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SLC12A5 / Solute carrier family 12 member 5 · IHC design guide
Design Immunohistochemistry for SLC12A5
Plan SLC12A5 paraffin IHC around the selective CNS staining reported in neurons and neuropil (HPA tissue IHC). Compare the catalog antibody’s staining with controls, especially when assessing tissue outside the CNS (datasheet A04629-1; HPA tissue IHC; standard IHC practice).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
SLC12A5 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 | CNS cytoplasm observed; membrane/dendrites annotated (HPA tissue IHC; UniProt) | |
| Staining pattern | Selective cytoplasmic CNS staining in neurons and neuropil (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A04629-1) | |
| Positive control | Cerebellum+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 sections. (standard IHC practice; not target-specific) | |
| Caveat | Extra-CNS catalog staining warrants specificity controls (datasheet A04629-1; HPA tissue IHC) | |
| Regulation | Brain-specific neuronal expression (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope differences unreported; map epitope side (UniProt) |
Section 1
Recommended SLC12A5 IHC & IF Protocols
The catalog antibody uses citrate pH 6 retrieval (datasheet A04629-1). Published SLC12A5 IHC protocols cover lung adenocarcinoma and bladder urothelial carcinoma tissue (PMC6811597; PMC5386524).
| Sample | Paraffin-embedded human tonsil tissues; fixative not specified (datasheet A04629-1) |
| Fixation | Image fixative and duration unreported (datasheet A04629-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet A04629-1) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A04629-1) |
| Primary antibody | Rabbit anti-SLC12A5, 0.5-1μg/ml (datasheet A04629-1) |
| Primary incubation | Overnight at 4 °C (datasheet A04629-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A04629-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | SLC12A5-positive staining in molecular layer - neuropil of cerebellum (HPA tissue IHC: Medium). HPA tissue profile: Selective cytoplasmic expression in CNS. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 heat retrieval (datasheet A04629-1); the bladder study used EDTA pH 8 microwave retrieval (PMC5386524).
Section 2
What Is the Expected SLC12A5 Staining Pattern?
SLC12A5 is a 12-pass cell-membrane protein detected on neuronal dendrites (UniProt Q9H2X9 topology and subcellular location). In paraffin-section IHC, expect selective CNS staining in neuronal cells and neuropil: HPA reports medium staining in cerebral cortex neurons and cerebellar molecular-layer neuropil, describing the tissue pattern as cytoplasmic (HPA tissue IHC). HPA rates the tissue pattern Enhanced for consistency with RNA expression (HPA tissue IHC).
| Medium staining outlines or fills neuronal profiles in cerebral cortex (HPA tissue IHC). | This fits the reported neuronal-cell signal (HPA tissue IHC) and membrane/dendritic location (UniProt Q9H2X9). Chromogenic IHC may not resolve a thin membrane rim from adjacent cytoplasm or processes (general IHC practice); require the expected cell distribution as well as a plausible compartment. |
| Medium staining extends through cerebellar molecular-layer neuropil (HPA tissue IHC). | This is a reported positive pattern (HPA tissue IHC). Neuronal processes can contribute to neuropil staining (general histology); UniProt detects SLC12A5 on dendrites (UniProt Q9H2X9). Do not require every positive profile to resemble a sharply outlined cell body. |
| Signal is predominantly nuclear, with little neuronal or neuropil staining. | A nuclear-only pattern conflicts with the reported membrane location (UniProt Q9H2X9) and selective CNS tissue pattern (HPA tissue IHC). Treat it as suspect staining, then compare controls and detection background before assigning a cause (general IHC practice). |
| Strong staining appears in adipocytes or caudate glial cells, both reported as not detected (HPA tissue IHC). | That distribution warrants a specificity check against the HPA cell-level observations (HPA tissue IHC). Cross-reactive antibody binding or endogenous detection activity are possible explanations, not diagnoses from the slide alone (general IHC practice). |
| Cortex neurons and cerebellar molecular-layer neuropil show no signal (HPA tissue IHC). | A blank result in these reported medium-staining sites does not establish absent SLC12A5 (HPA tissue IHC). Check tissue identity, assay controls, retrieval, antibody incubation and detection in sequence (general IHC practice); the supplied sources give no target-specific fixation sensitivity. |
💡Expected SLC12A5 appearanceCall a positive result when neuronal profiles in cerebral cortex or cerebellar molecular-layer neuropil show convincing medium chromogenic signal (HPA tissue IHC), compatible with neuronal membrane and dendritic localisation (UniProt Q9H2X9); isolated nuclear or broadly non-neuronal staining is suspect by comparison (HPA tissue IHC; UniProt Q9H2X9).
| Cell compartment and slide resolution | UniProt places SLC12A5 at the cell membrane and dendrites (UniProt Q9H2X9); HPA describes selective cytoplasmic CNS IHC staining (HPA tissue IHC). Read those descriptions at their respective resolution: chromogenic signal around somata and processes need not appear as a crisp membrane line (general IHC practice). |
| Positive tissue and cell choice | Cerebral cortex neuronal cells and cerebellar molecular-layer neuropil are reported at medium staining (HPA tissue IHC). They provide evidence-based positive comparators. HPA also reports no detection in adipocytes from adipose tissue and glial cells in caudate (HPA tissue IHC); use those observations only for those named cells and tissues. |
| Antibody validation | HPA rates tissue IHC reliability Enhanced and lists rabbit polyclonal HPA072058 as IHC Enhanced (HPA tissue IHC; HPA antibodies). This supports the reported pattern, but does not prove that every neuronal signal from another antibody is specific (general IHC interpretation). Check the antibody actually used against its own controls. |
| Topology and epitope access | SLC12A5 has 12 transmembrane segments plus extracellular and cytoplasmic regions (UniProt Q9H2X9 topology). Epitope position can matter when selecting staining conditions (general IHC practice), but the supplied record does not locate this antibody’s epitope or justify a particular retrieval method. |
| IF/ICC evidence boundary | HPA summarizes the ICC-IF location as membrane but supplies no cell-line ICC-IF images or main-location assignment (HPA subcellular). That summary is compatible with UniProt membrane localisation (UniProt Q9H2X9); it does not establish an IF staining pattern, antibody dilution or protocol for this IHC page. |
| Situation | Likely cause | Next action |
|---|---|---|
| No chromogenic signal in the reported medium-staining cortex neurons or cerebellar neuropil (HPA tissue IHC). | The assay may have failed at retrieval, antibody incubation or detection (general IHC practice); the slide alone cannot identify which step. | Verify tissue identity and a working positive control, then review the antibody instructions and detection reagents (general IHC practice). Do not attribute failure to SLC12A5 fixation sensitivity: none is reported in the supplied sources. |
| Only nuclei stain while neuronal profiles and neuropil remain blank. | This conflicts with SLC12A5 membrane/dendritic localisation (UniProt Q9H2X9) and the reported CNS distribution (HPA tissue IHC); background or off-target binding is possible (general IHC practice). | Inspect a no-primary control and reassess antibody and detection specificity before scoring the nuclear signal as positive (general IHC practice). |
| Adipocytes in adipose tissue stain despite HPA reporting them as not detected (HPA tissue IHC). | Unexpected signal may reflect cross-reactivity or endogenous chromogenic detection activity (general IHC practice); HPA’s observation alone cannot distinguish them. | Compare no-primary and reagent controls, examine the staining distribution, and review blocking appropriate to the detection system (general IHC practice). |
| The whole section has diffuse colour that obscures neuronal outlines. | Excess background may arise from detection reagents, insufficient blocking or inadequate washes (general IHC practice); it cannot be assigned to SLC12A5 topology. | Compare the no-primary control, then review blocking, washes and detection development under the assay instructions (general IHC practice). Score neuronal staining only when distinguishable from that background. |
| Cerebellar neuropil stains, but individual neuronal membranes are hard to trace (HPA tissue IHC). | The reported neuropil pattern (HPA tissue IHC) and finite resolution of chromogenic sections (general IHC practice) can make process-associated signal appear diffuse. | Compare the molecular layer with nearby tissue and a control section; assess regional and cellular distribution before calling the staining nonspecific (general IHC practice). |
| An IF image is being used to set an IHC result threshold. | HPA’s ICC-IF entry says membrane but includes no cell-line images (HPA subcellular), while its observed positive tissue levels come from IHC (HPA tissue IHC). | Score the paraffin IHC slide against the reported cortex neuronal and cerebellar neuropil patterns (HPA tissue IHC). Keep IF interpretation on its own guide page. |
Experiment evidence
IHC & IF images showing SLC12A5 staining
A04629-1 · IHCIHC analysis of SLC12A5 using anti-SLC12A5 antibody (A04629-1). SLC12A5 was detected in paraffin-embedded section of human tonsil tissues. H
A04629-1 · IHCIHC analysis of SLC12A5 using anti-SLC12A5 antibody (A04629-1). SLC12A5 was detected in paraffin-embedded section of rat brain tissues. Heat
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000124140-SLC12A5/tissue/cerebral+cortex#imid_19663591
HPA · CerebellumHuman Protein Atlas IHC, Cerebellum. Source: https://www.proteinatlas.org/ENSG00000124140-SLC12A5/tissue/cerebellum#imid_19663602
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000124140-SLC12A5/tissue/colon#imid_19663517
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000124140-SLC12A5/tissue/liver#imid_19663518
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for SLC12A5 IHC & IF
🧪Run cerebral cortex first and look for staining in neuronal cells (HPA: Medium in cerebral cortex neuronal cells). Use adipose tissue as the negative tissue and expect non-neuronal elements on the cortex slide to remain at background, while recognizing that HPA does not designate those internal cells as validated negatives (HPA: Not detected in adipose tissue adipocytes; HPA: Medium in cerebral cortex neuronal cells).
Positive control tissue: Cerebellum (Molecular layer - neuropil, 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 SLC12A5; derive a cell-line control from the positive tissue's cell type (Molecular layer - neuropil) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control, a rabbit isotype control matched to the primary antibody’s clonality, and SLC12A5 knockout tissue where available (caption: rabbit primary antibody; standard IHC controls). For chromogenic cortex sections, quench endogenous peroxidase and check for endogenous biotin if using the caption’s biotin-based detection; distinguish pigment deposits from specific staining (caption: biotinylated secondary and DAB; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A04629-1 paraffin-section caption does not state a fixative (selected-SKU caption: fixative not stated). That caption uses heat retrieval in citrate buffer at pH 6 for 20 minutes, but it does not establish that retrieval is required for every specimen (selected-SKU caption). Whether frozen sections or IF are easier is unreported; assess cortical pigment that could complicate chromogenic interpretation, and validate any tonsil staining independently given the neuronal distribution reported for SLC12A5 (standard IHC practice; selected-SKU caption: tonsil; HPA: Medium in cortical neuronal cells; UniProt: detected in neuronal cells).
HPA tissue IHC evidence for SLC12A5
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 |
|---|---|---|---|---|
| Cerebellum | Molecular layer - neuropil | Medium | Protein (IHC) | HPA → |
| Cerebral cortex | Neuronal 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 → |
| 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 SLC12A5 IHC Tips
Troubleshoot SLC12A5 staining by checking retrieval, tissue context, signal location and controls before comparing chromogenic IHC scores.
How should I retrieve SLC12A5 in paraffin sections when staining is weak?
Start with heat-mediated citrate retrieval at pH 6 for 20 minutes, matching the selected antibody’s tissue-IHC method (datasheet A04629-1). Keep section thickness, heating and cooling consistent across test and control slides so differences in staining can be judged against the same retrieval conditions (standard IHC practice). The documented section then received 10% goat serum block and 1 μg/mL primary antibody overnight at 4°C (datasheet A04629-1). If staining remains weak, compare a modestly longer citrate retrieval on adjacent sections, while checking whether tissue damage or increased background offsets any gain (standard IHC practice).
Could fixation explain weak or uneven SLC12A5 staining?
Target-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative, so it cannot establish how SLC12A5 responds to fixation (datasheet A04629-1). Record the fixative, fixation interval and tissue-processing history for every section before attributing a staining difference to the antibody (standard IHC practice). Compare adjacent sections processed together using the documented citrate pH 6, 20-minute retrieval and 1 μg/mL primary incubation (datasheet A04629-1). If a processing batch stains weakly, examine morphology and an independently processed positive control before changing retrieval or antibody concentration (standard IHC practice).
Where should convincing SLC12A5 staining appear in a tissue section?
Assess neuronal membranes and dendritic profiles because SLC12A5 is annotated at the cell membrane and on dendrites (UniProt Q9H2X9 localisation). The record reports detection on dendrites, but not axons, in spinal cord neurons and at GPHN-positive inhibitory synapses (UniProt Q9H2X9 localisation). In tissue IHC, medium staining was reported in cerebellar molecular-layer neuropil and cerebral-cortex neuronal cells, while the broader profile describes selective cytoplasmic expression in CNS (HPA tissue IHC). Because chromogenic staining may not resolve a thin membrane from nearby cytoplasm, inspect morphology and compare the same compartment across sections before scoring (standard IHC practice).
How do epitope location and isoforms affect interpretation of this antibody?
SLC12A5 has 2 annotated isoforms and 12 transmembrane segments, with a long cytoplasmic region at residues 633–1139 (UniProt Q9H2X9 isoforms and topology). Extracellular residues include 277–402, which contains annotated glycosylation sites at 314, 333, 351 and 362 (UniProt Q9H2X9 topology and glycosylation). The supplied antibody caption does not identify its epitope or establish isoform-specific recognition, so staining alone cannot assign a signal to either isoform (datasheet A04629-1; UniProt Q9H2X9 isoforms). Check the antibody’s epitope documentation before interpreting a changed staining pattern as altered isoform expression or epitope accessibility (standard IHC practice).
How should I assess SLC12A5 in a multiplex immunofluorescence experiment?
Pair SLC12A5 with a neuronal marker to test whether signal occupies the expected cell population, while keeping the chromogenic IHC pattern as the tissue reference (UniProt Q9H2X9 tissue specificity; HPA tissue IHC). Choose fluorophores after viewing unstained tissue, placing the weaker signal in a channel with low autofluorescence and checking each channel separately for bleed-through (standard IF practice). Select permeabilisation after establishing the antibody epitope: a cytoplasmic epitope needs access across the membrane, whereas an extracellular epitope may be assessable without that step (UniProt Q9H2X9 topology; standard IF practice). The supplied caption documents paraffin-section chromogenic IHC, not IF performance for this antibody (datasheet A04629-1).
What should I check when SLC12A5 DAB staining is widespread?
First compare a no-primary section with the test section, then inspect whether staining follows tissue edges, damaged areas or the expected neuronal structures (standard IHC practice; UniProt Q9H2X9 localisation). The documented detection used a biotinylated secondary antibody, an avidin-biotin complex and DAB, so run appropriate detection controls when background appears with this workflow (datasheet A04629-1; standard IHC practice). Check the peroxidase block, serum block, washing and chromogen development consistently across slides; the documented serum block was 10% goat serum (datasheet A04629-1; standard IHC practice). Diffuse staining in tissue compartments reported as undetected by HPA warrants particular scrutiny before it is scored as SLC12A5 (HPA tissue IHC).
How should I quantify SLC12A5 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the region and compartment before scoring: HPA reports medium signal in cerebellar molecular-layer neuropil and cerebral-cortex neuronal cells (HPA tissue IHC). For neuronal cell bodies, report the percentage positive and an intensity-based H-score; for neuropil, measure positive area or optical density within a defined region (standard IHC quantification). Normalise cell counts to the number of eligible neuronal cells, or area measurements to the sampled tissue area in mm², and apply one threshold across the comparison set (standard IHC quantification). Record retrieval and DAB development conditions with each batch because differences in those steps can shift measured intensity (standard IHC practice).
How can I distinguish true SLC12A5 staining from an artefact?
A plausible result tracks neuronal cells or neuropil and is consistent with membrane or dendritic localisation, while recognising that HPA describes a selective cytoplasmic CNS IHC profile (UniProt Q9H2X9 localisation; HPA tissue IHC). Treat staining concentrated at section edges, in necrotic areas or on a no-primary control as suspect, and check endogenous peroxidase signal before assigning DAB positivity to SLC12A5 (standard IHC practice). The selected antibody caption reports staining in a human tonsil paraffin section, whereas UniProt describes brain-specific neuronal expression and HPA reports selective CNS staining (datasheet A04629-1; UniProt Q9H2X9 tissue specificity; HPA tissue IHC). Resolve that discrepancy with independently supported controls before treating tonsil staining as evidence of SLC12A5 expression (standard IHC practice).
Boster reagents
Best SLC12A5 / Solute carrier family 12 member 5 IHC Antibodies
A04629-1 has IHC images from paraffin sections of human tonsil, ovarian cancer and placenta, and rat brain (catalog image captions). No IF/ICC image is supplied (catalog: IF images).
Real IHC data
Anti-KCC2/SLC12A5 Antibody ®
Cat # A04629-1
A04629-1 is listed for IHC and has paraffin-section images from human tonsil, ovarian cancer and placenta (catalog: applications; catalog image captions). Its rat brain image provides additional IHC evidence, while human, mouse and rat are listed as reactive species (catalog image caption; catalog: reactivity).
Which to pick: Choose A04629-1 for paraffin-section IHC; its human tonsil caption documents citrate retrieval at pH 6 for 20 minutes and antibody incubation at 1 μg/ml (catalog image caption). No supplied SKU has IF/ICC listed or an IF image, so an IF/ICC choice cannot be supported here (catalog: applications; catalog: IF images). For work across species, A04629-1 lists human, mouse and rat reactivity, although the supplied IHC images show human and rat samples only; the captions specify paraffin sections but do not report the fixative (catalog: reactivity; catalog image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9H2X9 (S12A5_HUMAN, Solute carrier family 12 member 5).
- Human Protein Atlas. SLC12A5 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. SLC12A5 subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. SLC12A5 antibody validation summary (1 antibodies).
- The TWIST1-centered competing endogenous RNA network promotes proliferation, invasion, and migration of lung adenocarcinoma. Oncogenesis 2019 — PMC6811597.
- Solute carrier family 12 member 5 promotes tumor invasion/metastasis of bladder urothelial carcinoma by enhancing NF-κB/MMP-7 signaling pathway. Cell death & disease 2017 — PMC5386524.
- ETV4‑mediated transcriptional activation of SLC12A5 exacerbates ferroptosis resistance and glucose metabolism reprogramming in breast cancer cells. Molecular medicine reports 2024 — PMC11465427.
- Increased expression of Solute carrier family 12 member 5 via gene amplification contributes to tumour progression and metastasis and associates with poor survival in colorectal cancer. Gut 2016 — PMC4819609.
- PubMed PMID:12106695 — UniProt-cited evidence.
- PubMed PMID:11780052 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for SLC12A5 (Solute carrier family 12 member 5). Expected localisation CNS cytoplasm observed; membrane/dendrites annotated (HPA tissue IHC; UniProt). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A04629-1). Validated IHC antibody: A04629-1 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Cerebellum (Molecular layer - neuropil); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human tonsil tissues; fixative not specified (datasheet A04629-1); 2. Fixation: Image fixative and duration unreported (datasheet A04629-1); 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: Citrate pH 6 , 20 min (datasheet A04629-1); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A04629-1); 8. Primary antibody: Rabbit anti-SLC12A5, 0.5-1μg/ml (datasheet A04629-1); 9. Primary incubation: Overnight at 4 °C (datasheet A04629-1). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Extra-CNS catalog staining warrants specificity controls (datasheet A04629-1; HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope differences unreported; map epitope side (UniProt); Regulation: Brain-specific neuronal expression (UniProt). Evidence: UniProt Q9H2X9, HPA tissue IHC · Enhanced, HPA ICC-IF · Membrane, Datasheet A04629-1, PMC6811597, PMC5386524. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/SLC12A5/immunohistochemistry-ihc-guide.