mutation and accumulated gene methylation in aberrant crypt foci (ACF), sessile serrated adenoma/polyp (SSA/P) and cancer in SSA/P. British journal of cancer 2015 — PMC4453443.
- PubMed PMID:10205266 — UniProt-cited evidence.
- PubMed PMID:15057823 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for GJA3 (Gap junction alpha-3 protein). Expected localisation Cell membrane and gap junctions (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A30696 at 1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Eye (Lens fiber cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung carcinoma tissue; fixative not specified (datasheet A30696); 2. Fixation: Image fixative and duration unreported (datasheet A30696); 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-GJA3, 1:100 (datasheet A30696); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Presumed off-target staining was disregarded (HPA tissue IHC); Isoform / epitope: No isoforms annotated; extracellular vs cytoplasmic epitope matters (UniProt); Regulation: Expression regulation is not annotated (UniProt). Evidence: UniProt Q9Y6H8, HPA tissue IHC · Supported, HPA ICC-IF · Membrane, Datasheet A30696, PMC13543546, PMC4453443. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/GJA3/immunohistochemistry-ihc-guide.