SLC12A5 / Solute carrier family 12 member 5 · IHC design guide

Design Immunohistochemistry for SLC12A5

Plan chromogenic IHC on paraffin sections using cortical neurons and cerebellar neuropil as positive staining references (HPA tissue IHC). The catalog antibody’s IHC-P dilution is 0.5–1 μg/mL (datasheet A04629-1).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for SLC12A5 (IHC for SLC12A5): expected localisation Cytoplasmic staining in CNS tissue (HPA tissue IHC), antibody A04629-1, validated IHC image, and IHC protocol steps — Printable **SLC12A5** IHC protocol sheet — expected localisation Cytoplasmic staining in CNS tissue (HPA tissue IHC), antibody A04629-1, controls and protocol steps. Open the full SLC12A5 IHC guide →

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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	Cytoplasmic staining in CNS tissue (HPA tissue IHC)
	Staining pattern	Cortical neurons and cerebellar neuropil show cytoplasmic staining (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). Selected-image fixative and duration unreported (datasheet A04629-1); verify before use.
	Caveat	Validate peripheral staining against the selective CNS profile (HPA tissue IHC)
	Regulation	No stimulus regulation reported (UniProt)
	Isoform / epitope	2 isoforms; check whether the epitope is extracellular or cytoplasmic (UniProt)

Section 1

Recommended SLC12A5 IHC & IF Protocols

The catalog antibody uses citrate pH 6 heat retrieval (datasheet A04629-1). Two published IHC protocols provide tissue-specific examples (PMC6811597; PMC5386524).

Recommended immunohistochemistry (IHC-P) protocol parameters
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 for the catalog antibody (datasheet A04629-1); the bladder carcinoma study used EDTA pH 8.0 with its own antibody (PMC5386524).

Section 2

What Is the Expected SLC12A5 Staining Pattern?

SLC12A5 is a 12-pass membrane transporter found in neuronal cells and dendrites (UniProt Q9H2X9 topology and subcellular location). In paraffin-section IHC, expect selective CNS staining, with medium signal in cerebral-cortex neuronal cells and cerebellar molecular-layer neuropil (HPA tissue IHC: Enhanced). HPA describes the tissue pattern as cytoplasmic; that appearance should be read alongside the membrane localisation reported by UniProt.

What am I looking at on my slide?
Medium staining in cerebral-cortex neuronal cells, with selective staining in the section.	This matches the observed cortex pattern (HPA tissue IHC: neuronal cells, Medium). A cytoplasmic-looking chromogenic signal can be assessed alongside SLC12A5's membrane and dendritic localisation (HPA tissue IHC: selective cytoplasmic expression in CNS; UniProt Q9H2X9 subcellular location).
Medium staining across the cerebellar molecular-layer neuropil.	This matches the recorded cerebellar compartment (HPA tissue IHC: molecular layer–neuropil, Medium). Neuropil signal need not resolve into a crisp cell outline in chromogenic sections (standard IHC interpretation); evaluate its location within the tissue rather than calling every stained profile a cell body.
Predominantly nuclear staining, or a pattern confined to axons.	These locations do not fit the reported membrane and dendritic distribution; UniProt reports detection on spinal-cord neuron dendrites, but not axons (UniProt Q9H2X9 subcellular location). Treat such a pattern as suspect and compare it with tissue morphology and controls (standard IHC practice).
Prominent staining of adipocytes, glandular cells, or caudate glial cells.	These are poor matches to the listed negative examples (HPA tissue IHC: adipose adipocytes, adrenal glandular cells, and caudate glial cells, Not detected). Consider nonspecific binding or detection-system activity before assigning the signal to SLC12A5 (standard IHC practice).
Diffuse color across tissue and blank spaces, or no staining in cortex and cerebellar neuropil.	Diffuse deposition obscures cellular localisation (standard IHC interpretation). An absent signal in both listed positive sites conflicts with their observed medium staining, but alone does not establish loss of SLC12A5; section quality and the staining run still need assessment (HPA tissue IHC; standard IHC practice).

💡Expected SLC12A5 appearanceCall a section positive when medium, selective signal marks cerebral-cortex neuronal cells or cerebellar molecular-layer neuropil (HPA tissue IHC); widespread nuclear or listed negative-cell staining is suspect (UniProt Q9H2X9 subcellular location; HPA tissue IHC).

How each factor affects the staining
Reference tissue and cell population	Cortex neuronal cells and cerebellar molecular-layer neuropil provide observed medium-signal references; adipocytes and specified glandular cells provide observed negatives (HPA tissue IHC). Score the named populations, since a whole-section positive or negative label can hide their distribution (standard IHC interpretation).
Membrane topology and apparent compartment	SLC12A5 has 12 transmembrane segments and a long cytoplasmic C terminus (UniProt Q9H2X9 topology). HPA nevertheless describes selective cytoplasmic CNS IHC staining (HPA tissue IHC). The supplied sources do not identify the IHC antibody epitope, so topology cannot establish its retrieval needs.
Strength of the tissue evidence	The HPA tissue profile has Enhanced reliability, described as high consistency between antibody staining and RNA expression (HPA tissue IHC). The listed HPA072058 antibody also has Enhanced IHC validation (HPA antibodies); neither designation makes every stained cell specific by itself (standard IHC interpretation).
Isoforms and modifications	Two isoforms, four glycosylation sites, and annotated phosphorylation sites are recorded (UniProt Q9H2X9). Without an antibody epitope or comparative staining data, their effects on this IHC signal cannot be assigned; do not use them to explain a failed section.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Neither cerebral-cortex neuronal cells nor cerebellar molecular-layer neuropil stain.	Both are observed medium-signal sites, so a failed staining run or unsuitable section is possible; the result alone does not identify the cause (HPA tissue IHC; standard IHC practice).	Check section integrity and the run's positive control, then review the established antibody-specific IHC conditions, including retrieval and detection (standard IHC practice). No SLC12A5-specific retrieval condition is supplied.
Color is widespread, including areas without a matching neuronal pattern.	Nonspecific binding, residual detection activity, or chromogen deposition can produce diffuse signal (standard IHC practice); this does not resemble selective CNS staining (HPA tissue IHC).	Compare a no-primary control, inspect background around the tissue, and review blocking, washes, and chromogen development (standard IHC practice). Interpret only staining that remains tied to the expected structures.
Adipocytes or glandular cells appear strongly positive.	Those populations are recorded as Not detected in listed tissues (HPA tissue IHC: adipose adipocytes; adrenal and appendix glandular cells). Cross-reactivity or detection-system activity is possible (standard IHC practice).	Compare the same populations with a no-primary control and with cortex or cerebellar reference sections in the run (standard IHC practice; HPA tissue IHC). Avoid assigning SLC12A5 from color intensity alone.
Signal appears mainly nuclear or restricted to axons.	That distribution conflicts with the reported membrane and dendritic location, including the stated absence from spinal-cord neuron axons (UniProt Q9H2X9 subcellular location).	Recheck compartment calls against morphology and counterstain, then assess control staining and nonspecific background (standard IHC practice). Do not score isolated nuclear color as the expected positive pattern.
Cerebellar neuropil stains, but discrete neuronal cell borders are hard to see.	The observed positive compartment is molecular-layer neuropil (HPA tissue IHC: Medium); chromogenic resolution may not separate every membrane profile (standard IHC interpretation).	Score the molecular-layer distribution and compare it with cortex neuronal staining in the same run (HPA tissue IHC). Record the compartment actually visible instead of inferring a sharply outlined membrane.
IF/ICC: should membrane fluorescence be expected?	Membrane localisation is reported for SLC12A5 (UniProt Q9H2X9 subcellular location; HPA subcellular summary: Membrane), but the supplied HPA ICC-IF record lists no image-bearing cell lines or main-location assignment (HPA subcellular ICC-IF).	Treat membrane localisation as a biological expectation, not as an image-validated ICC-IF pattern for this antibody (UniProt Q9H2X9; HPA subcellular ICC-IF). Use the separate IF/ICC guide for assay design.

Source: UniProt Q9H2X9 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing SLC12A5 staining

IHC analysis of SLC12A5 using anti-SLC12A5 antibody (A04629-1). SLC12A5 was detected in paraffin-embedded section of human tonsil tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-SLC12A5 Antibody (A04629-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

A04629-1 · IHCIHC analysis of SLC12A5 using anti-SLC12A5 antibody (A04629-1). SLC12A5 was detected in paraffin-embedded section of human tonsil tissues. H

IHC analysis of SLC12A5 using anti-SLC12A5 antibody (A04629-1). SLC12A5 was detected in paraffin-embedded section of rat brain tissues. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-SLC12A5 Antibody (A04629-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC)(Catalog # SA1022) with DAB as the chromogen.

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 cerebellum first and score staining in the molecular-layer neuropil (HPA: Medium). Run adipose tissue as the negative tissue, with adipocytes at background (HPA: Not detected); on the cerebellum slide, non-neuronal cells should provide an internal background comparison (UniProt Q9H2X9: detected in 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 no-primary (secondary-only) and concentration-matched rabbit IgG controls alongside knockout tissue, if available, as a biological negative (selected-SKU caption: rabbit primary antibody; standard IHC practice). Block endogenous peroxidase before chromogenic detection in cerebellum (standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the exact A04629-1 tissue-IHC caption does not state a fixative (selected-SKU caption). That caption uses citrate retrieval at pH 6 for 20 minutes, but does not establish that retrieval is required for every specimen (selected-SKU caption). Whether frozen sections or IF/ICC are easier is undetermined from the supplied evidence; dense cerebellar neuropil warrants careful background assessment when scoring the expected neuronal pattern (HPA: Medium in molecular-layer neuropil; UniProt Q9H2X9: cell membrane and dendrites).

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 chromogenic IHC in paraffin sections using the catalog antibody’s tissue image, reported localization, and matched controls.

What retrieval should I use when SLC12A5 staining is weak in paraffin sections?

Use heat-mediated citrate buffer at pH 6 for SLC12A5 paraffin-section IHC (datasheet A04629-1). The selected tissue image used 20 minutes of retrieval in that buffer before chromogenic detection (caption A04629-1). Process a known positive CNS section alongside the test section, because neuronal staining in cerebral cortex and neuropil staining in cerebellum provide useful comparison patterns (HPA tissue IHC). If staining remains weak, compare retrieval times on adjacent sections while holding antibody concentration and detection conditions constant (standard IHC practice). Check tissue integrity and background before accepting a stronger signal as an improvement (standard IHC practice).

Could fixation explain weak or uneven SLC12A5 staining?

Target-specific sensitivity to fixation is unknown from the supplied evidence, and the selected paraffin-section caption does not report its fixative (caption A04629-1). Record the fixative and fixation duration for each specimen before comparing SLC12A5 staining across cases (standard IHC practice). If processing varies, stain adjacent sections from the same block together and include a consistently processed positive control (standard IHC practice). Keep citrate retrieval at pH 6 and the antibody incubation constant while evaluating processing differences (datasheet A04629-1; caption A04629-1). Do not assign weak staining to fixation alone without a matched processing comparison (standard IHC practice).

How should membrane and cytoplasmic SLC12A5 staining be interpreted?

SLC12A5 is annotated at the cell membrane and dendrites, with detection at inhibitory synapses in the supplied record (UniProt Q9H2X9). Tissue IHC reports selective cytoplasmic expression in the CNS, including neuronal cells in cerebral cortex and molecular-layer neuropil in cerebellum (HPA tissue IHC). In chromogenic sections, assess staining around neuronal somata and within neuropil separately, since dense processes can obscure precise membrane boundaries (standard IHC practice). Compare each compartment with the same region in a negative-control section before calling diffuse pigment specific (standard IHC practice). A cytoplasmic IHC pattern alone does not establish intracellular transport or exclude membrane localization (HPA tissue IHC; UniProt Q9H2X9).

How can epitope placement alter SLC12A5 IHC results?

The record lists 2 SLC12A5 isoforms, but the supplied antibody caption does not identify its epitope (UniProt Q9H2X9; caption A04629-1). Ask for the antibody’s immunogen sequence and map it against both isoforms before interpreting selective staining as isoform specific (standard IHC practice). SLC12A5 has 12 transmembrane segments and a long cytoplasmic region spanning residues 633–1139 (UniProt Q9H2X9 topology). Its extracellular region includes annotated glycosylation sites at residues 314, 333, 351, and 362 (UniProt Q9H2X9). Epitope accessibility may vary with processing, so establish specificity using matched controls rather than inferring an epitope from staining alone (standard IHC practice).

How should I adapt SLC12A5 staining for multiplex IF?

For IF, pair SLC12A5 with a validated neuronal marker and inspect signal within marker-positive cells, consistent with reported neuronal expression (UniProt Q9H2X9; HPA tissue IHC). Select spectrally separated fluorophores and consider a far-red channel if the tissue has strong shorter-wavelength autofluorescence (standard IF practice). Check unstained and single-stained sections before assigning overlapping signals to colocalization (standard IF practice). Choose permeabilization after mapping the antibody epitope: a cytoplasmic epitope generally requires access through the membrane, whereas an accessible extracellular epitope may not (UniProt Q9H2X9 topology; standard IF practice). The supplied HPA subcellular record lists no ICC/IF images, so verify this antibody’s IF performance independently (HPA subcellular).

What should I check when SLC12A5 DAB staining is widespread?

The selected paraffin-section image used a biotinylated secondary, a streptavidin–biotin complex, and DAB development (caption A04629-1). Check a no-primary section for signal from detection reagents, and use appropriate endogenous peroxidase and biotin controls when troubleshooting that workflow (standard IHC practice). Compare questionable staining with reported neuronal and neuropil patterns in CNS tissue (HPA tissue IHC). Brown deposits at section edges, folds, or damaged areas should be evaluated separately from intact cells and processes (standard IHC practice). If background persists, compare blocking, primary-antibody concentration, wash conditions, and DAB development time one variable at a time (standard IHC practice).

How should I quantify SLC12A5 staining across brain sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the neuronal population and anatomical region before scoring, because the reported CNS staining includes cortical neuronal cells and cerebellar molecular-layer neuropil (HPA tissue IHC). For identifiable neuronal somata, record percent positive cells and intensity, or calculate an H-score from those categories (standard IHC practice). For neuropil, measure DAB-positive area or optical density per mm² within a fixed region of interest (standard IHC practice). Normalize cell counts to the total eligible neurons and neuropil measurements to the measured region area, using identical thresholds across sections (standard IHC practice). Report soma and neuropil results separately, with background subtraction from matched negative controls (standard IHC practice).

When is apparent SLC12A5 positivity likely to be artefactual?

Expect SLC12A5 findings to align with neuronal and neuropil expression in CNS tissue and with its membrane and dendritic annotation (HPA tissue IHC; UniProt Q9H2X9). Be cautious with strong staining confined to unrelated cell populations, because HPA reports selective CNS expression and several non-CNS tissues as not detected (HPA tissue IHC). The selected antibody image shows staining in paraffin-embedded human tonsil, so interpret that result against matched controls rather than treating it as proof of broad tissue expression (caption A04629-1; HPA tissue IHC). Exclude section edges, necrotic areas, and endogenous-enzyme signal using morphology and detection controls (standard IHC practice). Replicate a convincing cellular pattern across intact sections before assigning biological meaning (standard IHC practice).

Boster reagents

Best SLC12A5 / Solute carrier family 12 member 5 IHC Antibodies

The IHC-validated anti-SLC12A5 antibody A04629-1 has paraffin-section images from human tonsil, ovarian cancer and placenta, and rat brain (catalog image captions).

Real IHC data

Anti-KCC2/SLC12A5 Antibody ®

Cat # A04629-1

View product →

A04629-1 is the only SKU shown and lists IHC as an application (catalog: applications). Its IHC captions show staining in paraffin sections of human tonsil, ovarian cancer and placenta, and rat brain (catalog image captions).

Which to pick: Choose A04629-1 for paraffin-section IHC; its tonsil caption documents citrate retrieval and 1 μg/ml primary antibody (A04629-1 IHC image caption). No IF/ICC-validated SKU is listed (catalog: applications; if_image_alts empty). For cross-species work, A04629-1 lists human, mouse and rat reactivity, while its IHC images document human and rat samples; the fixative is unreported (catalog: reactivity; IHC 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.